Hongying Gang

Antagonism of E2F-1 regulated Bnip3 transcription by NF-κB is essential for basal cell survival

The transcription factor E2F-1 drives proliferation and death, but the mechanisms that differentially regulate these divergent actions are poorly understood. The hypoxia-inducible death factor Bnip3 is an E2F-1 target gene and integral component of the intrinsic mitochondrial death pathway. The mechanisms that govern Bnip3 gene activity remain cryptic. Herein we show that the transcription factor NF-κB provides a molecular switch that determines whether E2F-1 signals proliferation or death under physiological conditions. We show under basal nonapoptotic conditions that NF-κB constitutively occupies and transcriptionally silences Bnip3 gene transcription by competing with E2F-1 for Bnip3 promoter binding. Conversely, in the absence of NF-κB, or during hypoxia when NF-κB abundance is reduced, basal Bnip3 gene transcription is activated by the unrestricted binding of E2F-1 to the Bnip3 promoter. Genetic knock-down of E2F-1 or retinoblastoma gene product over-expression in cardiac and human pancreatic cancer cells deficient for NF-κB signaling abrogated basal and hypoxia-inducible Bnip3 transcription. The survival kinase PI3K/Akt inhibited Bnip3 expression levels in cells in a manner dependent upon NF-κB activation. Hence, by way of example, we show that the transcriptional inhibition of E2F-1-dependent Bnip3 expression by NF-κB highlights a survival pathway that overrides the E2F-1 tumor suppressor program. Our data may explain more fundamentally how cells, by selectively inhibiting E2F-1-dependent death gene transcription, avert apoptosis down-stream of the retinoblastoma/E2F-1 cell cycle pathway.

A Novel Hypoxia-Inducible Spliced Variant of Mitochondrial Death Gene Bnip3 Promotes Survival of Ventricular Myocytes

Rationale: Alternative splicing provides a versatile mechanism by which cells generate proteins with different or even antagonistic properties. Previously, we established hypoxia-inducible death factor Bnip3 as a critical component of the intrinsic death pathway. Objective: To investigate alternative splicing of Bnip3 pre-mRNA in postnatal ventricular myocytes during hypoxia. Methods and Results: We identify a novel previously unrecognized spliced variant of Bnip3 (Bnip3&Dgr;ex3) generated by alternative splicing of exon3 exclusively in cardiac myocytes subjected to hypoxia. Sequencing of Bnip3&Dgr;ex3 revealed a frame shift mutation that terminated transcription up-stream of exon5 and exon6 ablating translation of the BH3-like domain and critical carboxyl-terminal transmembrane domain crucial for mitochondrial localization and cell death. Notably, although the 26-kDa Bnip3 protein (Bnip3FL) encoded by full-length mRNA was localized to mitochondria and provoked cell death, the 8.2-kDa Bnip3&Dgr;ex3 protein encoded by the truncated spliced mRNA was defective for mitochondrial targeting but interacted with Bnip3FL resulting in less association of Bnip3FL with mitochondria and diminished apoptotic and necrotic cell death. Forced expression of Bnip3FL in cardiac myocytes or Bnip3−/− mouse embryonic fibroblasts triggered widespread cell death that was inhibited by coexpression of Bnip3&Dgr;ex3. Conversely, RNA interference targeted against sequences encompassing the unique exon2-exon4 junction of the Bnip3&Dgr;ex3 sensitized cardiac myocytes to mitochondrial perturbations and cell death induced by Bnip3FL. Conclusions: Given the otherwise lethal consequences of deregulated Bnip3FL expression in postmitotic cells, our findings reveal a novel intrinsic defense mechanism that opposes the mitochondrial defects and cell death of ventricular myocytes that is obligatorily linked and mutually dependent on alternative splicing of Bnip3FL during hypoxia or ischemic stress.

p53 Mediates Autophagy and Cell Death by a Mechanism Contingent On Bnip3

Myocardial ischemia and angiotensin II activate the tumor suppressor p53 protein, which promotes cell death. Previously, we showed that the Bcl-2 death gene Bnip3 is highly induced during ischemia, where it triggers mitochondrial perturbations resulting in autophagy and cell death. However, whether p53 regulates Bnip3 and autophagy is unknown. Herein, we provide new compelling evidence for a novel signaling axis that commonly links p53 and Bnip3 for autophagy and cell death. p53 overexpression increased endogenous Bnip3 mRNA and protein levels resulting in mitochondrial defects leading to loss of mitochondrial &Dgr;&PSgr;m. This was accompanied by an increase in autophagic flux and cell death. Notably, genetic loss of function studies, such as Atg7 knock-down or pharmacological inhibition of autophagy with 3-methyl adenine, suppressed cell death induced by p53—indicating that p53 induces maladaptive autophagy. Our previous work demonstrated that Bnip3 induces mitochondrial defects and autophagic cell death. Conversely, loss of function of Bnip3 in cardiac myocytes or Bnip3−/− mouse embryonic fibroblasts prevented mitochondrial targeting of p53, autophagy, and cell death. To our knowledge, these data provide the first evidence for the dual regulation of autophagy and cell death of cardiac myocytes by p53 that is mutually dependent on and obligatorily linked to Bnip3 gene activation. Hence, our findings may explain more fundamentally, how, autophagy and cell death are dually regulated during cardiac stress conditions where p53 is activated.

Bi-Directional Regulation of NF-κB and mTOR Signaling Functionally Links Bnip3 Gene Repression and Cell Survival of Ventricular Myocytes

Background—Tumor necrosis factor-&agr; and other proinflammatory cytokines activate the canonical Nuclear Factor (NF)-&kgr;B pathway through the kinase IKK&bgr;. Previously, we established that IKK&bgr; is also critical for Akt-mediated NF-&kgr;B activation in ventricular myocytes. Akt activates the kinase mammalian target of rapamycin (mTOR), which mediates important processes such as cardiac hypertrophy. However, whether mTOR regulates cardiac myocyte cell survival is unknown. Methods and Results—Herein, we demonstrate bidirectional regulation between NF-&kgr;B signaling and mTOR, the balance which determines ventricular myocyte survival. Overexpression of IKK&bgr; resulted in mTOR activation and conversely overexpression of mTOR lead to NF-&kgr;B activation. Loss of function approaches demonstrated that endogenous levels of IKK&bgr; and mTOR also signal through this pathway. NF-&kgr;B activation by mTOR was mediated by phosphorylation of the NF-&kgr;B p65 subunit increasing p65 nuclear translocation and activation of gene transcription. This circuit was also important for NF-&kgr;B activation by the canonical tumor necrosis factor-&agr; pathway. Our previous work has shown that NF-&kgr;B signaling suppresses transcription of the death gene Bnip3 resulting in ventricular myocyte survival. Inhibition of mTOR with rapamycin decreased NF-&kgr;B activation resulting in increased Bnip3 expression and cell death. Conversely, mTOR overexpression suppressed Bnip3 levels and cell death of ventricular myocytes in response to hypoxia. Conclusions—To our knowledge, these data provide the first evidence for a bidirectional link between NF-&kgr;B signaling and mTOR that is critical in the regulation of Bnip3 expression and cardiac myocyte death. Hence, modulation of this axis may be cardioprotective during ischemia.

Bidirectional Regulation of Nuclear Factor-κB and Mammalian Target of Rapamycin Signaling Functionally Links Bnip3 Gene Repression and Cell Survival of Ventricular Myocytes

Background—Tumor necrosis factor-&agr; and other proinflammatory cytokines activate the canonical Nuclear Factor (NF)-&kgr;B pathway through the kinase IKK&bgr;. Previously, we established that IKK&bgr; is also critical for Akt-mediated NF-&kgr;B activation in ventricular myocytes. Akt activates the kinase mammalian target of rapamycin (mTOR), which mediates important processes such as cardiac hypertrophy. However, whether mTOR regulates cardiac myocyte cell survival is unknown. Methods and Results—Herein, we demonstrate bidirectional regulation between NF-&kgr;B signaling and mTOR, the balance which determines ventricular myocyte survival. Overexpression of IKK&bgr; resulted in mTOR activation and conversely overexpression of mTOR lead to NF-&kgr;B activation. Loss of function approaches demonstrated that endogenous levels of IKK&bgr; and mTOR also signal through this pathway. NF-&kgr;B activation by mTOR was mediated by phosphorylation of the NF-&kgr;B p65 subunit increasing p65 nuclear translocation and activation of gene transcription. This circuit was also important for NF-&kgr;B activation by the canonical tumor necrosis factor-&agr; pathway. Our previous work has shown that NF-&kgr;B signaling suppresses transcription of the death gene Bnip3 resulting in ventricular myocyte survival. Inhibition of mTOR with rapamycin decreased NF-&kgr;B activation resulting in increased Bnip3 expression and cell death. Conversely, mTOR overexpression suppressed Bnip3 levels and cell death of ventricular myocytes in response to hypoxia. Conclusions—To our knowledge, these data provide the first evidence for a bidirectional link between NF-&kgr;B signaling and mTOR that is critical in the regulation of Bnip3 expression and cardiac myocyte death. Hence, modulation of this axis may be cardioprotective during ischemia.

Regulation of Autophagy in the Heart: “You Only Live Twice”

Autophagy is a highly orchestrated cellular process by which proteins and organelles are degraded via an elaborate lysosomal pathway to generate free amino acids and sugars for ATP during metabolic stress. At present, the exact role of autophagy in the heart is highly debated but suggested to play a key role in regulating cell turnover in cardiomyopathies and heart failure. The signaling pathways and molecular effectors that govern autophagy are incomplete, as are the mechanisms that determine whether autophagy promotes or prevents cell death. The mitochondrion has been identified as a key organelle centrally involved in regulating autophagy. Certain members of the Bcl-2 gene family, including Beclin-1, Bcl-2 nineteen kilodaltons interacting protein (Bnip3), and Nix/Bnip3L, provoke mitochondrial perturbations leading to permeability transition pore opening, resulting in apoptosis, autophagy, or both. These and other aspects of autophagy processes have been discussed.

PDK2-mediated alternative splicing switches Bnip3 from cell death to cell survival

In hypoxia, the survival property of cancer cells mediated by the glycolytic enzyme PDK2 is obligatorily linked to alternative splicing and generation of a novel isoform of death gene Bnip3, which suppresses mitochondrial injury and promotes survival.

Epigenetic regulation of canonical TNFα pathway by HDAC1 determines survival of cardiac myocytes

Gene transcription is regulated by post-translation modifications. Histone deacetylases (HDACs) remove acetyl groups from histone and non-histone factors inhibiting transcription. Proinflammatory cytokines such as TNFα activate the canonical nuclear factor-κB (NF-κB) pathway. Earlier we established a cytoprotective role for NF-κB in the heart. Though a causal relationship for HDAC1 and NF-κB has been established, the impact of HDAC1 on TNFα signaling is unknown. Herein, we demonstrate that HDAC1 provides a molecular switch for determining cell survival in the TNFα pathway. In contrast to vehicle-treated control cells, TNFα-treated cells displayed a marked increase in NF-κB gene transcription. Notably, cells treated with TNFα were indistinguishable from vehicle controls cells with respect to viability. Interestingly, HDAC activity was reduced in cells treated with TNFα. Conversely, in the presence of HDAC1, NF-κB gene transcription by TNFα was repressed, resulting in mitochondrial perturbations and widespread cell death. Heterologous fusion proteins comprised of yeast Gal4 DNA binding domain fused in frame to the NF-κB p65 transactivation domain were preferentially repressed by HDAC1. Moreover, transcription mediated by Gal4VP16 protein from herpes virus was unaffected by HDAC1 in cardiac myocytes. Mutations that abrogate known catalytic activities of HDAC1, small interference RNA, or pharmacological inhibition of HDAC1 restored NF-κB signaling and suppressed cell death induced by TNFα. These data provide the first evidence for an obligate link between HDAC1 and canonical TNFα pathway for cell survival of cardiac myocytes.

Reductions in the Cardiac Transient Outward K+ Current Ito Caused by Chronic β-Adrenergic Receptor Stimulation Are Partly Rescued by Inhibition of Nuclear Factor κB

The fast transient outward potassium current (Ito,f) plays a critical role in the electrical and contractile properties of the myocardium. Ito,f channels are formed by the co-assembly of the pore-forming α-subunits, Kv4.2 and Kv4.3, together with the accessory β-subunit KChIP2. Reductions of Ito,f are common in the diseased heart, which is also associated with enhanced stimulation of β-adrenergic receptors (β-ARs). We used cultured neonatal rat ventricular myocytes to examine how chronic β-AR stimulation decreases Ito,f. To determine which downstream pathways mediate these Ito,f changes, adenoviral infections were used to inhibit CaMKIIδc, CaMKIIδb, calcineurin, or nuclear factor κB (NF-κB). We observed that chronic β-AR stimulation with isoproterenol (ISO) for 48 h reduced Ito,f along with mRNA expression of all three of its subunits (Kv4.2, Kv4.3, and KChIP2). Inhibiting either CaMKIIδc nor CaMKIIδb did not prevent the ISO-mediated Ito,f reductions, even though CaMKIIδc and CaMKIIδb clearly regulated Ito,f and the mRNA expression of its subunits. Likewise, calcineurin inhibition did not prevent the Ito,f reductions induced by β-AR stimulation despite strongly modulating Ito,f and subunit mRNA expression. In contrast, NF-κB inhibition partly rescued the ISO-mediated Ito,f reductions in association with restoration of KChIP2 mRNA expression. Consistent with these observations, KChIP2 promoter activity was reduced by p65 as well as β-AR stimulation. In conclusion, NF-κB, and not CaMKIIδ or calcineurin, partly mediates the Ito,f reductions induced by chronic β-AR stimulation. Both mRNA and KChIP2 promoter data suggest that the ISO-induced Ito,f reductions are, in part, mediated through reduced KChIP2 transcription caused by NF-κB activation.