Regulation of mitochondrial ceramide distribution by members of the BCL-2 family

Apoptosis is an intricately regulated cellular process that proceeds through different cell type- and signal-dependent pathways. In the mitochondrial apoptotic program, mitochondrial outer membrane permeabilization by BCL-2 proteins leads to the release of apoptogenic factors, caspase activation, and cell death. In addition to protein components of the mitochondrial apoptotic machinery, an interesting role for lipids and lipid metabolism in BCL-2 family-regulated apoptosis is also emerging. We used a comparative lipidomics approach to uncover alterations in lipid profile in the absence of the proapoptotic proteins BAX and BAK in mouse embryonic fibroblasts (MEFs). We detected over 1,000 ions in these experiments and found changes in an ion with an m/z of 534.49. Structural elucidation of this ion through tandem mass spectrometry revealed that this molecule is a ceramide with a 16-carbon N-acyl chain and sphingadiene backbone (d18:2/16:0 ceramide). Targeted LC/MS analysis revealed elevated levels of additional sphingadiene-containing ceramides (d18:2-Cers) in BAX, BAK-double knockout MEFs. Elevated d18:2-Cers are also found in immortalized baby mouse kidney epithelial cells lacking BAX and BAK. These results support the existence of a distinct biochemical pathway for regulating ceramides with different backbone structures and suggest that sphingadiene-containing ceramides may have functions that are distinct from the more common sphingosine-containing species.     

Mechanisms of cytochrome c release by proapoptotic BCL-2 family members

A crucial amplificatory event in several apoptotic cascades is the nearly complete release of cytochrome c from mitochondria. Proteins of the BCL-2 family which include both anti- and proapoptotic members control this step. Here, we review the proposed mechanisms by which proapoptotic BCL-2 family members induce cytochrome c release. Data support a model in which the apoptotic pathway bifurcates following activation of a "BH3 only" family member. BH3 only molecules induce the activation of the multidomain proapoptotics BAX and BAK, resulting in the permeabilization of the outer mitochondrial membrane and the efflux of cytochrome c. This is coordinated with the activation of a distinct pathway characterized by profound changes of the inner mitochondrial membrane morphology and organization. This mitochondrial remodelling insures complete release of cytochrome c and the onset of mitochondrial dysfunction that is a typical feature of many apoptotic deaths.     

Caspase-2-mediated cleavage of Mdm2 creates a p53-induced positive feedback loop

Caspase-2 is an evolutionarily conserved caspase, yet its biological function and cleavage targets are poorly understood. Caspase-2 is activated by the p53 target gene product PIDD (also known as LRDD) in a complex called the Caspase-2-PIDDosome. We show that PIDD expression promotes growth arrest and chemotherapy resistance by a mechanism that depends on Caspase-2 and wild-type p53. PIDD-induced Caspase-2 directly cleaves the E3 ubiquitin ligase Mdm2 at Asp 367, leading to loss of the C-terminal RING domain responsible for p53 ubiquitination. As a consequence, N-terminally truncated Mdm2 binds p53 and promotes its stability. Upon DNA damage, p53 induction of the Caspase-2-PIDDosome creates a positive feedback loop that inhibits Mdm2 and reinforces p53 stability and activity, contributing to cell survival and drug resistance. These data establish Mdm2 as a cleavage target of Caspase-2 and provide insight into a mechanism of Mdm2 inhibition that impacts p53 dynamics upon genotoxic stress.     

Regulation of mitochondrial membrane permeabilization by BCL-2 family proteins and caspases

Mitochondria play an important role in the integration and transmission of cell death signals, activating caspases and other cell death execution events by releasing apoptogenic proteins from the intermembrane space. The BCL-2 family of proteins localize (or can be targeted) to mitochondria and regulate the permeability of the mitochondrial outer membrane to these apoptotic factors. Recent evidence suggests that multiple mechanisms may regulate the release of mitochondrial factors, some of which depend on the action of caspases.     

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.     

Human embryonic stem cells express elevated levels of multiple pro-apoptotic BCL-2 family members

Two of the greatest challenges in regenerative medicine today remain (1) the ability to culture human embryonic stem cells (hESCs) at a scale sufficient to satisfy clinical demand and (2) the ability to eliminate teratoma-forming cells from preparations of cells with clinically desirable phenotypes. Understanding the pathways governing apoptosis in hESCs may provide a means to address these issues. Limiting apoptosis could aid scaling efforts, whereas triggering selective apoptosis in hESCs could eliminate unwanted teratoma-forming cells. We focus here on the BCL-2 family of proteins, which regulate mitochondrial-dependent apoptosis. We used quantitative PCR to compare the steady-state expression profile of all human BCL-2 family members in hESCs with that of human primary cells from various origins and two cancer lines. Our findings indicate that hESCs express elevated levels of the pro-apoptotic BH3-only BCL-2 family members NOXA, BIK, BIM, BMF and PUMA when compared with differentiated cells and cancer cells. However, compensatory expression of pro-survival BCL-2 family members in hESCs was not observed, suggesting a possible explanation for the elevated rates of apoptosis observed in proliferating hESC cultures, as well as a mechanism that could be exploited to limit hESC-derived neoplasms.     

Regulation of the mitochondrial apoptosis-induced channel, MAC, by BCL-2 family proteins

Programmed cell death or apoptosis is central to many physiological processes and pathological conditions such as organogenesis, tissue homeostasis, cancer, and neurodegenerative diseases. Bcl-2 family proteins tightly control this cell death program by regulating the permeabilization of the mitochondrial outer membrane and, hence, the release of cytochrome c and other pro-apoptotic factors. Control of the formation of the mitochondrial apoptosis-induced channel, or MAC, is central to the regulation of apoptosis by Bcl-2 family proteins. MAC is detected early in apoptosis by patch clamping the mitochondrial outer membrane. The focus of this review is on the regulation of MAC activity by Bcl-2 family proteins. The role of MAC as the putative cytochrome c release channel during early apoptosis and insights concerning its molecular composition are also discussed.     

Mitochondrial BCL-2 inhibits AMBRA1-induced autophagy

BECLIN 1 is a central player in macroautophagy. AMBRA1, a BECLIN 1-interacting protein, positively regulates the BECLIN 1-dependent programme of autophagy. In this study, we show that AMBRA1 binds preferentially the mitochondrial pool of the antiapoptotic factor BCL-2, and that this interaction is disrupted following autophagy induction. Further, AMBRA1 can compete with both mitochondrial and endoplasmic reticulum-resident BCL-2 (mito-BCL-2 and ER-BCL-2, respectively) to bind BECLIN 1. Moreover, after autophagy induction, AMBRA1 is recruited to BECLIN 1. Altogether, these results indicate that, in normal conditions, a pool of AMBRA1 binds preferentially mito-BCL-2; after autophagy induction, AMBRA1 is released from BCL-2, consistent with its ability to promote BECLIN 1 activity. In addition, we found that the binding between AMBRA1 and mito-BCL-2 is reduced during apoptosis. Thus, a dynamic interaction exists between AMBRA1 and BCL-2 at the mitochondria that could regulate both BECLIN 1-dependent autophagy and apoptosis.     

Caspase-9 mediates Puma activation in UCN-01-induced apoptosis

The protein kinase inhibitor 7-hydroxystaurosporine (UCN-01) is one of the most potent and frequently used proapoptotic stimuli. The BH3-only molecule of Bcl-2 family proteins has been reported to contribute to UCN-01-induced apoptosis. Here we have found that UCN-01 triggers Puma-induced mitochondrial apoptosis pathway. Our data confirmed that Akt-FoxO3a pathway mediated Puma activation. Importantly, we elucidate the detailed mechanisms of Puma-induced apoptosis. Our data have also demonstrated that caspase-9 is a decisive molecule of Puma induction after UCN-01 treatment. Caspase-9 mediates apoptosis through two kinds of feedback loops. On the one hand, caspase-9 enhances Puma activation by cleaving Bcl-2 and Bcl-xL independent of caspase-3. On the other hand, caspase-9 directly activated caspase-3 in the presence of caspase-3. Caspase-3 could cleave XIAP in an another positive feedback loop to further sensitize cancer cells to UCN-01-induced apoptosis. Therefore, caspase-9 mediates Puma activation to determine the threshold for overcoming chemoresistance in cancer cells.The apoptosis pathway is closely related to the Bcl-2 family proteins in which antiapoptotic members sequester multidomain proapoptotic proteins, thereby inhibiting their active role in apoptosis. In contrast, BH3-only proteins that are considered stress sensors can dissociate Bax-like proteins from their antiapoptotic sequestrators, and thus leading to apoptosis.¹The expression of Bcl-2 family proteins is regulated during carcinogenesis,¹ and the expression of both the Bcl-2 and Bcl-xL antiapoptotic proteins is associated with resistance to antitumor agents such as cisplatin (CP).² The inhibition of the protective function of antiapoptotic Bcl-2 members can either restore the normal apoptotic process in cancer cells or circumvent resistance to chemotherapy.^3,4 In this regard, enhanced expression of BH3-only proteins can effectively bind the antiapoptotic members and prevent the function of these proteins.Some reports suggest that the BH3-only protein Puma has important roles in p53-dependent and -independent apoptosis in human cancer cells and mediates cell death through the Bcl-2 family proteins Bax/Bak and the mitochondrial pathway.^5,6 Our studies also reveal that Puma upregulation induces cell apoptosis in chemoresistant ovarian cancer cells,^7,8 confirming the requisite role of Puma in chemosensitivity.7-Hydroxystaurosporine (UCN-01) is a protein kinase C-selective inhibitor that is successfully used in phase I and II clinical trials.^9,10 As a modulator, UCN-01 enhances the cytotoxicity of other anticancer drugs such as DNA-damaging agents and antimetabolite drugs by putative abrogation of G2- and/or S-phase accumulation induced by these anticancer agents.¹¹ As a single agent, UCN-01 exhibits two key biochemical effects, namely accumulation of cells in the G1 phase of the cell cycle and induction of apoptosis.¹² Both these effects may be important for its anticancer activity. Previous studies have demonstrated that UCN-01 potently decreased the levels of activated the phosphorylation level of Akt (p-Akt) in in vitro or in in vivo systems.^{12, 13, 14} Some researchers have also approved that UCN-01 can modulate Bcl-2 family members to potentiate apoptosis in cancer cells.^15,16 These reports suggest that Akt and Bcl-2 family proteins may be the potent targets of UCN-01 to trigger cancer cell apoptosis.In this study, we also investigate the role of Puma in UCN-01-induced apoptosis and confirm that p53-independent Puma induction is pivotal for the anticancer effects of UCN-01. Moreover, we first elucidate the detailed mechanism of Puma-induced apoptosis after UCN-01 treatment. We found that Puma expression mediated caspase-9 and caspase-3 activation. Among the caspase proteins, caspase-9 has a key role in Puma-induced apoptosis. Our data demonstrated that caspase-9 could mediate Puma-induced apoptosis through two feedback pathways. On the one hand, activated caspase-9 was initiated followed by caspase-3 activity, and activated caspase-3 cleaved XIAP in a positive feedback loop to strengthen Puma expression. On the other hand, caspase-9 itself cleaved antiapoptotic Bcl-2 and Bcl-xL to positively enhance Puma induction. These results provide the detailed mechanistic insight into therapeutic response to UCN-01 and the theoretical basis for its applications.     

Proapoptotic BCL-2 family members and mitochondrial dysfunction during ischemia/reperfusion injury, a study employing cardiac HL-1 cells and GFP biosensors

The objective of this study was to evaluate mitochondrial alterations in a cell-based model of myocardial ischemia/reperfusion (I/R) injury. Using GFP-biosensors and fluorescence deconvolution microscopy, we investigated mitochondrial morphology in relation to Bax and Bid activation in the HL-1 cardiac cell line. Mitochondria underwent extensive fragmentation during ischemia. Bax translocation from cytosol to mitochondria was initiated during ischemia and proceeded during reperfusion. However, Bax translocation was not sufficient to induce cell death or mitochondrial dysfunction. Bid processing was caspase-8 dependent, and Bid translocation to mitochondria occurred after Bax translocation and clustering, and minutes before cell death. Clustering of Bax into distinct regions on mitochondria could be prevented by CsA, an inhibitor of the mitochondrial permeability transition pore, and also by SB203580, an inhibitor of p38 MAPK. Surprisingly, mitochondrial fragmentation which occurred during ischemia and before Bax translocation could be reversed by the addition of the p38 inhibitor SB203580 at reperfusion. Taken together, these results implicate p38 MAPK in the mitochondrial remodeling response to I/R that facilitates Bax recruitment to mitochondria.     

Caspase-2-induced apoptosis requires bid cleavage: a physiological role for bid in heat shock-induced death

The mechanisms through which Caspase-2 leads to cell death are controversial. Here we show, using a combination of cell-free and cell culture-based approaches, that cleavage of the Bcl-2-family protein Bid is required for the induction of apoptosis by Caspase-2. Caspase-2 promoted cytochrome c release from mitochondria in the presence of cytosol from wild-type, but not Bid-deficient, mouse embryonic fibroblasts (MEFs). Recombinant wild-type Bid, but not a noncleavable mutant (D59E), restored cytochrome c release. Similarly, Bid-null MEFs were relatively resistant to apoptosis triggered by active Caspase-2, and apoptosis was restored in Bid-null cells by the expression of wild-type, but not D59E, Bid. Finally, Bid-null MEFs were substantially more resistant to apoptosis induced by heat shock, which has been shown to be dependent on apical activation of Caspase-2. The data are consistent with a model in which Caspase-2 induces apoptosis via cleavage of Bid at D59 and the subsequent engagement of the mitochondrial (intrinsic) pathway.     

Caspase 8-mediated cleavage of the pro-apoptotic BCL-2 family member BID in p53-dependent apoptosis

The objective of this study was to characterize the apoptotic pathways activated by fast neutrons in the human lymphoblastoid cell line TK6 and in its p53 −/− derivative. Our results demonstrate that while p53 is not required for neutron-induced apoptosis, as previously shown, it does affect the kinetics of apoptosis and the molecular pathways leading to the activation of effector caspases. Indeed, rapid p53-dependent apoptosis was associated with the activation of caspase 9, 8, 3, and 7 and the cleavage of BID by caspase 8. In contrast, the slow-occurring p53-independent apoptotic process, mediated by caspase 7, took place without BID cleavage and loss of transmembrane mitochondrial potential. Altogether, our findings highlight an essential role for caspase 8-mediated BID cleavage, in the course of p53-dependent apoptosis triggered by fast neutrons in lymphoid cells. They also demonstrate that this mechanism is not involved in p53-independent apoptosis.     

Control of mitochondrial integrity by Bcl-2 family members and caspase-independent cell death

Programmed cell death (PCD) is essential for normal development and maintenance of tissue homeostasis in multicellular organisms. While it is now evident that PCD can take many different forms, apoptosis is probably the most well-defined cell death programme. The characteristic morphological and biochemical features associated with this highly regulated form of cell death have until recently been exclusively attributed to the caspase family of cysteine proteases. As a result, many investigators affiliate apoptosis with its pivotal execution system, i.e. caspase activation. However, it is becoming increasingly clear that PCD or apoptosis can also proceed in a caspase-independent manner and maintain key characteristics of apoptosis. Mitochondrial integrity is central to both caspase-dependent and-independent cell death. The release of pro-apoptotic factors from the mitochondrial intermembrane space is a key event in a cell's commitment to die and is under the tight regulation of the Bcl-2 family. However, the underlying mechanisms governing the efflux of these pro-death molecules are largely unknown. This review will focus on the regulation of mitochondrial integrity by Bcl-2 family members with particular attention to the controlled release of factors involved in caspase-independent cell death.     

Caspase cleavage of members of the amyloid precursor family of proteins

The synapse loss and neuronal cell death characteristic of Alzheimer's disease (AD) are believed to result in large part from the neurotoxic effects of beta-amyloid peptide (Abeta), a 40-42 amino acid peptide(s) derived proteolytically from beta-amyloid precursor protein (APP). However, APP is also cleaved intracellularly to generate a second cytotoxic peptide, C31, and this cleavage event occurs in vivo as well as in vitro and preferentially in the brains of AD patients (Lu et al. 2000). Here we show that APPC31 is toxic to neurons in primary culture, and that like APP, the APP family members APLP1 and possibly APLP2 are cleaved by caspases at their C-termini. The carboxy-terminal peptide derived from caspase cleavage of APLP1 shows a degree of neurotoxicity comparable to APPC31. Our results suggest that even though APLP1 and APLP2 cannot generate Abeta, they may potentially contribute to the pathology of AD by generating peptide fragments whose toxicity is comparable to that of APPC31.     

Caspase-6 gene disruption reveals a requirement for lamin A cleavage in apoptotic chromatin condensation

To study the role of caspase-6 during nuclear disassembly, we generated a chicken DT40 cell line in which both alleles of the caspase-6 gene were disrupted. No obvious morphological differences were observed in the apoptotic process in caspase-6- deficient cells compared with the wild type. However, examination of apoptosis in a cell-free system revealed a block in chromatin condensation and apoptotic body formation when nuclei from HeLa cells expressing lamin A or lamin A-transfected Jurkat cells were incubated in caspase-6-deficient apoptotic extracts. Transfection of exogenous caspase-6 into the clone reversed this phenotype. Lamins A and C, which are caspase-6-only substrates, were cleaved by the wild-type and heterozygous apoptotic extracts but not by the extracts lacking caspase-6. Furthermore, the caspase-6 inhibitor z-VEID-fmk mimicked the effects of caspase-6 deficiency and prevented the cleavage of lamin A. Taken together, these observations indicate that caspase-6 activity is essential for lamin A cleavage and that when lamin A is present it must be cleaved in order for the chromosomal DNA to undergo complete condensation during apoptotic execution.     

BCL-2-induced glioma cell invasiveness depends on furin-like proteases

Migration and invasion are prerequisites for the neoplastic phenotype of malignant glioma. Ectopic expression of BCL-2 enhances migration and invasion of glioma cells and promotes their synthesis of transforming growth factor-beta2 (TGF-beta2). We here report that BCL-2-expressing cells show enhanced expression and activity of the proprotein convertase, furin, which processes metalloproteinases (MMP) and TGF-beta. Consistent with a biological role for a BCL-2-dependent increase in furin-like protease (FLP) activity, BCL-2-expressing cells exhibit enhanced MMP activity. Both a pseudosubstrate furin inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk), or alpha 1-anti-trypsin Portland (PDX), a recombinant furin-inhibitory protein, suppress constitutive and BCL-2-mediated MMP activity and invasion. This inhibition is not overcome by TGF-beta or hepatocyte growth factor (HGF). A neutralizing TGF-beta antibody attenuates, but not abrogates, the invasive properties conferred by exogenous expression of BCL-2, whereas the MMP inhibitor o-phenantroline (o-PA) abolishes the pro-invasive action of BCL-2. Exogenous HGF results in enhanced, and expression of dominant-negative ezrin in reduced, FLP activity, and dec-RVKR-cmk blunts the HGF-induced expression of mature TGF-beta2. Consequently, HGF and BCL-2 family proteins use a furin-dependent pathway to promote invasion via TGF-beta and MMP in human malignant glioma cells and the pro-invasive properties of TGF-beta require furin- dependent MMP activity.