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Understanding the role of BNIP3 in the systemic response to hypoxia has been complicated by conflicting results that indicate on the one hand that BNIP3 promotes cell death, and other data, including our own that BNIP3 is not sufficient for cell death, but rather plays a critical role in hypoxia-induced autophagy. This work suggests that rather than promoting death, BNIP3 may actually allow survival either by preventing ATP depletion or by eliminating damaged mitochondria. However, the function of BNIP3 may be subverted under unusual conditions associated with acidosis that arise following extended periods of hypoxia and anaerobic glycolysis. Despite this novel insight into BNIP3 function, much remains to be done in terms of pinning down a molecular activity for BNIP3 that explains both its role in autophagy and how this may be subverted to induce cell death. As a target of the RB tumor suppressor, our work also places BNIP3 at the center of efforts to exploit autophagy to better treat human cancers in which tumor hypoxia is implicated as a progression factor.
Oxygen deprivation is a unique form of nutrient stress that induces a specialized cellular response through stabilization of the hypoxia-inducible factors (HIF) that turn on HIF-response genes,1 and also elicits HIF-independent effects through activation of AMPK and repression of mTOR.2 The overall effect of these responses to hypoxia is to maximize ATP generation in tissues through increased glycolysis and blood transport,1 but also to limit ATP consumption through suppression of energy-intensive processes, such as protein translation and the cell cycle.2 Importantly, many HIF target genes are translated through cap-independent mechanisms2 that are not inhibited by hypoxia. The diverse functions of known HIF target genes speaks to the importance of a systemic response to hypoxia, and among the major classes of hypoxia-inducible genes are regulators of glucose metabolism, angiogenesis and red blood cell production, iron homeostasis, cellular adhesion and extracellular matrix integrity.1
BNIP3 has been a part of the HIF target gene repertoire for several years but its role in the hypoxic response has remained ambiguous. Originally identified as an adenovirus E1B(19K)/Bcl-2 interacting protein,3 it has been widely reported as promoting non-apoptotic cell death when induced or overexpressed in cells.4–6 However, whereas anoxia kills cells, hypoxia does not routinely lead to cell death despite high levels of BNIP3 expression.7 Furthermore, BNIP3 is expressed in healthy adult heart without evidence of cell death.8 Our recent work provides further evidence that overexpression of BNIP3 is not sufficient to induce cell death and defines a role for BNIP3 in promoting autophagy in response to hypoxia.9 Consistent with previous reports showing elevated autophagic vesicle formation and processing of LC3 in cardiomyocytes overexpressing BNIP38 and in glioma cells treated with ceramide,10 we observe that overexpression of BNIP3 in a variety of different tumor cell lines results in increased autophagy whereas knockdown of BNIP3 inhibits hypoxia-induced autophagy.9
What purpose does autophagy serve in hypoxic cells? Hypoxia limits ATP production by oxidative phosphorylation and although glycolysis is elevated, the cell remains much less efficient at generating ATP. Thus, hypoxia-induced autophagy may increase the efficiency of ATP production through catabolism of macromolecules at the autolysosome. Hypoxia also results in elevated levels of reactive oxygen species (ROS) with consequent effects on oxidative damage to nucleic acids, proteins and the lipid membranes of organelles, such as mitochondria.11 Thus, autophagy may promote survival under hypoxic conditions by ridding the cell of damaged mitochondria, a process known as mitophagy, thereby limiting ROS production and preventing cytochrome c release that could potentiate caspase activation and apoptosis. Our work identifies BNIP3 as being essential for autophagy induced by hypoxia and shows that knockdown of BNIP3 in hypoxic cells reduces the cellular levels of ATP, consistent with BNIP3-dependent autophagy contributing to maintaining the energy balance in hypoxic cells.
The autophagic machinery is highly conserved between species and involves the critical function of Atg genes to regulate membrane trafficking from the ER to generate autophagosomes that engulf cytoplasmic constituents and target them for degradation upon fusion with lysosomes.12 These components are constitutively expressed in most cells and BNIP3 represents a novel class of autophagy gene that is induced by a specific stress or signal. This raises the question as to whether there are other stress-specific autophagy regulators that only participate in autophagy under unique circumstances. For example, Dram is a p53 target gene that promotes autophagy in response to DNA damage.13 Additional and outstanding questions that emerge from our work include determining how BNIP3 promotes autophagy at the molecular level and whether other components of the autophagy machinery are also upregulated by hypoxia, or indeed whether BNIP3-induced autophagy depends on the canonical autophagy machinery.
BNIP3 forms stable homodimers that integrate into the outer mitochondrial membrane; due to the charged nature of the helical interface of the transmembrane domain, dimerization is required for integration into the lipid membrane.14 BNIP3 dimers are highly stable with a dissociation constant of 50 nM in lipid membrane and are not readily dissociated by detergent. Based on the resistance of BNIP3 dimers to SDS and DTT, early studies suggest that it forms covalent dimers,4 but structural analysis reveals that this is more likely explained by very strong and stable intermolecular hydrogen bond pairing between key residues in the helical interface that mediates dimerization.15 The role of the amino terminal domain, the putative BH3 domain and the conserved domain of BNIP3 is less clear, but elucidating the molecular interactions and functions of these domains are key to defining how BNIP3 functions in autophagy.
As a mitochondrial protein that promotes autophagy, a number of hypotheses have been put forward to explain how BNIP3 functions (Fig. 1). Overexpression of BNIP3 in cardiomyocytes exposed to ischema/reperfusion (I/R) conditions induces mitochondrial fragmentation as well as autophagy, and the authors suggest that BNIP3 dimers cause damage to the mitochondria (Fig. 1, model 1).16 However, these studies did not address whether elevated ROS induced by I/R stress was the key damaging agent, rather than BNIP3.
An alternative model is that as opposed to being the cause of the damage, BNIP3 is a part of the “mitochondrial quality control” mechanism11 that gets rid of damaged mitochondria by targeting them to the autophagosome for degradation (Fig. 1, model 2). According to this hypothesis, BNIP3 dimers at the mitochondrial membrane would be predicted to interact with some “docking” molecule at the phagophore thereby targeting the mitochondria to the autophagosome. However, this model does not explain how BNIP3 distinguishes between functional mitochondria and damaged mitochondria, nor indeed is there any evidence that it does distinguish between them in any way. Fragmentation of the mitochondrial reticulum may promote autophagy indirectly by reducing mitochondria to a size that is readily taken up by autophagosomes. However, it remains to be determined whether it is ROS, membrane lipid or protein oxidation, or indeed BNIP3 dimer integration that induces mitochondrial fragmentation. Finally, BNIP3 interacts with both Bcl-2 and Bcl-XL, and thus a third hypothesis to explain how BNIP3 promotes autophagy is by titrating Bcl-2 and/or Bcl-XL away from Beclin-1, a critical mediator of autophagy (Fig. 1, model 3).16
A primary role for BNIP3 in autophagy rather than cell death makes more biological sense in terms of the cellular response to hypoxia but the question remains whether there are unique conditions under which BNIP3 plays a subversive role in promoting cell death. Although our work shows that BNIP3 expression is not sufficient to kill cells, work examining the role of BNIP3 in cardiovascular disease clearly points to additional factors altering or subverting the activity of BNIP3 such that it promotes cell death.17,18 Importantly, it was suggested that acidosis promotes the ability of BNIP3 to kill by increasing its association with the mitochondria.17 Acidosis is associated with extended periods of hypoxia, ischemia/reperfusion injury and with anaerobic glycolysis. Thus, while BNIP3 may initially protect cells from death by promoting autophagy, it may ultimately induce cell death as lactic acid builds up following extended periods of hypoxia and nutrient deprivation. However, the molecular basis of this switch in BNIP3 activity is still not defined.
In addition to BNIP3, there is a BNIP3-related molecule called Nix that diverges significantly from other BH3-only domain proteins and is also induced by hypoxia.19 Thus, the question arises whether there is redundancy of function between BNIP3 and Nix, in a manner akin to the redundancy between Bak and Bax. However, Nix has not yet been implicated in autophagy and the phenotype of Nix null mice in which there is an accumulation of immature erythroblasts and a striking resistance to apoptosis induced by erythropoietin deprivation20 indicates that Nix may function more like a canonical BH3-only protein than does BNIP3. Nevertheless, it remains an open question as to whether BNIP3 and Nix may have overlapping functions in the systemic response to hypoxia.
A key component of our work was the identification of BNIP3 as a target of transcriptional repression by the RB tumor suppressor.9 We characterized the importance of a conserved E2F binding site in the human and mouse BNIP3 promoters with respect to attenuation of BNIP3 induction by HIF, mediated through a juxtaposed HIF response element, with the overall effect of limiting BNIP3 levels within a range that promotes autophagy and prevents autophagic cell death. As already mentioned, the p53 tumor suppressor also modulates the expression of the autophagy regulator Dram13 and the BNIP3-related molecule, Nix.19 The role of oncogenes and tumor suppressors in modulating autophagy has been previously recognized21 but by identifying BNIP3 as a target of pRB, we are proposing that different tumor suppressors may play different roles in regulating autophagy depending on the specific stress. Thus, as alluded to above, pRB may promote autophagy under conditions of hypoxia whereas p53-dependent autophagy would come into play in response to DNA damage.
Recent work has identified a role for autophagy in mitigating against necrosis induced by nutrient starvation in mouse models of tumorigenesis22,23 and future work will determine whether the RB-BNIP3 axis or the p53-Dram/Nix axis plays a more important role in a specific tumor type or disease stage, depending on whether there is tumor hypoxia or genome instability, or some other unique stress. Of further importance is the role of autophagy in determining more efficacious treatments for cancer. Recent work has indicated that tumor cells remaining resistant to apoptosis induced by cyclophosamide are dependent on autophagy for survival and that inhibition of autophagy with chloroquine elicited a more effective reduction in tumor volume when combined with cyclophosamide than with cyclophosamide or chloroquine alone.24 An equally valid approach at this point in time may be to promote autophagic cell death in tumors with drugs, such as rapamycin or so-called rapa-logs that promote autophagy through repression of mTOR.25 Autophagic cell death would get rid of tumor cells that likely evolved to be resistant to apoptosis, but importantly would circumvent necrotic cell death that correlates with inflammation, tumor metastasis and a generally poor clinical outcome.26
BNIP3 is frequently inactivated in human cancers at late stages of disease that coincide with progression to metastasis. For example, the epigenetic silencing of BNIP3 in pancreatic cancer is associated with drug resistance27 whereas in mouse models of breast cancer, knockdown of BNIP3 promoted tumor growth and metastasis.28 Thus, reactivation of BNIP3 in human cancers, possibly through use of drugs such as 5-aza-cytidine that reverse promoter methylation and gene silencing, may offer another opportunity to exploit autophagy to arrest or kill tumor cells in a more effective manner than is currently available.