Nuclear Factor-κB–Mediated Cell Survival Involves Transcriptional Silencing of the Mitochondrial Death Gene BNIP3 in Ventricular Myocytes
Background— A survival role for the transcription factor nuclear factor-κB (NF-κB) in ventricular myocytes has been reported; however, the underlying mechanism is undefined. In this report we provide new mechanistic evidence that survival signals conferred by NF-κB impinge on the hypoxia-inducible death factor BNIP3.
Methods and Results— Activation of the NF-κB signaling pathway by IKKβ in ventricular myocytes suppressed mitochondrial permeability transition pore (PTP) opening and cell death provoked by BNIP3. Expression of IKKβ or p65 NF-κB suppressed basal and hypoxia-inducible BNIP3 gene activity. Deletion analysis of the BNIP3 promoter revealed the NF-κB elements to be crucial for inhibiting basal and inducible BNIP3 gene activity. Cells derived from p65−/−-deficient mice or ventricular myocytes rendered defective for NF-κB signaling with a nonphosphorylative IκB exhibited increased basal BNIP3 gene expression, mitochondrial PTP, and cell death. Genetic or functional ablation of the BNIP3 gene in NF-κB–defective myocytes rescued them from mitochondrial defects and cell death.
Conclusions— The data provide new compelling evidence that NF-κB suppresses mitochondrial defects and cell death of ventricular myocytes through a mechanism that transcriptionally silences the death gene BNIP3. Collectively, our data provide new mechanistic insight into the mode by which NF-κB suppresses cell death and identify BNIP3 as a key transcriptional target for NF-κB–regulated expression in ventricular myocytes.
Received July 6, 2005; revision received September 2, 2005; accepted October 3, 2005.
Emerging evidence suggests that mitochondria play a central role in the integration and execution of apoptotic signals.1 Perturbations to mitochondria from the opening of a large multiprotein conductance channel referred to as permeability transition pore (PTP) have been reported.2 Earlier work by our laboratory demonstrated that hypoxia triggers mitochondrial perturbations and cell death of adult and neonatal ventricular myocytes.3,4 This was attributed to the induction of the death protein BNIP3 in hypoxic cells.3,3a Mutations of BNIP3 defective for mitochondrial targeting suppressed hypoxia-induced cell death, identifying BNIP3 as a key regulator of mitochondrial function and cell death of ventricular myocytes.
The cellular factor nuclear factor-κB (NF-κB) regulates a variety of processes including inflammation, differentiation, and cell survival. In unstimulated cells, NF-κB is composed of p50- and p65-kDa protein subunits bound to the cytoplasmic inhibitor protein IκBα. Signal-induced activation of NF-κB involves the phosphorylation-dependent degradation of IκBα mediated by the IKK signaling complex. Ostensibly, degradation of IκBα permits NF-κB to translocate to the nucleus and affect gene transcription. However, the ability of NF-κB to shuttle freely between cytoplasm and nucleus in unstimulated cells has been reported,5 suggesting that NF-κB may regulate basal gene expression. In this context, the known biological actions of NF-κB, including cell survival, have been attributed to the well-established and acknowledged property of the p65 NF-κB subunit as a transcriptional coactivator.6 However, a less defined but emerging role for NF-κB includes transcriptional repression.7,8
Clinical Perspective p 3785
Recently, we demonstrated that IKKβ-mediated NF-κB activation was sufficient to suppress hypoxia-induced mitochondrial perturbations and cell death of ventricular myocytes9; however, the underlying mechanism was undefined. In this report we provide novel mechanistic insight into the mode by which NF-κB suppresses cell death and show that NF-κB transcriptionally silences basal and inducible expression of the death gene BNIP3.
Cell Culture and Transfection
Postnatal ventricular myocytes from 2-day-old Sprague-Dawley rat hearts were isolated and submitted to primary culture as previously described.10 BNIP3 luciferase reporter constructs (BNIPLuc) wild-type and mutant were created by subcloning 2.3 kb of the human BNIP3 promoter into the HindIII/BglII sites of the luciferase reporter plasmid PGL3 (Promega, Inc).11 Deletion or point substitution mutations were introduced into the NF-κB sequences 5′-GGGACGC-3′ (base pairs −1075 to −1069) of the BNIP3 promoter by primer extension PCR (Quick Change, Stratagene Inc). Myocytes were transfected with the NF-κBLuc or herpes simplex virus thymidine kinase promoter (TKLuc) or BNIP3 promoter luciferase reporter constructs as reported.10,12 Wild-type 3T3 and p65−/−-derived 3T3 cell lines were generously provided by Dr D. Baltimore.13 Expression plasmids encoding wild-type p65 and transactivation-defective mutations (p65S529A, p65S36A) were kindly provided by Dr A. Baldwin.14 Small hairpin interference RNA against BNIP3 was generated as described15 (Block-iT U6 RNAi, Invitrogen Inc). Myocytes were harvested 24 to 48 hours after transfection. Luciferase activity was normalized to β-galactosidase activity to control for potential differences in transfection efficiency and expressed as relative light units.10
Normoxic control and hypoxic myocytes were infected with adenoviruses encoding wild-type IKKβ16 (AdIKKβwt), a kinase-defective IKKβ (AdIKKβmt), nonphosphorylative IκBα (AdIκBαSA),17 wild-type BNIP3 (AdBNIP3), mutant BNIP3 (AdBNIP3ΔTM),18,19 or an “empty” control adenovirus containing the CMV promoter (AdCMV), as previously described.10 Myocytes were infected at multiplicity of infection of 10, which achieves >90% of gene delivery to ventricular myocytes.10,18
Cell Culture and Hypoxia
Postnatal ventricular myocytes from 1- to 2-day-old Sprague-Dawley rats were subjected to hypoxia for 24 hours in an air-tight chamber under serum-free culture conditions and continually gassed with 95% N2/5% CO2, Po2 ≤10 mm Hg, as previously described.4,18,20
RNA Isolation and Semiquantitative Reverse Transcriptase–Polymerase Chain Reaction
Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed with the use of 0.5 μg of total RNA with the Promega Access RT-PCR System (Promega Corporation) on a PTC-100 thermocycler (MJ Research, Inc) for BNIP3 or housekeeping control gene L32, respectively; BNIP3: forward 5′-GGGTAGAACTGCAC-TTCAGCAA-3′ and reverse 5′-CCCTGTTGGTATCTTGTGGTGT-3′; L32 gene: forward 5′-TAAGCGAAACTGGCGGAAAC-3′ and reverse 5′-GCTGCTCTTTCTACGATGGCTT-3′. RT-PCR products were analyzed by 2% gel electrophoresis. Relative band intensity was quantified by fluorescence scanning densitometry and normalized to L32 gene on a Storm gel analysis system (Molecular Dynamics).
Western Blot Analysis
Cardiac myocytes were harvested in 1.0% NP-40 lysis buffer and resolved on a 10% sodium dodecyl sulfate–polyacrylamide gel at 140 V for 4 hours. For detection of p65 NF-κB or BNIP3 proteins, the nitrocellulose membrane (Roche Diagnostics) was incubated with a murine antibody directed toward BNIP319 or p65 NF-κB subunit (Santa Cruz Inc). Bound proteins were detected by chemiluminescence reaction with horseradish peroxidase–conjugated antibodies with the use of enhanced ECL reagents (Amersham Pharmacia).
Cell viability was determined with the use of the vital dyes calcein-acetoxymethylester (calcein-AM) (2 μmol/L) and ethidium homodimer-1 (2 μmol/L) to determine the number of live and dead cells, respectively (Molecular Probes), as reported.10,18 Cells were analyzed from at least n=3 to n=4 independent myocyte cultures counting ≥200 cells from replicates of n=3 for each condition tested. Data are expressed as mean±SE percent reduction from control.
Mitochondrial Permeability Transition Pore
Mitochondrial PTP opening was determined with 0.4 μmol/L calcein-AM (Molecular Probes) in the presence of 5 μmol/L cobalt chloride, as previously reported.4,18,21 Change in fluorescence intensity is an index of PTP opening. Data are expressed as mean±SE. Integrated optical density was determined.3,22
Multiple comparisons between groups were determined by 2-way ANOVA. Unpaired 2-tailed Student t test was used to compare mean differences between groups. Differences were considered statistically significant to a level of P<0.05. In all cases the data were obtained from at least n=3 to n=4 independent myocyte isolations, with n=3 replicates for each condition tested.
To formally address the mode by which NF-κB suppresses mitochondrial defects and cell death of ventricular myocytes during hypoxia, we focused our attention on the death protein BNIP3 because previous work from our laboratory established BNIP3 as a critical factor for provoking mitochondrial perturbations and cell death of ventricular myocytes during hypoxia.18
To establish whether IKKβ-mediated activation of NF-κB is sufficient to suppress mitochondrial perturbations and cell death provoked by BNIP3, cells were infected with recombinant adenoviruses encoding BNIP3 or IKKβ and assessed for cell viability.9,23 As shown in Figure 1A, myocytes overexpressing IKKβwt were indistinguishable from uninfected control cells (P=0.14, control versus IKKβwt) with respect to cell viability. In contrast, a 6.7-fold increase (P<0.001) in myocyte death was observed in cells overexpressing BNIP3, a finding concordant with our previous data verifying that BNIP3 provokes apoptosis of ventricular myocytes3 (Figure 1B). Importantly, cell death induced by BNIP3 was suppressed by the IKKβwt but not by the catalytically inactive IKKβ (IKKβmt) defective for activating NF-κB.
Because perturbations to mitochondria resulting from mitochondrial PTP opening are an underlying feature of BNIP3-induced cell death, we next ascertained whether IKKβ-mediated NF-κB activation was sufficient to suppress BNIP3-induced mitochondrial PTP changes.18,21 As shown in Figure 2A, NF-κB activation resulted in a significant 2.5-fold reduction (P<0.001) in mitochondrial green fluorescence in cells overexpressing BNIP3, a finding indicative of the PTP regulation by NF-κB. Importantly, BNIP3-induced mitochondrial PTP opening was suppressed in cells expressing IKKβ (P=0.08, BNIP3+IKKβ versus control), a finding consistent with the cell death data and concordant with our earlier work demonstrating suppression of mitochondrial PTP opening by IKKβ during hypoxia.9 Together the data establish that IKKβ-mediated NF-κB activation is sufficient to suppress mitochondrial defects and cell death of ventricular myocytes provoked by BNIP3.
Because a critical role for BNIP3 has been reported for provoking mitochondrial defects in ventricular myocytes during hypoxia, we ascertained whether NF-κB activation influences BNIP3 activity in hypoxic myocytes. As shown by semiquantitative RT-PCR analysis, a 2.6-fold increase in BNIP3 gene transcription was observed in cardiac myocytes subjected to hypoxia compared with normoxic control cells (Figure 3A). However, hypoxia-induced BNIP3 gene expression was repressed in cells overexpressing IKKβ. Similarly, BNIP3 protein expression was increased in cells subjected to hypoxia, a finding concordant with the BNIP3 gene transcription data but repressed in myocytes overexpressing IKKβ (Figure 3B). Collectively, the data raise the intriguingly possibility that the BNIP3 gene may be transcriptionally repressed by NF-κB.
Interestingly, sequence analysis of the BNIP3 promoter revealed canonical elements for NF-κB (base pairs −1075 to −1069). Therefore, to test the possibility that BNIP3 may be transcriptionally regulated by NF-κB, postnatal ventricular myocytes were transfected with luciferase reporter constructs for NF-κB or BNIP3 in the presence and absence of IKKβwt or a kinase-inactive IKKβmt. As shown in Figure 3C, a 7.9-fold increase (P<0.001) in NF-κB luciferase gene activity was observed in cells transfected with IKKβwt compared with vector-transfected cells or cells transfected with a kinase-defective mutant, IKKβmt.24 Moreover, IKKβ-mediated NF-κB–induced gene transcription was inhibited with a nonphosphorylative form of IκBα (IκBαSA), verifying that the observed increase in NF-κB promoter activity by IKKβ was contingent on the activation of p65 NF-κB. In contrast, a 1.5-fold decrease (P<0.001) in BNIP3 promoter activity was observed in cells transfected with IKKβwt compared with cells transfected with vector alone (Figure 3D). A 2.5- and 2.7-fold increase (P<0.001) in basal BNIP3 gene expression was observed in cells transfected with the kinase-inactive IKKβmt or IκBαSA, respectively (Figure 3D). The inhibitory effects of IKKβwt on BNIP3 promoter activity could be abrogated by inhibiting NF-κB signaling with IκBαSA. The data indicate that BNIP3 promoter is repressed by IKKβ-mediated NF-κB activation.
Because the biological properties of NF-κB are related to the p65 subunit, we next determined whether the inhibitory effects of IKKβ on BNIP3 promoter activity were mediated by p65 NF-κB subunit. As shown in Figure 4, a 5.6-fold increase (P<0.001) in NF-κB gene transcription was observed in cells transfected with the p65 compared with vector-transfected cells or cells transfected with the constitutively active TKLuc promoter, which lacks consensus NF-κB elements, verifying that p65-dependent gene activation was contingent on the presence of NF-κB response elements. Furthermore, the increased NF-κB promoter activity by p65 was inhibited with IκBαSA. In contrast, a 2.9-fold decrease (P<0.001) in BNIP3 gene transcription was observed in cells in the presence of p65 NF-κB. Furthermore, the inhibitory effects of p65 on BNIP3 promoter activity were alleviated by IκBαSA (Figure 4C), confirming that p65 NF-κB was responsible for repressing BNIP3 promoter activity.
To verify that the negative regulation of BNIP3 gene expression by p65 was contingent on the NF-κB elements within the BNIP3 promoter, we generated BNIP3 promoter luciferase reporter constructs in which the NF-κB consensus elements had been deleted or mutated. As shown in Figure 5A, compared with the wild-type BNIP3 promoter, a 62-fold increase (P<0.0001) in basal BNIP3 gene transcription was observed after deletion of the NF-κB elements. Similarly, point substitution mutations that disrupted the integrity of NF-κB elements within the BNIP3 increased basal BNIP3 expression, confirming that the NF-κB elements are essential for repressing BNIP3 gene expression (K. Ens, MSc, and L.A. Kirshenbaum, PhD, unpublished data, 2003). Importantly, the ability of IKKβ or p65 to repress BNIP3 gene activation was lost after deletion of the NF-κB elements (Figure 5B). Together, the data substantiate the importance of the NF-κB elements for repressing basal expression of BNIP3.
To determine whether the observed repression of BNIP3 gene expression by NF-κB was contingent on de novo activation by p65 NF-κB, we tested the impact of mutations of the p65 previously shown to be defective for transactivation on BNIP3 promoter activity. As shown in Figure 4D, BNIP3 promoter activity was suppressed in a manner comparable to that of wild-type p65 by either of the transactivation-defective p65 mutants tested, ruling out the possibility that repressive effects of the p65 on BNIP3 promoter activity were contingent on de novo gene activation by NF-κB.
Given that earlier work by our laboratory established a critical role for IKKβ-mediated NF-κB activation for suppression of mitochondrial perturbation during hypoxic injury of ventricular myocytes,9 we next tested whether NF-κB influences BNIP3 activity during hypoxia. For these experiments, ventricular myocytes were transfected with BNIP3 luciferase promoter reporter constructs and subjected to hypoxia in the presence and absence of p65. As shown in Figure 5C, in the absence of the p65, a significant 5.6-fold increase (P<0.001) in BNIP3 promoter activity was observed in cells subjected to hypoxia compared with normoxic control cells. However, in the presence of p65, BNIP3 gene expression during hypoxia was markedly repressed compared with vector-transfected control cells, a finding consistent with the repression of the endogenous BNIP3 protein in cells expressing IKKβ. Consistent with the increased BNIP3 expression was a marked reduction in NF-κB–dependent gene transcription and endogenous p65 protein levels in ventricular myocytes during hypoxia (Figure 5D, 5E).
To test the functional significance of these observations, we rendered ventricular myocytes defective for NF-κB activation with IκBαSA, previously shown by our laboratory to functionally block NF-κB–dependent DNA binding and gene activation.25 A 2.3-fold increase (P<0.01) in basal BNIP3 gene expression was observed in ventricular myocytes overexpressing IκBαSA compared with vector-transfected control cells (Figure 6A). Furthermore, cells rendered defective for NF-κB activation with either IκBαSA or IKKβmt displayed a 1.9- and 1.8-fold increase, respectively, in BNIP3 gene expression as determined by semiquantitative RT-PCR analysis (Figure 6C). In addition, a 2.9-fold increase (P<0.001) in basal BNIP3 gene transcription was observed in cells derived from p65−/− mice compared with wild-type cells (Figure 6B). Consistent with a cytoprotective role for NF-κB, the p65−/− mouse embryonic fibroblast cells were found to be more sensitive to hypoxic stress, displaying a 2.1-fold increase (P<0.01) in cell death compared with wild-type control cells (J. Shaw, BSc, and L.A. Kirshenbaum, PhD, unpublished data, 2005). Furthermore, perturbations to mitochondria consistent with PTP opening were observed in ventricular myocytes rendered defective for NF-κB activation (Figure 6D, 6E), a finding concordant with the increased expression of BNIP3 in the absence of NF-κB activation. Vital staining revealed a significant 4.2-fold increase (P<0.001) in cell death in cells defective for NF-κB activation (Figure 7A). Moreover, staining of cardiac nuclei with Hoechst 33258 dye indicated the mode of cell death as apoptosis (Figure 7B). This observation is consistent with our earlier work as well as that of others demonstrating a survival role for NF-κB in ventricular myocytes.9,17,26 Collectively, these findings suggest strongly that deregulated expression of BNIP3 provokes cell death in the absence of NF-κB signaling.
To verify that BNIP3 was the underlying cause of apoptosis in cells defective for NF-κB activation, we determined whether genetic or functional ablation of BNIP3 would suppress cell death in NF-κB–deficient myocytes. As shown in Figure 7C and 7D, cells treated with small-interfering RNA (siRNA) directed against BNIP3 were indistinguishable from control cells treated with an irrelevant siRNA with respect to viability. Interestingly, myocytes rendered defective for NF-κB activation with IκBαSA and treated with BNIP3 siRNA displayed a dose-dependent reduction in the incidence of cell death compared with NF-κB–defective myocytes treated with irrelevant siRNA (Figure 7C, 7D). To substantiate these novel findings, we used an alternative strategy to inactivate BNIP3. For these experiments, we used a dominant-negative mutation of BNIP3, previously shown by our laboratory to suppress cell death induced by BNIP3.18 As shown in Figure 7C and 7D, the incidence of cell death was markedly suppressed by the dominant-negative BNIP3 (3.7-fold decrease; P<0.001) in cells rendered defective for NF-κB activation with IκBαSA, a finding concordant with our siRNA BNIP3 data.
The underlying mechanism by which oxygen deprivation triggers cell death of ventricular myocytes is poorly understood. Recently, we demonstrated that IKKβ-mediated activation of the NF-κB signaling pathway was sufficient to suppress hypoxia-induced mitochondrial defects and cell death of ventricular myocytes.9 As a step toward elucidating the mode by which NF-κB averts cell death, we reasoned that NF-κB likely modulates the activity of factor(s) that would otherwise provoke these changes during hypoxia. In this report we provide new compelling evidence that NF-κB transcriptionally silences basal and hypoxia-inducible expression of the mitochondrial death gene BNIP3.
Previously, we identified BNIP3 as a critical component of the mitochondrial death pathway during hypoxic and ischemic injury in ventricular myocytes.3 The closest homologue to BNIP3 is Nix/BNIP3L,27 which can reportedly provoke mitochondrial defects and cell death in a Gq model of heart failure.27,28 However, unlike BNIP3, the Nix promoter does not contain hypoxia-inducible response elements and consequently does not appear to be regulated during ischemia or hypoxia like BNIP3.18,29 BNIP3 is readily distinguished from other Bcl-2 family members known to provoke cell death by at least 2 important features. First, unlike other Bcl-2 family members, BNIP3 appears to be directly activated by hypoxia or ischemic injury in ventricular myocytes.18 The second and perhaps most compelling feature is the presence of NF-κB consensus elements within the BNIP3 promoter that are absent from other factors. These unique properties of BNIP3 underscore its importance as a key regulator of mitochondrial function during hypoxic injury and suggest that it must be regulated, at least transcriptionally, in a manner distinct from the other death factors. Although the mechanisms that regulate basal and inducible BNIP3 gene expression are poorly defined, the present data strongly suggest that BNIP3 gene expression is negatively regulated by NF-κB. Furthermore, our data demonstrate that NF-κB elements within the BNIP3 are crucial for repressing BNIP3 under basal and inducible conditions.
The fact that deregulated and uncontrolled expression of BNIP3 would otherwise provoke mitochondrial defects and cell death implies that BNIP3 must be highly regulated and under tight transcriptional control. In this report we show that disruption of NF-κB signaling pathway by not 1 but 3 independent approaches resulted in increased basal BNIP3 expression and cell death. Furthermore, the fact that cells defective for NF-κB activation displayed mitochondrial perturbations implies that NF-κB prevents cell death by suppressing untimely or inappropriate activation of the intrinsic mitochondrial death pathway. This finding is consistent with previous work by our laboratory and others demonstrating that functional inactivation of NF-κB increases susceptibility to cell death.9,26,30,31 Indeed, genetic or functional inactivation of BNIP3 in myocytes defective for NF-κB activation suppressed mitochondrial PTP and apoptosis. That p65 NF-κB activity was diminished in hypoxic myocytes is consistent with a paradigm shift from cell survival toward cell death through increased expression levels of BNIP3. This is in agreement with our earlier work demonstrating a crucial role for IKKβ-mediated NF-κB activation for the suppression of mitochondrial perturbations and cell death of ventricular myocytes during hypoxic injury.9,17 Collectively, our data support a model in which NF-κB averts cell death by repressing basal expression of BNIP3.
The counterintuitive and unexpected repression of BNIP3 by NF-κB, despite its well-established and acknowledged role as a transcriptional activator,6,32–35 highlights a less defined but emerging role for NF-κB as transcriptional repressor.7,8,36,37 The fact that deletion of the NF-κB elements within the BNIP3 promoter resulted in a greater basal and hypoxia-induced expression of BNIP3 supports our contention that NF-κB negatively regulates BNIP3 gene expression. Although the mode by which NF-κB regulates BNIP3 gene activity was not determined here, it appears to be independent of de novo gene activation given that mutations of the p65 subunit defective for transactivation repressed BNIP3 gene activity. Whether NF-κB represses BNIP3 promoter by signaling through inhibitory or accessory proteins7 is currently unknown and remains an area of active investigation. Furthermore, the fact that mitochondrial defects provoked by BNIP3 protein overexpression were suppressed by NF-κB suggests that NF-κB may also influence posttranscriptional events that impinge on the ability of BNIP3 to trigger cell death.
To date, the mechanism by which NF-κB averts cell death has not been established. In this report we provide the first direct evidence that NF-κB averts cell death by repressing basal activation of BNIP3. That BNIP3 gene expression was increased in cells defective for NF-κB signaling is interesting, and, although not proven, it is tempting to speculate that it may underlie the phenotype of the p65−/− mice that die embryonically from excessive apoptosis.30,38
Nevertheless, under the conditions tested, our data provide the first evidence that NF-κB prevents cell death through a mechanism that involves the transcriptional silencing of the mitochondrial death protein BNIP3. Furthermore, our data contribute to the dichotomous actions of NF-κB and extend the paradigm for its cytoprotective properties, which now include repression of the death gene BNIP3.
This work was supported by grants to Dr Kirshenbaum from the Canadian Institutes of Health Research (CIHR). Dr Kirshenbaum holds a Canada Research Chair in Molecular Cardiology. Dr Baetz holds a Postdoctoral fellowship from the CIHR-IMPACT, and Dr Regula holds a CIHR studentship. We are grateful to the support and friendship of the late Dr Arnold H. Greenberg and to Dr H. Weisman for critical comments on the manuscript. Expert technical assistance was provided by F. Aguliar, J. Nychek, and T. Zhang.
Crow MT, Mani K, Nam YJ, Kitsis RN. The mitochondrial death pathway and cardiac myocyte apoptosis. Circ Res. 2004; 95: 957–970.
Zamzami N, Marchetti P, Castedo M, Decaudin D, Macho A, Hirsch T, Susin SA, Petit PX, Mignotte B, Kroemer G. Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J Exp Med. 1995; 182: 367–377.
Regula KM, Ens K, Kirshenbaum LA. Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes. Circ Res. 2002; 91: 226–231.
Kubasiak LA, Hernandez OM, Bishopric NH, Webster KA. Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNIP3. Proc Natl Acad Sci U S A. 2002; 99: 12825–12830.
Gurevich RM, Regula KM, Kirshenbaum LA. Serpin protein CrmA suppresses hypoxia-mediated apoptosis of ventricular myocytes. Circulation. 2001; 103: 1984–1991.
Carlotti F, Dower SK, Qwarnstrom EE. Dynamic shuttling of nuclear factor kappa B between the nucleus and cytoplasm as a consequence of inhibitor dissociation. J Biol Chem. 2000; 275: 41028–41034.
Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS Jr. NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science. 1998; 281: 1680–1683.
Ashburner BP, Westerheide SD, Baldwin AS Jr. The p65 (RelA) subunit of NF-kappaB interacts with the histone deacetylase (HDAC) corepressors HDAC1 and HDAC2 to negatively regulate gene expression. Mol Cell Biol. 2001; 21: 7065–7077.
McCullough KD, Martindale JL, Klotz LO, Aw TY, Holbrook NJ. Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and perturbing the cellular redox state. Mol Cell Biol. 2001; 21: 1249–1259.
Regula KM, Baetz D, Kirshenbaum LA. Nuclear factor-kappaB represses hypoxia-induced mitochondrial defects and cell death of ventricular myocytes. Circulation. 2004; 110: 3795–3802.
Kirshenbaum LA, Schneider MD. Adenovirus E1A represses cardiac gene transcription and reactivates DNA synthesis in ventricular myocytes, via alternative pocket protein- and p300-binding domains. J Biol Chem. 1995; 270: 7791–7794.
de Moissac D, Mustapha S, Greenberg AH, Kirshenbaum LA. Bcl-2 activates the transcription factor NFkappaB through the degradation of the cytoplasmic inhibitor IkappaBalpha. J Biol Chem. 1998; 273: 23946–23951.
Hoffmann A, Leung TH, Baltimore D. Genetic analysis of NF-kappaB/Rel transcription factors defines functional specificities. EMBO J. 2003; 22: 5530–5539.
Madrid LV, Mayo MW, Reuther JY, Baldwin AS Jr. Akt stimulates the transactivation potential of the RelA/p65 subunit of NF-kappa B through utilization of the Ikappa B kinase and activation of the mitogen-activated protein kinase p38. J Biol Chem. 2001; 276: 18934–18940.
O’Mahony A, Lin X, Geleziunas R, Greene WC. Activation of the heterodimeric IkappaB kinase alpha (IKKalpha)-IKKbeta complex is directional: IKKalpha regulates IKKbeta under both basal and stimulated conditions. Mol Cell Biol. 2000; 20: 1170–1178.
Regula KM, Ens K, Kirshenbaum LA. IKK beta is required for Bcl-2-mediated NF-kappa B activation in ventricular myocytes. J Biol Chem. 2002; 277: 38676–38682.
Regula KM, Ens K, Kirshenbaum LA. Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes. Circ Res. 2002; 91: 226–231.
Vande VC, Cizeau J, Dubik D, Alimonti J, Brown T, Israels S, Hakem R, Greenberg AH. BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore. Mol Cell Biol. 2000; 20: 5454–5468.
Mustapha S, Kirshner A, de Moissac D, Kirshenbaum LA. A direct requirement of nuclear factor-kappa B for suppression of apoptosis in ventricular myocytes. Am J Physiol. 2000; 279: H939–H945.
Li ZW, Chu W, Hu Y, Delhase M, Deerinck T, Ellisman M, Johnson R, Karin M. The IKKbeta subunit of IkappaB kinase (IKK) is essential for nuclear factor kappaB activation and prevention of apoptosis. J Exp Med. 1999; 189: 1839–1845.
de Moissac D, Zheng H, Kirshenbaum LA. Linkage of the BH4 domain of Bcl-2 and the nuclear factor kappaB signaling pathway for suppression of apoptosis. J Biol Chem. 1999; 274: 29505–29509.
Misra A, Haudek SB, Knuefermann P, Vallejo JG, Chen ZJ, Michael LH, Sivasubramanian N, Olson EN, Entman ML, Mann DL. Nuclear factor-kappaB protects the adult cardiac myocyte against ischemia-induced apoptosis in a murine model of acute myocardial infarction. Circulation. 2003; 108: 3075–3078.
Chen G, Cizeau J, Vande VC, Park JH, Bozek G, Bolton J, Shi L, Dubik D, Greenberg A. Nix and Nip3 form a subfamily of pro-apoptotic mitochondrial proteins. J Biol Chem. 1999; 274: 7–10.
Bruick RK. Expression of the gene encoding the proapoptotic Nip3 protein is induced by hypoxia. Proc Natl Acad Sci U S A. 2000; 97: 9082–9087.
Wang CY, Mayo MW, Baldwin AS Jr. TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-kappaB. Science. 1996; 274: 784–787.
Beg AA, Baltimore D. An essential role for NF-kappaB in preventing TNF-alpha-induced cell death. Science. 1996; 274: 782–784.
Sen R, Baltimore D. In vitro transcription of immunoglobulin genes in a B-cell extract: effects of enhancer and promoter sequences. Mol Cell Biol. 1987; 7: 1989–1994.
Madrid LV, Wang CY, Guttridge DC, Schottelius AJ, Baldwin AS Jr, Mayo MW. Akt suppresses apoptosis by stimulating the transactivation potential of the RelA/p65 subunit of NF-kappaB. Mol Cell Biol. 2000; 20: 1626–1638.
Stein B, Cogswell PC, Baldwin AS Jr. Functional and physical associations between NF-kappa B and C/EBP family members: a Rel domain-bZIP interaction. Mol Cell Biol. 1993; 13: 3964–3974.
Zhang W, Kone BC. NF-kappaB inhibits transcription of the H(+)-K(+)-ATPase alpha(2)-subunit gene: role of histone deacetylases. Am J Physiol. 2002; 283: F904–F911.
Heart failure remains a major cause of morbidity and mortality in North America and is approaching pandemic proportions worldwide. The limited and meager capacity of the heart for repair, coupled with the inordinate cell death after injury, is believed to be a contributing factor to ventricular remodeling and diminished pump performance in patients after myocardial infarction. The molecular signaling pathways that govern cell death during oxygen deprivation are poorly understood. In this report we decipher the molecular mechanisms that control activation of the death gene BNIP3. Our earlier work identified BNIP3 to be a central factor in the cell death process during hypoxic injury. We demonstrate that under normal oxygen tension, BNIP3 poses no threat to the viability of the cell because it is “switched off” by the cellular factor nuclear factor-κB (NF-κB). The biological actions of NF-κB work in a way analogous to the brakes of a car, which prevent the car from moving. We show that during hypoxia NF-κB is inactivated, releasing the “brakes” and “switching on” BNIP3. This results in elevated BNIP3 levels and cell death of heart cells. Furthermore, we show that genetic interventions that “mimic” the braking actions of NF-κB suppress BNIP3 gene activation and cell death of ventricular myocytes during hypoxia. The clinical importance of this study holds promise for the design of new therapies that specifically target components of the cell death pathway as a means to prevent inordinate cell loss and improve ventricular function in patients after infarction.
↵*The first 2 authors contributed equally to this work.
The online-only Data Supplement, which contains a supplemental figures, can be found with this article at http://circ.ahajournals.org/cgi/content/full/112/24/3777/DC1.