Published online before print December 20, 2007, doi: 10.1161/CIRCRESAHA.107.164731
© 2008 American Heart Association, Inc.
Cellular Biology |
The Cell Cycle Factor E2F-1 Activates Bnip3 and the Intrinsic Death Pathway in Ventricular Myocytes
From the Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Departments of Physiology (N.Y., K.B., D.W., R.J., B.F., L.A.K.), Pharmacology & Therapeutics (J.S., L.A.K.), Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
Correspondence to Dr Lorrie A. Kirshenbaum, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Room 3016, 351 Taché Ave, Winnipeg, Manitoba, Canada R2H 2A6. E-mail Lorrie{at}sbrc.ca
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Abstract |
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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.
Key Words: Bnip3 • Bnip3L/Nix • E2F-1 • apoptosis • hypoxia • mitochondria • myocyte
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Introduction |
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Defects in the regulatory pathways that govern apoptosis have been linked to a variety of human pathologies, including neurodegenerative diseases, cancer, and cardiovascular disease. Mitochondrial perturbations have been postulated to be an underlying feature of the intrinsic cell death pathway (reviewed previously1). Oxygen deprivation provokes mitochondrial permeability transition pore (PTP) opening, loss of mitochondrial membrane potential (
m), cytochrome c release, and cell death of ventricular myocytes.2,3 Previously, we have established the mitochondrial death protein Bnip3 as an integral component of the intrinsic death pathway during hypoxic injury of postnatal ventricular myocytes.4,5 However, the signaling pathways and transcriptional processes that regulate Bnip3 transcription remain poorly defined. The cellular factor E2F-1 is the archetypal member of a family of transcription factors first characterized for their ability to activate genes required for G1 exit and DNA synthesis.6 To date, at least 6 homologs of E2F (E2F-1 to E2F-6), together with their dimerization partners DRFT1-polypeptides 1 and 2 (DP-1 and DP-2), have been identified.7,8 Notably, E2F-1 is uniquely distinguished from other E2F proteins by its propensity to provoke apoptosis,7–9 yet the underlying mechanism for this property of E2F-1 is, at best, poorly understood. In cells, E2F-1 interacts with the retinoblastoma gene product Rb via its L-X-C-X-E motif, which is crucial for suppressing cellular E2F activity and E2F-1–dependent promoters.10 Accordingly, loss-of-function mutations or germ line deletion of Rb in mice results in increased E2F-1 activity and apoptosis.11 Furthermore, the unexpected and counterintuitive tumorigenesis in E2F-1–/– mice underscores the importance of E2F-1 as a key regulator of apoptosis.12
Previously, we have shown that displacement of E2F-1 from Rb by viral oncoproteins or overexpression of E2F-1 alone was sufficient to provoke apoptosis of ventricular myocytes; however, the underlying mechanism(s) was not determined.13 In this report, we provide new compelling evidence that E2F-1 provokes cell death of ventricular myocytes through a mechanism that directly impinges on the intrinsic death pathway. We further show that the death factor Bnip3 is a direct transcriptional target of E2F-1 that elicits death signals downstream of E2F-1 during hypoxia. Our data provide new evidence that operationally links E2F-1 to the intrinsic death pathway via the death factor Bnip3.
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Materials and Methods |
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Cell Culture, Transfection, and Hypoxia
Ventricular myocytes were isolated from 1- to 2-day-old Sprague–Dawley rats and submitted to primary culture as reported.13 After overnight incubation in DMEM/Ham’s nutrient mixture F-12 (1:1), 17 mmol/L HEPES, 3 mmol/L NaHCO3, 2 mmol/L L-glutamine, 50 µg/mL gentamicin, and 10% FBS, cells were transferred to serum-free medium for 24 to 48 hours as reported previously.13 Cells were transfected with Bnip3 luciferase reporter plasmids (0.5 µg), along with an expression plasmid for β-galactosidase or small interference (si)RNA (10 nmol/L) using Effectine Transfection Reagent (Qiagen Inc) under serum-free conditions as reported previously.4,13 Luciferase reporter activity was normalized to β-galactosidase activity to control for potential differences in transfection efficiency between myocyte cultures. Details for 2.3 kb of the human Bnip3 promoter luciferase reporter (Bnip3 Luc) have been reported previously.4 Eukaryotic expression plasmids encoding the wild-type Rb (Rb) and caspase resistant Rb (RbMI) were kindly provided by Dr J. Wang (University of California at San Diego).14 Expression plasmids encoding cDNAs for wild-type E2F-1 and mutant E2F-1E138 proteins have been described previously.15 Cells were subjected to hypoxia for 24 hours in an air-tight chamber under serum-free culture conditions continually gassed with 95% N2, 5% CO2, with a PO2 of
5 mm Hg, as described previously.2,4,5
Recombinant Adenovirus and siRNA
Myocytes were infected with a control adenovirus (AdCMV), adenoviruses encoding E2F-1 (AdE2F-1), or carboxyl-terminal transmembrane domain deletion mutant of Bnip3 defective for mitochondrial targeting (AdBnip3TM), as reported previously.4,16 siRNA directed against E2F-1 was obtained from Santa Cruz Biotechnology (catalog no. cs-29297). Our preliminary findings verified the specificity of siRNA directed against E2F-1 for silencing of the E2F-1gene without influencing other E2F proteins or the housekeeping control gene L32. Earlier, we described the generation of short hairpin interference RNA (shRNAi) directed against Bnip3.5 Adenoviral delivery of shRNAi against Bnip3 was generated using the BLOCK-iT adenoviral RNAi expression system (Invitrogen) using a target sequence of 5'-GATCTACATTGGAAGGCGTCT-3'; we previously characterized the authenticity and specificity of the shRNAi knockdown sequence in ventricular myocytes, which achieves 
80% inhibition of endogenous Bnip3 gene expression.5 All adenovirus were used at a multiplicity of infection of 10.4,13
Apoptosis and Cell Viability
Cell viability was determined using the vital dyes calcein–acetoxymethyl ester (calcein-AM) (2 µmol/L; green) to determine the number of living cells and ethidium homodimer-1 (2 µmol/L; red) to identify the number of dead cells as reported previously.2,18 Following interventions, cell viability was assessed by counting 200 cells from at least 3 to 4 independent myocyte isolations from 3 different glass coverslips for each condition tested. Apoptotic cell death was assessed by visually inspecting individual myocytes from at least 3 to 4 independent myocyte isolations counting 200 cells for each condition tested from 3 different glass coverslips. Individual cells were assessed for evidence of nucleosomal DNA fragmentation characterized by pyknotic hyperchromatic staining nuclei by Hoechst 33258 dye (Molecular Probes, Eugene Ore), as reported previously.2,18 All fluorescent images were captured using an Olympus AX70 Research grade epifluorescence microscope.
Immunocytochemistry
Immunocytochemistry was performed on fixed cells using a primary murine antibody directed against E2F-1 (clone KH95, Santa Cruz Biotechnology)18 that was detected by subsequent incubation with a secondary antibody conjugated to the fluorochrome Alexa Fluor 488 (Molecular Probes). Nuclei were visualized with the fluorescent dye TO-PRO-3 (Molecular Probes).
Mitochondrial PTP
Mitochondrial PTP opening was determined by loading ventricular myocytes with 5 µmol/L calcein-AM (Molecular Probes) in the presence of 2 to 5 mmol/L cobalt chloride, as reported previously.2,17 Changes in integrated fluoresce intensity is an index of PTP opening. Data are expressed as mean percentages change±SE.
Western Blot Analysis
For detection of the Rb 68-kDa cleavage fragment, total cardiac cell lysate (50 µg) was subjected to Western blot analysis using a murine antibody directed toward the 68-kDa Rb cleavage fragment (cleaved [Ab-1], clone 172C1094, catalog no. OP198; Oncogene Research Products).14 The filter was stained with Ponceau-S dye (0.1%) to verify equivalent protein loading of the Western Blot. Bound proteins were detected by enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia Inc).
Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation (ChIP) assays were performed using a ChIP assay kit (Upstate, Lake Placid, NY) with slight modifications involving a 2-step cross-linking step, as described by Nowak et al.19 DNA and protein were cross-linked in cells by incubation with 2 mmol/L disuccinimidyl glutarate for 45 minutes at room temperature and washed 3 times with PBS, followed by incubation with 1% formaldehyde at room temperature for 15 minutes. Sonicated lysates were immunoprecipitated with 2 µg of antibody directed against E2F-1 (clone KH95, Santa Cruz Biotechnology) and incubated overnight at 4°C with rotation. Purified DNA was amplified by PCR using a primer pair designed to amplify a 278-bp segment spanning the E2F-1 elements within the Bnip3 promoter: forward, 5'-AAAGCGGGAAATGAGAAAGC-3'; reverse, 5'-TCGAGCAGAGTCGAAAGAGTC-3'. PCR products were analyzed on a 2% agarose gel.
RNA and Quantitative Real-Time PCR Analysis
Total RNA was extracted from cells using TRIzol Reagent (Invitrogen), followed by treatment with DNase I as reported previously.5 RNA (1 µg) was reverse-transcribed with oligo(dT) primers using SuperScript III Reverse Transcriptase (Invitrogen). Gene-specific primers were designed to amplify Bnip3, Nix, and the housekeeping gene L32 by quantitative PCR on a Bio-Rad iCycler thermal cycler using iQSYBR Green Supermix (Bio-Rad). Specific primers were designed using Primer3 version 4.0 software from the GenBank sequence for the rat Bnip3, Nix, and L32, respectively,5: Bnip3 forward, 5'-TGCACTTCAGCAATGGGAAT-3'; Bnip3 reverse, 5'-ACATTTTCTGGCCGAATTGA-3' (accession number no. NM_053420); Nix forward, 5'-CATCCACAATGGAGACATGGAG-3'; Nix reverse, 5'-CTGATACCCAGTCCGCACTTTT-3' (accession no. NM_080888); L32 gene forward, 5'-TAAGCGAAACTGGCGGAAAC-3'; L32 gene reverse, 5'-GCTGCTCTTTCTACGATGGCTT-3' (accession no. XO 6483). All amplifications were performed in the final reaction mixture (25 µL) containing 1x final concentration of SYBR Green Supermix, 500 nmol/L gene-specific primers, and 0.5 µL of template under the following conditions: 95°C for 5 minutes, followed by 40 cycles consisting of 95.0°C for 10 seconds and 60°C for 30 seconds. Following amplification, iCycler 3.1 software was used to establish the baseline and threshold for each reaction. A cycle threshold (Ct) was assigned at the beginning of the logarithmic phase of PCR amplification. The 2–CT (Livak) method was used to calculate the relative gene expression. Expression of each gene relative to the L32 housekeeping gene was calculated as the difference between the Ct values of the 2 genes (Ct). The difference in the Ct of the control and experimental sample (Ct) was used to determine the relative expression of the gene in each sample. The generation of specific PCR products was confirmed by melting curve analysis and agarose gel electrophoresis. The values are means±SE from 3 separate experiments in duplicate for each condition tested.
Semiquantitative RT-PCR
RT-PCR was performed using 0.5 µg of total RNA using the Access RT-PCR System (Promega Corp, Madison, Wis) on an MBS Satellite 0.2G Thermo Fisher Scientific for Bnip3, E2F-1, or housekeeping control gene L32, respectively; the forward and reverse primer set for Bnip3 and L32 genes are described above. The primers for E2F-1 were as follows: forward, 5'-ACGCTATGAAACCTCACTAAA-3'; reverse, 5'-AGGACATTGGTGATGTCATA-3'. RT-PCR products were analyzed by 2% gel electrophoresis as reported earlier.5
Statistical Analysis
Multiple comparisons between groups were determined by 1-way ANOVA. Unpaired 2-tailed Student’s t test was used to compare mean differences between groups. Differences were considered to be statistically significant at P<0.05. In all cases, unless otherwise stated, the data were obtained from at least 3 to 4 independent myocyte isolations, using replicates (n=3 for each condition tested).
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Results |
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As a step toward understanding the underlying mechanism by which E2F-1 provokes apoptosis in cells, postnatal ventricular myocytes were infected with recombinant adenovirus encoding E2F-1 and assessed for nucleosomal DNA fragmentation by Hoechst 33258 nuclear DNA staining. Apoptotic cell death was determined by visually inspecting individual myocyte nuclei for nucelosomal DNA fragmentation and the appearance of pyknotic hyperchromatic staining nuclei, as reported previously.2,18 As shown in Figure 1A and 1B, cells overexpressing E2F-1 displayed a significantly greater percentage of apoptotic nuclei compared with control cells, verifying that E2F-1 is toxic to myocytes and provokes apoptosis. Because perturbations to mitochondria from the opening of the mitochondrial PTP have been suggested to be an underlying feature of the intrinsic death pathway, we assessed whether cell death provoked by E2F-1 involves mitochondrial PTP opening. For these experiments, ventricular myocytes were loaded with calcein-AM in the presence of cobalt chloride to quench the cytoplasmic signal in the presence and absence of E2F-1. The loss of green fluorescence by mitochondria is an index of PTP opening. As shown in Figure 1C and 1D, control cells displayed punctate green staining mitochondria indicative of the PTP in closed configuration; however, a marked reduction in mitochondrial green fluorescence was observed in cells expressing E2F-1, a finding consistent with PTP opening. These findings establish that E2F-1 provokes mitochondrial perturbations, consistent with the intrinsic apoptotic pathway.
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Earlier work from our laboratory and by others established the BH3-only protein Bnip3 as a central component of the intrinsic death pathway in ventricular myocytes during hypoxic injury.4,20 Interestingly, sequence analysis of the Bnip3 promoter revealed the presence of canonical DNA elements for E2F-1, raising the intriguing possibility that Bnip3 may be a transcriptional target of E2F-1. To test the possibility that Bnip3 expression was transcriptionally regulated by E2F-1, we monitored Bnip3 gene expression in ventricular myocytes in the presence and absence of E2F-1. As shown in Figure 2A, overexpression of E2F-1 in ventricular myocytes resulted in a 1.5-fold induction (P<0.001) of Bnip3 luciferase reporter gene activity compared with cells expressing a mutation of E2F-1 (E2F-1E138) defective for DNA binding or vector control cells. Concordant with this finding was a marked 6.2-fold increase (P<0.001) in endogenous Bnip3 gene expression in cells expressing E2F-1, as indicated by quantitative real-time PCR (Figure 2B). Interestingly, no apparent change in the expression levels of the Bnip3 homolog protein Nix/Bnip3L was observed in myocytes expressing E2F-1 (Figure 2B), verifying that Bnip3 and not Nix/Bnip3L is transcriptionally activated by E2F-1. To further validate the importance of E2F-1 in the transcriptional activation of Bnip3, we conducted additional experiments in which we assessed the level of Bnip3 gene activation using 2 independent approaches that interfere with cellular E2F-1 activity.
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statistically different from E2F-1. B, Quantitative real-time PCR analysis for endogenous Bnip3 (
) and Nix (
) genes, respectively, in the presence and absence of Rb and E2F-1 proteins. *Statistically different from control;
Because cellular E2F-1 is regulated by Rb, we first tested whether expression of Rb itself as a means to sequester E2F-1 activity would influence Bnip3 gene transcription. As shown in Figure 2B, E2F-1–induced Bnip3 gene transcription was markedly suppressed in cells in the presence of Rb compared with E2F-1 alone, supporting our contention for the involvement of E2F-1 in the regulation of Bnip3. To further verify the importance of E2F-1 in the regulation of Bnip3, we next assessed by an alternative method whether siRNA directed against E2F-1, as a means to genetically knock down E2F-1, would impair Bnip3 gene expression. Previously we demonstrated the utility and authenticity RNAi knockdown in ventricular myocytes, which achieves 80% efficiency of inhibition of endogenous gene expression.5 As shown in Figure 2C, in contrast to vector control cells, genetic knockdown of E2F-1 resulted in a significant reduction in endogenous Bnip3 gene transcription, a finding consistent with the repression of Bnip3 gene expression with Rb (Figure 2B). Collectively, our data strongly suggest that E2F-1 is important for the transcriptional regulation of Bnip3.
To test the physiological significance of these findings and to substantiate the possibility that E2F-1–induced cell death involves Bnip3, we next tested whether loss-of-function mutations of Bnip3 would abrogate the cytotoxic actions of E2F-1. For these experiments, we used a carboxyl-terminal mutant of Bnip3 (Bnip3TM), which has been shown previously by our laboratory to be defective for mitochondrial membrane insertion and for provoking cell death. As shown in Figure 3A and 3B, in the presence of the Bnip3 TM mutant, E2F-1–induced cell death was dramatically suppressed compared with cells expressing E2F-1 alone. Similarly, E2F-1–induced cell death was strongly inhibited by genetic knockdown of Bnip3 using RNAi directed against Bnip3. These findings confirm and support our contention that E2F-1–induced cell death is obligatorily linked and mutually dependent on Bnip3.
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Because earlier work established that Bnip3 is transcriptionally activated in ventricular myocytes during hypoxia,4,20 we next ascertained whether E2F-1 influences Bnip3 transcription during hypoxia. To test this possibility, postnatal ventricular myocytes were subjected to 24 hours of hypoxia and assessed for E2F-1 activity. As shown in Figure 4A, in contrast to normoxic control cells, a marked increase in endogenous E2F-1 gene expression was observed in ventricular myocytes subjected to hypoxia. In accordance with these findings, immunostaining of ventricular myocytes with an antibody directed against E2F-1 protein verified that E2F-1 protein was localized to nuclei of hypoxic cells compared to the predominant cytoplasmic distribution observed in normoxic control cells (Figure 4B).
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Previous work from our laboratory established the involvement of the death effector caspases as an underlying feature of hypoxic injury in cardiac myocytes.2,21 This observation, together with the recent finding of a caspase cleavage site in Rb,22,23 prompted us to test whether Rb is proteolytically cleaved in ventricular myocytes during hypoxia. For these studies, cardiac cell lysates derived from normoxic and hypoxic myocytes were subjected to Western blot and analyzed for the presence of the 68-kDa Rb caspase cleavage product.23 As shown in Figure 4C, in contrast to normoxic control cells, a marked increase in the 68-kDa Rb cleavage product was detected in myocytes subjected to hypoxia, a finding consistent with the increased E2F-1 activity in hypoxic cells.
To assess whether E2F-1 is involved in the hypoxia-induced activation of Bnip3, we next monitored Bnip3 promoter activity under normoxic and hypoxic conditions in the presence of interventions that suppress or inhibit E2F-1 activity. As shown in Figure 5, a 3.8-fold increase (P<0.001) in Bnip3 luciferase reporter activity was observed in myocytes during hypoxia compared with normoxic control cells. However, hypoxia-induced expression of the Bnip3 luciferase reporter was markedly suppressed by a caspase-resistant form of Rb (RbMI). Furthermore, genetic knockdown of E2F-1 similarly repressed hypoxia-induced Bnip3 promoter luciferase activity, substantiating our contention that E2F-1 is required for activating the Bnip3 promoter during hypoxia.
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To verify that the induction of Bnip3 gene transcription by E2F-1 was directly related to E2F-1 occupying the Bnip3 promoter during hypoxia, we performed ChIP analysis of the Bnip3 promoter under normoxic and hypoxic conditions. As shown in Figure 6, E2F-1 was relatively undetectable at the Bnip3 promoter under normoxic control conditions; however, it readily bound the Bnip3 promoter during hypoxia. Importantly, hypoxia-induced E2F-1 promoter binding was abrogated in cells expressing of Rb. These findings are concordant with our transcription data for Bnip3 and confirm that E2F-1 directly engages the Bnip3 promoter during hypoxia to affect Bnip3 gene transcription. Collectively our data support our contention that E2F-1 is necessary and sufficient for induction of Bnip3 transcription.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously we reported that E2F-1 was sufficient to provoke apoptosis of postnatal ventricular myocytes; however, the underlying mechanism was not determined.24,25 In this report, we provide new evidence that E2F-1 provokes cell death of ventricular myocytes through a mechanism that impinges on the intrinsic death pathway. We further show that the death factor Bnip3 is a direct transcriptional target of E2F-1 that is necessary for E2F-1–induced cell death. Bnip3 was first identified as an adenovirus E1B 19-kDa–interacting protein26,27; however, its role in the context of the viral oncoprotein E1A- or E2F-1–induced cell death has not been addressed.13 Since these initial observations, our laboratory established Bnip3 as a critical component of the intrinsic death pathway during hypoxic injury.4,5 Nix/Bnip3L is the closest homolog to Bnip3,28 but, unlike Bnip3, Nix appears to be regulated by hypertrophic growth signals and not by hypoxia or ischemic injury,29,30 highlighting the diversity of these factors. Bnip3 is further distinguished from other Bcl-2 family members by at least 2 important features. The first is that Bnip3 is transcriptionally activated by hypoxic or ischemic stress4; the second is the presence of consensus elements for E2F-1 within the Bnip3 promoter, raising the strong possibility that Bnip3 may be a transcriptional target of E2F-1. Indeed, our finding that endogenous Bnip3 gene expression and not Nix/Bnip3L was increased in ventricular myocytes in the presence of E2F-1 supports this contention.
Furthermore, this appears to be a restricted feature of E2F-1, given that neither E2F-2 nor E2F-3 had any influence on Bnip3 gene transcription (N.Y. and L.A.K., unpublished data, 2007). Hence, the unique ability of E2F-1 to regulate Bnip3 may explain, in part, the propensity of E2F-1 for provoking apoptosis over the other E2F factors.8,31,32 For this reason, the findings of the present study are intriguing and suggest that E2F-1 induces cell death through a mechanism that involves de novo Bnip3 gene transcription.
The interrelationship between E2F-1 and Bnip3 is profound because we show by not 1 but by 2 independent approaches that functional inhibition of Bnip3 was sufficient to suppress the cytotoxic actions of E2F-1. These findings substantiate the importance of Bnip3 as a downstream target of E2F-1 during hypoxia-induced apoptosis of cardiac myocytes. The fact that deregulated Bnip3 would otherwise be lethal to cells and provoke apoptosis implies that it must be under highly regulated and tight transcriptional control. Indeed, earlier work by our laboratory established that the Bnip3 promoter is strongly repressed under basal conditions.5,33 Therefore, we believe that an inappropriate or untimely Bnip3 gene transcription would have catastrophic consequences on cell survival. The limited E2F-1 binding to the Bnip3 promoter under basal normoxic conditions is consistent with this view. Although unproven, it is tempting to speculate that the inordinate apoptosis and midgestational lethality observed in the Rb–/– mice is a result of increased Bnip3 activation from deregulated E2F-1 activity. This notion is founded on relative resistance of the E2F-1–/– mice to apoptotic signals34 and our own findings demonstrating that interventions that suppressed E2F-1 activity similarly suppressed Bnip3 gene transcription. The finding that Rb was proteolytically cleaved during hypoxia is intriguing and exemplifies the genetic and functional diversity among other pocket protein members, p107 and p130, that do not contain caspase cleavage sites and do not appear to be involved in apoptosis.23 The finding that overexpression of a caspase-resistant Rb suppressed hypoxia-induced E2F-1 binding to the Bnip3 promoter and Bnip3 gene transcription strongly supports the importance of E2F-1 as a transcriptional activator of Bnip3 and the putative role of Rb as an antideath factor.35
To our knowledge, our data provide the first direct evidence that Bnip3 is transcriptionally activated in ventricular myocytes by E2F-1 and underlies E2F-1–induced mitochondrial defects and cell death. Whether E2F-1 acts in concert with transcription factors such as hypoxia-inducible factor-1
to activate Bnip3 transcription during hypoxia in ventricular myocytes is unknown and an active area of investigation.
Importantly, our finding that Bnip3 is transcriptionally activated by E2F-1 is in complete agreement with a recent report documenting the regulation of Bnip3 by E2F-1 in nonmuscle Rb-deficient cells,36 supporting our contention that Bnip3 is a transcriptional target of E2F-1 in ventricular myocytes. Although we cannot exclude the possibility that other factors regulated by E2F-1 may contribute to cell death induced by Bnip3,37 the notion that E2F-1 causes cell death directly via increased Bnip3 expression is favorable based on our finding that E2F-1 engages the Bnip3 promoter coincidentally with the activation of Bnip3 transcription. Additionally, the fact that loss-of-function mutations of Bnip3 or genetic knockdown of Bnip3 with siRNA abrogated the ability of E2F-1 to provoke cell death would argue that other death factors are not likely involved in E2F-1 induced cell death seen here. The findings of the present study are intriguing and provide new mechanistic evidence that E2F-1 is necessary and sufficient for induction of Bnip3 gene transcription. Based on the present findings, our data establish a novel paradigm for regulation of the intrinsic death pathway by E2F-1 that is operationally linked and mutually dependent on the transcriptional activation of Bnip3.
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Acknowledgments |
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We thank Dr H. Weisman for critical comments on the manuscript; Dr Jean Wang (University of California at San Diego) for the reagents cited; Dr Jacqueline Lees (Massachusetts Institute of Technology Center for Cancer Research, Boston) for insightful comments and advice; and Dr Tong Zhang and Floribeth Aguilar for technical assistance.
Sources of Funding
J.S. is supported by a Studentship award from the Manitoba Health Research Council. This work was supported by grants from the Canadian Institutes for Health Research (to L.A.K.). L.A.K. is a Canada Research Chair in Molecular Cardiology.
Disclosures
None.
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Footnotes |
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Original received September 21, 2007; revision received November 7, 2007; accepted December 12, 2007.
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References |
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