Involvement of oxidative stress and caspase 2-mediated intrinsic pathway signaling in age-related increase in muscle cell apoptosis in mice

Apoptosis has been implicated as a mechanism of loss of muscle cells in normal aging and plays an important role in age-related sarcopenia. To test the hypothesis that caspase 2 and c-Jun NH₂-terminal kinase (JNK)-mediated intrinsic pathway signaling contribute to skeletal muscle cell apoptosis in aging, we compared activation of caspase 2 and JNK and the in vivo expression of 4-hydroxynonenal protein adducts (4-HNE), inducible nitric oxide synthase (iNOS), glucose-6-phosphate dehydrogenase (G6PDH), B-cell lymphoma-2 (BCL-2), BAX, and phospho-BCL-2 in gastrocnemius muscles of young (5 months old) and old (25 months old) mice. A distinct age-related increase in 4-HNE and iNOS expression was readily detected in mice. Increased oxidative stress and iNOS induction were further accompanied by a decrease in G6PDH expression, activation of caspase 2 and JNK, and inactivation of BCL-2 through phosphorylation at serine 70, and caspase 9 activation. Regression analysis further revealed that increased muscle cell death in aging was significantly correlated with changes in the levels of these molecules. Taken together, our data indicate that caspase 2 and JNK-mediated intrinsic pathway signaling is one of the mechanisms involved in age-related increase in muscle cell apoptosis.     

Involvement of p38 mitogen-activated protein kinase and inducible nitric oxide synthase in apoptotic signaling of murine and human male germ cells after hormone deprivation

This study investigates the role of p38 MAPK, inducible nitric oxide synthase (iNOS), and the intrinsic pathway signaling in male germ cell death in rats after hormonal deprivation by a potent GnRH antagonist treatment. Germ cell apoptosis, involving exclusively middle (VII-VIII) stages, was activated by d 5 after GnRH antagonist treatment. Initiation of germ cell apoptosis was preceded by p38 MAPK activation and induction of iNOS. p38 MAPK activation and iNOS induction were further accompanied by a marked perturbation of the BAX/BCL-2 rheostat, cytochrome c, and DIABLO release from mitochondria, caspase activation, and poly(ADP-ribose) polymerase cleavage. Concomitant administration of aminoguanidine, a selective iNOS inhibitor, significantly prevented hormone deprivation-induced germ cell apoptosis. Inhibitors of iNOS or p38 MAPK were also effective in preventing human male germ cell apoptosis induced by hormone-free culture conditions. Together, these results establish a new signal transduction pathway involving p38 MAPK and iNOS that, through activation of the intrinsic pathway signaling, promotes male germ cell death in response to a lack of hormonal stimulation across species.     

Inhibition of apoptotic signalling in spermine‐treated vascular smooth muscle cells by a novel glutathione precursor

CKD (chronic kidney disease) is a public health problem, mediated by haemodynamic and non‐haemodynamic events including oxidative stress. We investigated the effect of two GSH (glutathione) precursors, NAC (N‐acetylcysteine) and cystine as the physiological carrier of cysteine in GSH with added selenomethionine (F1) in preventing spermine (uraemic toxin)‐induced apoptosis in cultured human aortic VSMC (vascular smooth muscle cells). VSMCs exposed to spermine (15 μM) with or without antioxidants (doses 50, 100, 200 and 500 μg/ml) were assessed for apoptosis, JNK (c‐Jun‐NH₂‐terminal kinase) activation and iNOS (inducible nitric oxide synthase) induction and activation of intrinsic pathway signalling. Spermine exposure resulted in activation of JNK and iNOS induction and apoptosis. NAC and F1 (dose range 50–500 μg/ml) attenuated spermine‐induced acceleration of VSMC apoptosis but only F1 (at 200 and 500 μg/ml) maintained spermine‐induced apoptosis at control levels. Spermine‐induced JNK activation was prevented by 200 μg/ml of both NAC and F1, while iNOS induction was blocked only by F1. Notably, the adverse effects of spermine on BAX/BCL‐2 ratio, cytochrome c release and caspase activation was fully attenuated by F1. In conclusion, F1 was more effective than NAC in preventing spermine‐induced apoptosis and downstream changes in related signal transduction pathways in VSMCs. Further studies are needed to examine the effect of these compounds in preventing CKD‐associated vascular disease.     

LPS/IFN-γ-Induced RAW 264.7 Apoptosis is Regulated by Both Nitric Oxide–Dependent and –Independent Pathways Involving JNK and the Bcl-2 Family

Lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) stimulate macrophages to produce nitric oxide (NO) via inducible nitric oxide synthase (iNOS) and activate stress signaling cascades including the c-jun-N-terminal kinase (JNK) pathway. These events trigger an apoptotic cascade that ultimately results in death. Since JNK regulates pro-apoptotic and anti-apoptotic Bcl-2 family members, the role of NO in LPS/IFN-γ-induced activation of JNK and its effects on the Bcl-2 family was examined in RAW 264.7 macrophage-like cells. Inhibition of JNK by siRNA verified a role for JNK in LPS/IFN-γ-induced apoptosis. Suppression of NO production by a pharmacologic agent, i.e. iNOS inhibitor L-NIL, altered the kinetics of JNK activation by LPS/IFN-γ. Examination of mitochondrial and nuclear compartments of RAW 264.7 cells demonstrated NO-dependent activation of mitochondrial JNK by LPS/IFN-γ, but NO-independent, cytokine-induced phosphorylation of Bim. NO did not affect phosphorylation, but did inhibit Bax phosphorylation. These results suggest a novel mechanism of LPS/IFN-γ-induced apoptosis in macrophages involving NO-independent phosphorylation of Bim and NO-dependent dephosphorylation of Bax.     

Prevention of TNF-alpha-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2

It has been documented that polyamines play a critical role in the regulation of apoptosis in intestinal epithelial cells. We have recently reported that protection from TNF-alpha/cycloheximide (CHX)-induced apoptosis in epithelial cells depleted of polyamines is mediated through the inactivation of a proapoptotic mediator, JNK. In this study, we addressed the involvement of the MAPK pathway in the regulation of apoptosis after polyamine depletion of IEC-6 cells. Polyamine depletion by alpha-difluromethylornithine (DFMO) resulted in the sustained activation of ERK in response to TNF-alpha/CHX treatment. Pretreatment of polyamine-depleted IEC-6 cells with a cell membrane-permeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-alpha/CHX-induced ERK phosphorylation and significantly increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-alpha/CHX. Moreover, the dose dependency of U-0126-mediated inhibition of TNF-alpha/ CHX-induced ERK phosphorylation correlated with the reversal of the antiapoptotic effect of DFMO. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-alpha/CHX-induced JNK phosphorylation and were significantly protected from apoptosis. Conversely, a dominant-negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant-negative MEK1 cells did not prevent JNK activation or cytochrome c release and failed to confer protection from both TNF-alpha/CHX and camptothecin-induced apoptosis. Finally, expression of a dominant-negative mutant of JNK significantly protected IEC-6 cells from TNF-alpha/CHX-induced apoptosis. These data indicate that polyamine depletion results in the activation of ERK, which inhibits JNK activation and protects cells from apoptosis.     

Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site

The BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Pro-apoptotic Bcl-2-associated X protein (BAX) is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. Following cellular stresses that induce apoptosis, cytosolic BAX is activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore. How the BAX activation pathway proceeds and how this may be inhibited is not yet completely understood. Here we describe synthetic antibody fragments (Fabs) as structural and biochemical probes to investigate the potential mechanisms of BAX regulation. These synthetic Fabs bind with high affinity to BAX and inhibit its activation by the BH3-only protein tBID (truncated Bcl2 interacting protein) in assays using liposomal membranes. Inhibition of BAX by a representative Fab, 3G11, prevented mitochondrial translocation of BAX and BAX-mediated cytochrome c release. Using NMR and hydrogen-deuterium exchange mass spectrometry, we showed that 3G11 forms a stoichiometric and stable complex without inducing a significant conformational change on monomeric and inactive BAX. We identified that the Fab-binding site on BAX involves residues of helices α1/α6 and the α1-α2 loop. Therefore, the inhibitory binding surface of 3G11 overlaps with the N-terminal activation site of BAX, suggesting a novel mechanism of BAX inhibition through direct binding to the BAX N-terminal activation site. The synthetic Fabs reported here reveal, as probes, novel mechanistic insights into BAX inhibition and provide a blueprint for developing inhibitors of BAX activation.     

MCL-1 inhibits BAX in the absence of MCL-1/BAX Interaction

The BCL-2 family of proteins plays a major role in the control of apoptosis as the primary regulator of mitochondrial permeability. The pro-apoptotic BCL-2 homologues BAX and BAK are activated following the induction of apoptosis and induce cytochrome c release from mitochondria. A second class of BCL-2 homologues, the BH3-only proteins, is required for the activation of BAX and BAK. The activity of both BAX/BAK and BH3-only proteins is opposed by anti-apoptotic BCL-2 homologues such as BCL-2 and MCL-1. Here we show that anti-apoptotic MCL-1 inhibits the function of BAX downstream of its initial activation and translocation to mitochondria. Although MCL-1 interacted with BAK and inhibited its activation, the activity of MCL-1 against BAX was independent of an interaction between the two proteins. However, the anti-apoptotic function of MCL-1 required the presence of BAX. These results suggest that the pro-survival activity of MCL-1 proceeds via inhibition of BAX function at mitochondria, downstream of its activation and translocation to this organelle.     

Two distinct signaling pathways regulate peroxynitrite-induced apoptosis in PC12 cells

The mechanisms of peroxynitrite-induced apoptosis are not fully understood. We report here that peroxynitrite-induced apoptosis of PC12 cells requires the simultaneous activation of p38 and JNK MAP kinase, which in turn activates the intrinsic apoptotic pathway, as evidenced by Bax translocation to the mitochondria, cytochrome c release to the cytoplasm and activation of caspases, leading to cell death. Peroxynitrite induces inactivation of the Akt pathway. Furthermore, overexpression of constitutively active Akt inhibits both peroxynitrite-induced Bax translocation and cell death. Peroxynitrite-induced death was prevented by overexpression of Bcl-2 and by cyclosporin A, implicating the involvement of the intrinsic apoptotic pathway. Selective inhibition of mixed lineage kinase (MLK), p38 or JNK does not attenuate the decrease in Akt phosphorylation showing that inactivation of the Akt pathway occurs independently of the MLK/MAPK pathway. Together, these results reveal that peroxynitrite-induced activation of the intrinsic apoptotic pathway involves interactions with the MLK/MAPK and Akt signaling pathways.     

Apoptosis regulators

Over the last decade, a great deal of attention has been directed at elucidating the role of apoptosis regulators in governing survival decisions in neoplastic cells, particularly those of hematopoietic origin. A major focus of this work has involved investigation of the function of pro- and anti-apoptotic members of the BCL-2 family, and the relationship between these proteins and mitochondrial integrity. Currently, these proteins can be classified into two broad categories: those that modulate mitochondrial function and those that regulate the activation of caspases responsible for activation and execution of the apoptotic cascade. Within the first category, certain proteins (e.g., BCL-2, BCL-xL) act to preserve mitochondrial integrity by preventing loss of mitochondrial membrane potential and/or release of pro-apoptotic proteins such as cytochrome C into the cytosol. Other proapoptotic proteins (e.g., BAX, BAK, BIM) promote release of cytochrome C. These proteins are therefore primarily involved in regulation of the intrinsic, mitochondrial apoptotic pathway. Within the second category, proteins such as the inhibitors of apoptosis proteins (e.g., XIAP) or FLIP block the activation of caspases, particularly those involved in engagement of the receptor-related, extrinsic apoptotic pathway. Cross-talk between the intrinsic and extrinsic pathways exists. For example, the BH3-domain only protein BID is cleaved by the activation of pro-caspase-8 through the extrinsic pathway, and translocates to the mitochondrion to promote cytochrome C release. Apoptosis is also regulated by various signal transduction pathways, possibly through post-translational modifications in BCL-2 family proteins. For example, phosphorylation of BCL-2 through a JNK-dependent mechanism has been postulated to contribute to apoptosis induced by the taxane class of cytotoxic agents. Finally, attempts to modulate apoptotic pathways with small molecules have recently received much attention. For example, small molecule inhibitors of BCL-2 or mimetics of SMAC/DIABLO, which opposes the actions of XIAP, have recently been shown to promote the antineoplastic activity of conventional cytotoxic agents. It is likely that an improved understanding of apoptosis regulation will lead to new insights into neoplastic transformation, and may also provide important leads for the development of novel antileukemic strategies.     

A BAX/BAK and cyclophilin D-independent intrinsic apoptosis pathway

Most intrinsic death signals converge into the activation of pro-apoptotic BCL-2 family members BAX and BAK at the mitochondria, resulting in the release of cytochrome c and apoptosome activation. Chronic endoplasmic reticulum (ER) stress leads to apoptosis through the upregulation of a subset of pro-apoptotic BH3-only proteins, activating BAX and BAK at the mitochondria. Here we provide evidence indicating that the full resistance of BAX and BAK double deficient (DKO) cells to ER stress is reverted by stimulation in combination with mild serum withdrawal. Cell death under these conditions was characterized by the appearance of classical apoptosis markers, caspase-9 activation, release of cytochrome c, and was inhibited by knocking down caspase-9, but insensitive to BCL-X(L) overexpression. Similarly, the resistance of BIM and PUMA double deficient cells to ER stress was reverted by mild serum withdrawal. Surprisingly, BAX/BAK-independent cell death did not require Cyclophilin D (CypD) expression, an important regulator of the mitochondrial permeability transition pore. Our results suggest the existence of an alternative intrinsic apoptosis pathway emerging from a cross talk between the ER and the mitochondria.     

Direct and selective small-molecule activation of proapoptotic BAX

BCL-2 family proteins are key regulators of the apoptotic pathway. Antiapoptotic members sequester the BCL-2 homology 3 (BH3) death domains of proapoptotic members such as BAX to maintain cell survival. The antiapoptotic BH3-binding groove has been successfully targeted to reactivate apoptosis in cancer. We recently identified a geographically distinct BH3-binding groove that mediates direct BAX activation, suggesting a new strategy for inducing apoptosis by flipping BAX's 'on switch'. Here we applied computational screening to identify a BAX activator molecule that directly and selectively activates BAX. We demonstrate by NMR and biochemical analyses that the molecule engages the BAX trigger site and promotes the functional oligomerization of BAX. The molecule does not interact with the BH3-binding pocket of antiapoptotic proteins or proapoptotic BAK and induces cell death in a BAX-dependent fashion. To our knowledge, we report the first gain-of-function molecular modulator of a BCL-2 family protein and demonstrate a new paradigm for pharmacologic induction of apoptosis.     

The hierarchical relationship between MAPK signaling and ROS generation in human leukemia cells undergoing apoptosis in response to the proteasome inhibitor Bortezomib

The hierarchy of events accompanying induction of apoptosis by the proteasome inhibitor Bortezomib was investigated in Jurkat lymphoblastic and U937 myelomonocytic leukemia cells. Treatment of Jurkat or U937 cells with Bortezomib resulted in activation of c-Jun-N-terminal kinase (JNK) and p38 MAPK (mitogen-activated protein kinase), inactivation of extracellular signal-regulating kinase 1/2 (ERK1/2), cytochrome c release, caspase-9, -3, and -8 activation, and apoptosis. Bortezomib-mediated cytochrome c release and caspase activation were blocked by the pharmacologic JNK inhibitor SP600125, but lethality was not diminished by the p38 MAPK inhibitor SB203580. Inducible expression of a constitutively active MEK1 construct blocked Bortezomib-mediated ERK1/2 inactivation, significantly attenuated Bortezomib lethality, and unexpectedly prevented JNK activation. Conversely, pharmacologic MEK/ERK1/2 inhibition promoted Bortezomib-mediated JNK activation and apoptosis. Lastly, the antioxidant N-acetyl-l-cysteine (LNAC) attenuated Bortezomib-mediated reactive oxygen species (ROS) generation, ERK inactivation, JNK activation, mitochondrial dysfunction, and apoptosis. In contrast, enforced MEK1 and ERK1/2 activation or JNK inhibition did not modify Bortezomib-induced ROS production. Together, these findings suggest that in human leukemia cells, Bortezomib-induced oxidative injury operates at a proximal point in the cell death cascade to antagonize cytoprotective ERK1/2 signaling, promote activation of the stress-related JNK pathway, and to trigger mitochondrial dysfunction, caspase activation, and apoptosis. They also suggest the presence of a feedback loop wherein Bortezomib-mediated ERK1/2 inactivation contributes to JNK activation, thereby amplifying the cell death process.     

AKT/protein kinase B regulation of BCL family members during oxysterol-induced apoptosis

Cells of the vasculature, including macrophages, smooth muscle cells, and endothelial cells, exhibit apoptosis in culture upon treatment with oxidized low density lipoprotein, as do vascular cells of atherosclerotic plaque. Several lines of evidence support the hypothesis that the apoptotic component of oxidized low density lipoprotein is one or more oxysterols, which have been shown to induce apoptosis through the mitochondrial pathway. Activation of the mitochondrial pathway of apoptosis is regulated by members of the BCL family of proteins. In this study, we demonstrate that, in the murine macrophage-like cell line P388D1, oxysterols (25-hydroxycholesterol and 7-ketocholesterol) induced the degradation of the prosurvival protein kinase AKT (protein kinase B). This led, in turn, to the activation of the BCL-2 homology-3 domain-only proteins BIM and BAD and down-regulation of the anti-apoptotic multi-BCL homology domain protein BCL-xL. These responses would be expected to activate the pro-apoptotic multi-BCL homology domain proteins BAX and BAK, leading to the previously reported release of cytochrome c observed during oxysterol-induced apoptosis. Somewhat surprisingly, small interfering RNA knockdown of BAX resulted in a complete block of the induction of apoptosis by 25-hydroxycholesterol.     

Hyperglycemia induces apoptosis of pancreatic islet endothelial cells via reactive nitrogen species-mediated Jun N-terminal kinase activation

Hyperglycemia significantly stimulates pancreatic islet endothelial cell apoptosis; however, the precise mechanisms are not fully understood. In the present study, treating pancreatic islet endothelial (MS-1) cells with high glucose (30 mmol/l) but not mannitol significantly increased the number of apoptotic cells as compared with a physiological glucose concentration (5.5 mmol/l). Hyperglycemia significantly stimulated the expression of inducible nitric oxide synthase (iNOS) and production of NO and peroxynitrite (ONOO⁻), relevant to MS-1 cell apoptosis. Moreover, induced reactive nitrogen species (RNS) significantly increased the expression of bax, cleaved caspase-3 and poly adenosine diphosphate (ADP)-ribose polymerase (PARP) via JNK activation, but the expression of bcl-2 was not altered. Furthermore, SP600125 (a specific inhibitor of JNK) and 1400W (a specific inhibitor of iNOS) significantly attenuated cell apoptosis induced by high glucose. Therefore, hyperglycemia triggers MS-1 cell apoptosis by activating an intrinsic-dependent apoptotic pathway via RNS-mediated JNK activation.     

Pro-apoptotic complexes of BAX and BAK on the outer mitochondrial membrane

In multicellular organisms the regulated cell death apoptosis is critically important for both ontogeny and homeostasis. Mitochondria are indispensable for stress-induced apoptosis. The BCL-2 protein family controls mitochondrial apoptosis and initiates cell death through the pro-apoptotic activities of BAX and BAK at the outer mitochondrial membrane (OMM). Cellular survival is ensured by the retrotranslocation of mitochondrial BAX and BAK into the cytosol by anti-apoptotic BCL-2 proteins. BAX/BAK-dependent OMM permeabilization releases the mitochondrial cytochrome c (cyt c), which initiates activation of caspase-9. The caspase cascade leads to cell shrinkage, plasma membrane blebbing, chromatin condensation, and apoptotic body formation. Although it is clear that ultimately complexes of active BAX and BAK commit the cell to apoptosis, the nature of these complexes is still enigmatic. Excessive research has described a range of complexes, varying from a few molecules to several 10,000, in different systems. BAX/BAK complexes potentially form ring-like structures that could expose the inner mitochondrial membrane. It has been suggested that these pores allow the efflux of small proteins and even mitochondrial DNA. Here we summarize the current state of knowledge for mitochondrial BAX/BAK complexes and the interactions between these proteins and the membrane.     

BOK and NOXA are essential mediators of p53-dependent apoptosis

Cellular stress leads to DNA damage and activation of the intrinsic apoptotic pathway in which translocation of mitochondrial cytochrome c to the cytosol plays a critical role. Previous studies have suggested alternative mechanisms responsible for this process. We examined initiation mechanisms of the intrinsic apoptotic pathway using human neuroblastoma and breast cancer cells. Results indicated that translocation of cytochrome c does not require prior activation of caspases but rather depends on activation of specific BCL-2 family members, depending upon the type of death signal. Thus, DNA damage-induced apoptosis requires new protein synthesis, accumulation of p53 tumor suppressor protein, and p53-dependent induction of BOK and NOXA genes, while a role for BAX in this pathway is not essential. In contrast, apoptosis induced by staurosporine does not require protein synthesis but is characterized by translocation of BAX. Based on these findings, we propose a model of the intrinsic apoptotic cascade induced by DNA damage where proapoptotic BOK substitutes for a function of BAX.     

S-palmitoylation represents a novel mechanism regulating the mitochondrial targeting of BAX and initiation of apoptosis

The intrinsic pathway of apoptotic cell death is mainly mediated by the BCL-2-associated X (BAX) protein through permeabilization of the mitochondrial outer membrane (MOM) and the concomitant release of cytochrome c into the cytosol. In healthy, non-apoptotic cells, BAX is predominantly localized in the cytosol and exhibits a dynamic shuttle cycle between the cytosol and the mitochondria. Thus, the initial association with mitochondria represents a critical regulatory step enabling BAX to insert into MOMs, promoting the release of cytochrome c and ultimately resulting in apoptosis. However, the molecular mode of how BAX associates with MOMs and whether a cellular regulatory mechanism governs this process is poorly understood. Here we show that in both primary tissues and cultured cells, the association with MOMs and the proapoptotic action of BAX is controlled by its S-palmitoylation at Cys-126. A lack of BAX palmitoylation reduced BAX mitochondrial translocation, BAX oligomerization, caspase activity and apoptosis. Furthermore, ectopic expression of specific palmitoyl transferases in cultured healthy cells increases BAX S-palmitoylation and accelerates apoptosis, whereas malignant tumor cells show reduced BAX S-palmitoylation consistent with their reduced BAX-mediated proapoptotic activity. Our findings suggest that S-palmitoylation of BAX at Cys126 is a key regulatory process of BAX-mediated apoptosis.     

Survival factor-mediated BAD phosphorylation raises the mitochondrial threshold for apoptosis

Growth factor suppression of apoptosis correlates with the phosphorylation and inactivation of multiple proapoptotic proteins, including the BCL-2 family member BAD. However, the physiological events required for growth factors to block cell death are not well characterized. To assess the contribution of BAD inactivation to cell survival, we generated mice with point mutations in the BAD gene that abolish BAD phosphorylation at specific sites. We show that BAD phosphorylation protects cells from the deleterious effects of apoptotic stimuli and attenuates death pathway signaling by raising the threshold at which mitochondria release cytochrome c to induce cell death. These findings establish a function for endogenous BAD phosphorylation, and elucidate a mechanism by which survival kinases block apoptosis in vivo.