The autophagy protein Atg12 associates with antiapoptotic Bcl-2 family members to promote mitochondrial apoptosis

Autophagy and apoptosis constitute important determinants of cell fate and engage in a complex interplay in both physiological and pathological settings. The molecular basis of this crosstalk is poorly understood and relies, in part, on "dual-function" proteins that operate in both processes. Here, we identify the essential autophagy protein Atg12 as a positive mediator of mitochondrial apoptosis and show that Atg12 directly regulates the apoptotic pathway by binding and inactivating prosurvival Bcl-2 family members, including Bcl-2 and Mcl-1. The binding occurs independently of Atg5 or Atg3 and requires a unique BH3-like motif in Atg12, characterized by interaction studies and computational docking. In apoptotic cells, knockdown of Atg12 inhibited Bax activation and cytochrome c release, while ectopic expression of Atg12 antagonized the antiapoptotic activity of Mcl-1. The interaction between Atg12 and Bcl-2 family members may thus constitute an important point of convergence between autophagy and apoptosis in response to specific signals.     

Bcl-2 family members and disease

Apoptosis plays an important role during development and in the maintenance of multicellular organisms. Bcl-2 family members affect cell death in either a positive or negative fashion. Although some redundancy exists between family members, expression of certain family members is important during development in an organ-specific manner. The founding family member bcl-2 tends to be highly expressed in the embryo and declines postnatally following differentiation and maturation. Altered expression of bcl-2, as well as other family members, has been observed in disease states potentially affecting treatment modalities. Here we examine the distribution and role death repressors bcl-2, bcl-x(L) and bcl-w as well as death effectors bax and bak play regulating apoptosis in a tissue-specific manner. Understanding the normal role of these proteins during embryogenesis and in the mature organ will give us important insight into what goes awry in various disease states.     

Ionomycin-activated calpain triggers apoptosis. A probable role for Bcl-2 family members

Ubiquitous calpains (mu- and m-calpain) have been repeatedly implicated in apoptosis, but the underlying mechanism(s) remain(s) to be elucidated. We examined ionomycin-induced cell death in LCLC 103H cells, derived from a human large cell lung carcinoma. We detected hallmarks of apoptosis such as membrane blebbing, nuclear condensation, DNA ladder formation, caspase activation, and poly-(ADP-ribose)polymerase cleavage. Apoptosis was prevented by preincubation of the cells with the calpain inhibitor acetyl-calpastatin 27-peptide and the caspase inhibitor Z-DEVD-fmk, implicating both the calpains and caspases in the apoptotic process. The apoptotic events correlated in a calpastatin-inhibitable manner with Bid and Bcl-2 decrease and with activation of caspases-9, -3, and -7. In vitro both ubiquitous calpains cleaved recombinant Bcl-2, Bid, and Bcl-x(L) at single sites truncating their N-terminal regions. Binding studies revealed diminished interactions of calpain-truncated Bcl-2 and Bid with immobilized intact Bcl-2 family proteins. Moreover, calpain-cleaved Bcl-2 and Bid induced cytochrome c release from isolated mitochondria. We conclude that ionomycin-induced calpain activation promotes decrease of Bcl-2 proteins thereby triggering the intrinsic apoptotic pathway.     

Control of proliferation by Bcl-2 family members

The anti-proliferative effect of Bcl-2 acts mainly at the level of the G0/G1 phase of the cell cycle. Deletions and point mutations in the bcl-2 gene show that the anti-proliferative activity of Bcl-2, can in some cases, be dissociated from its anti-apoptotic function. This indicates that the effect of Bcl-2 on cell cycle progression can be a direct effect and not only a consequence of its anti-apoptotic activity. Bcl-2 appears to mediate its anti-proliferative effect by acting on both signal transduction pathways (NFAT, ERK) and on specific cell cycle regulators (p27, p130).     

Role of Bcl-2 family members in invertebrates

Proteins belonging to the Bcl-2 family function as regulators of 'life-or-death' decisions in response to various intrinsic and extrinsic stimuli. In mammals, cell death is controlled by pro- and anti-apoptotic members of the Bcl-2 family, which function upstream of the caspase cascade. Structural and functional homologues of the Bcl-2 family proteins also exist in lower eukaryotes, such as nematodes and flies. In nematodes, an anti-apoptotic Bcl-2 family protein, CED-9, functions as a potent cell death inhibitor, and a BH3-only protein, EGL-1, acts as an inhibitor of CED-9 to facilitate the spatio-temporal regulation of programmed cell death. On the other hand, the Drosophila genome encodes two Bcl-2 family proteins, Drob-1/Debcl/dBorg-1/dBok and Buffy/dBorg-2, both of which structurally belong to the pro-apoptotic group, despite abundant similarities in the cell death mechanisms between flies and vertebrates. Drob-1 acts as a pro-apoptotic factor in vitro and in vivo, and Buffy/dBorg-2 exhibits a weak anti-apoptotic function. The ancestral role of the Bcl-2 family protein may be pro-apoptotic, and the evolution of the functions of this family of proteins may be closely linked with the contribution of mitochondria to the cell death pathway.     

Sphingolipids: regulators of crosstalk between apoptosis and autophagy

Apoptosis and autophagy are two evolutionarily conserved processes that maintain homeostasis during stress. Although the two pathways utilize fundamentally distinct machinery, apoptosis and autophagy are highly interconnected and share many key regulators. The crosstalk between apoptosis and autophagy is complex, as autophagy can function to promote cell survival or cell death under various cellular conditions. The molecular mechanisms of crosstalk are beginning to be elucidated and have critical implications for the treatment of various diseases, such as cancer. Sphingolipids are a class of bioactive lipids that mediate many key cellular processes, including apoptosis and autophagy. By targeting several of the shared regulators, sphingolipid metabolites differentially regulate the induction of apoptosis and autophagy. Importantly, individual sphingolipid species appear to “switch” autophagy toward cell survival (e.g., sphingosine-1-phosphate) or cell death (e.g., ceramide, gangliosides). This review assesses the current understanding of sphingolipid-induced apoptosis and autophagy to address how sphingolipids mediate the “switch” between the cell survival and cell death. As sphingolipid metabolism is frequently dysregulated in cancer, sphingolipid-modulating agents, or sphingomimetics, have emerged as a novel chemotherapeutic strategy. Ultimately, a greater understanding of sphingolipid-mediated crosstalk between apoptosis and autophagy may be critical for enhancing the chemotherapeutic efficacy of these agents.     

The role of antiapoptotic Bcl-2 family members in endothelial apoptosis elucidated with antisense oligonucleotides

In this study, we utilized potent antisense oligonucleotides to examine the role of two Bcl-2 family members found in human umbilical vein endothelial cells (HUVEC). The first, A1, is thought to be a TNF-alpha-inducible cytoprotective gene, and the second, Bcl-XL, is constitutively expressed. Inhibition of the constitutive levels of Bcl-XL caused 10-25% of the cell population to undergo apoptosis and increased the susceptibility of cells to treatment with low concentrations of staurosporin or ceramide. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2 prevented DNA fragmentation and DeltaYm loss caused by Bcl-XL inhibition or Bcl-XL inhibition combined with staurosporin. However, disruption of DeltaYm caused by Bcl-XL inhibition combined with ceramide treatment was not inhibited by benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2, although DNA fragmentation was completely prevented. Taken together, these results demonstrate a direct protective role for Bcl-XL under normal resting conditions and under low level apoptotic challenges to HUVEC. Furthermore, Bcl-XL protects cells from caspase-dependent and -independent mechanisms of DeltaYm disruption. In contrast to Bcl-XL, A1 inhibition did not show a marked effect on the susceptibility of HUVEC to undergo apoptosis in response to TNF-alpha, ceramide, or staurosporin. These results demonstrate that although A1 may be a cytoprotective gene induced by TNF-alpha, it is not primarily responsible for HUVEC resistance to this cytokine.     

miR-181 targets multiple Bcl-2 family members and influences apoptosis and mitochondrial function in astrocytes

Mitochondria are central to the execution of apoptosis, and the Bcl-2 protein family of pro- and anti-apoptotic proteins interacts with mitochondria to regulate apoptosis. Using bioinformatics we predicted that miR-181, a microRNA expressed in brain, could target the 3'UTRs of Bcl-2 family members Bcl-2-L11/Bim, Mcl-1, and Bcl-2. Using the luciferase reporter assay we confirmed these targets. We used mimic and inhibitor to alter miR-181a levels in primary astrocyte cultures and found miR-181a reduction was associated with increased Bcl-2 and Mcl-1 protein levels. Decreased miR-181a levels reduced glucose deprivation induced apoptosis, mitochondrial dysfunction, and loss of mitochondrial membrane potential in astrocytes.     

The Bcl-2 family of proteins: regulators of cell death and survival

The Bcl-2 protein inhibits apoptosis induced by a variety of signals, in a range of cell types and in diverse organisms, and it is implicated in both normal development and oncogenesis. Despite this central role, the mechanism of action of Bcl-2 is not yet clear. Recent studies have uncovered a number of Bcl-2-related gene products that regulate apoptosis either negatively or positively, and Bcl-2 forms heterodimers with at least one of these proteins, Bax. This article discusses the role of the Bcl-2 family of proteins in the light of these findings.     

Role of Bcl-2 family members in immunity and disease

The different members of the Bcl-2 family are essential regulators of programmed cell death. These different members share one or more Bcl-2 homology domains, required for their ability to regulate each other. In this review, we describe current knowledge of the functions of different Bcl-2 members and their potential roles in disease and immunity.     

The role of Bcl-2 family members in tumorigenesis

The Bcl-2 family consists of about 20 homologues of important pro- and anti-apoptotic regulators of programmed cell death. The established mode of function of the individual members is to either preserve or disturb mitochondrial integrity, thereby inducing or preventing release of apoptogenic factors like Cytochrome c (Cyt c) from mitochondria. Recent findings also indicate further Bcl-2-controlled mitochondria-independent apoptosis pathways. Bcl-2 represents the founding member of the new and growing class of cell death inhibiting oncoproteins. In this review, we try to briefly summarize current models of Bcl-2 family function and to outline the work demonstrating the influence of deregulated Bcl-2 family member expression on tumorigenesis and cancer therapy. Since several Bcl-2 homologues, in addition to influencing apoptotic behaviour, also impinge on cell cycle progression, we discuss possible implications of this additional role for the expression of Bcl-2 family members in tumor cells.     

Control of mitochondrial permeability by Bcl-2 family members

Programmed cell death (apoptosis) is regulated by the Bcl-2 family of proteins. Although it remains unclear how these family members control apoptosis, they clearly have the capacity to regulate the permeability of intracellular membranes to ions and proteins. Proapoptotic members of the Bcl-2 family, especially Bax and Bid, have been extensively analyzed for the ability to form channels in membranes and to regulate preexisting channels. Anti-apoptotic members of the family tend to have the opposing effects on membrane channel formation. The molecular mechanisms of the different models for the permeabilization of membranes by the Bcl-2 family members and the regulation of Bcl-2 family member subcellular localizations are discussed.     

Bcl-2 and Bax mammalian regulators of apoptosis are functional in Drosophila

Studies of apoptosis in C. elegans have allowed the identification of three genes, ced-3, ced-4 and ced-9. Their products constitute the components of an induction pathway of apoptosis conserved in the nematode and mammals. In Drosophila, homologues have been found for CED-3, CED-4 and CED-9. CED-9 belongs to the Bcl-2 family which includes negative (Bcl-2) and positive (Bax) regulators of apoptosis. The recently discovered Bcl-2 family member named Drob-1 acts as a positive regulator of cell death. To address whether a Bcl-2 anti-apoptotic pathway exists in the fly, we studied the effects of expressing the mammalian genes bcl-2 in Drosophila. In embryos, expression of bcl-2 inhibits developmental and X-ray-induced apoptosis. Expressing bcl-2 or the pro-apoptotic mammalian bax in the developing eye and wing alters these structures, bcl-2 increasing the number of cells, while bax reduces the number of cells. In addition, the functional interaction between Bcl-2 and Bax is conserved. These results indicate that factors necessary for the activity of bcl-2 and bax are present in Drosophila. Therefore, a Bcl-2 pathway for inhibition of cell death may exist in the fly.     

Bcl-2 protein family: Implications in vascular apoptosis and atherosclerosis

Apoptosis has been recognized as a central component in the pathogenesis of atherosclerosis, in addition to the other human pathologies such as cancer and diabetes. The pathophysiology of atherosclerosis is complex, involving both apoptosis and proliferation at different phases of its progression. Oxidative modification of lipids and inflammation differentially regulate the apoptotic and proliferative responses of vascular cells during progression of the atherosclerotic lesion. Bcl-2 proteins act as the major regulators of extrinsic and intrinsic apoptosis signalling pathways and more recently it has become evident that they mediate the apoptotic response of vascular cells in response to oxidation and inflammation either in a provocative or an inhibitory mode of action. Here we address Bcl-2 proteins as major therapeutic targets for the treatment of atherosclerosis and underscore the need for the novel preventive and therapeutic interventions against atherosclerosis, which should be designed in the light of molecular mechanisms regulating apoptosis of vascular cells in atherosclerotic lesions.     

Bcl-2-regulated calcium signals as common mediators of both apoptosis and autophagy

The calcium ion, a major intracellular second messenger, is a known mediator of apoptosis and is regulated by the antiapoptotic protein Bcl-2. A paper by H?yer-Hasen et al. (2007) in the current issue of Molecular Cell indicates that calcium also mediates the induction of macroautophagy in a Bcl-2 regulated fashion and identifies a signaling pathway through which calcium exerts its action. These intriguing findings provoke speculation as to how a cell decides to undergo either apoptosis or macroautophagy in response to calcium signals.