Neuronal Apoptosis: BH3-Only Proteins the Real Killers?

At present there is a poor understanding of the events that lead up to neuronal apoptosis that occurs in neurodegenerative diseases and following acute ischemic episodes. Apoptosis is critical for the elimination of unwanted neurons within the developing nervous system. The Bcl-2 family of proteins contains pro- and anti-apoptotic proteins that regulate the mitochondrial pathway of apoptosis. There is increasing interest in a subfamily of the Bcl-2 family, the BH3-only proteins, and their pro-apoptotic effects within neurons. Recently ischemic and seizure-induced neuronal injury has been shown to result in the activation of the BH3-only protein, Bid. This protein is cleaved and the truncated protein (tBid) translocates to the mitochondria. The translocation of tBid to the mitochondria is associated with the activation of outer mitochondrial membrane proteins Bax/Bak and the release of cytochrome C from the mitochondria. ER stress also has been implicated as a factor for the induction of apoptosis in ischemic neuronal injury. The induction of ER stress in hippocampal neurons has been shown to activate expression of bb3/PUMA, a member of the BH3-only gene family. Activation of PUMA is associated with the activation and clustering of the pro-apoptotic Bcl-2 family member Bax and the loss of cytochrome C from the mitochondria.     

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.     

Endoplasmic reticulum stress-induced apoptosis requires bax for commitment and Apaf-1 for execution in primary neurons

Apoptosis triggered by endoplasmic reticulum (ER) stress is associated with various pathophysiological conditions including neurodegenerative diseases and ischemia. However, the mechanism by which ER stress induces neuronal apoptosis remains controversial. Here we identify the pathway of apoptosis carried out in sympathetic neurons triggered to die by ER stress-inducing agent tunicamycin. We find that ER stress induces a neuronal apoptotic pathway which upregulates BH3-only genes DP5 and Puma. Importantly, we show that ER stress commits neurons to die before cytochrome c release and this commitment requires Bax activation and c-jun N-terminal kinase signaling. Furthermore, ER stress engages the mitochondrial pathway of death as neurons release cytochrome c and Apaf-1 deficiency is sufficient to block apoptosis. Our findings identify a critical function of Bax in committing neurons to ER stress-induced apoptosis and clarify the importance of the apoptosome as the non-redundant caspase activation pathway to execute neuronal apoptosis in response to ER stress.     

A common BIM deletion polymorphism mediates intrinsic resistance and inferior responses to tyrosine kinase inhibitors in cancer

Tyrosine kinase inhibitors (TKIs) elicit high response rates among individuals with kinase-driven malignancies, including chronic myeloid leukemia (CML) and epidermal growth factor receptor-mutated non-small-cell lung cancer (EGFR NSCLC). However, the extent and duration of these responses are heterogeneous, suggesting the existence of genetic modifiers affecting an individual's response to TKIs. Using paired-end DNA sequencing, we discovered a common intronic deletion polymorphism in the gene encoding BCL2-like 11 (BIM). BIM is a pro-apoptotic member of the B-cell CLL/lymphoma 2 (BCL2) family of proteins, and its upregulation is required for TKIs to induce apoptosis in kinase-driven cancers. The polymorphism switched BIM splicing from exon 4 to exon 3, which resulted in expression of BIM isoforms lacking the pro-apoptotic BCL2-homology domain 3 (BH3). The polymorphism was sufficient to confer intrinsic TKI resistance in CML and EGFR NSCLC cell lines, but this resistance could be overcome with BH3-mimetic drugs. Notably, individuals with CML and EGFR NSCLC harboring the polymorphism experienced significantly inferior responses to TKIs than did individuals without the polymorphism (P = 0.02 for CML and P = 0.027 for EGFR NSCLC). Our results offer an explanation for the heterogeneity of TKI responses across individuals and suggest the possibility of personalizing therapy with BH3 mimetics to overcome BIM-polymorphism-associated TKI resistance.     

Coupling endoplasmic reticulum stress to the cell-death program: a novel HSP90-independent role for the small chaperone protein p23

The endoplasmic reticulum (ER) is the principal organelle for the biosynthesis of proteins, steroids and many lipids, and is highly sensitive to alterations in its environment. Perturbation of Ca(2+) homeostasis, elevated secretory protein synthesis, deprivation of glucose or other sugars, altered glycosylation and/or the accumulation of misfolded proteins may all result in ER stress, and prolonged ER stress triggers cell death. Studies from multiple laboratories have identified the roles of several ER stress-induced cell-death modulators and effectors through the use of biochemical, pharmacological and genetic tools. In the present work, we describe the role of p23, a small chaperone protein, in preventing ER stress-induced cell death. p23 is a highly conserved chaperone protein that modulates HSP90 activity and is also a component of the steroid receptors. p23 is cleaved during ER stress-induced cell death; this cleavage, which occurs close to the carboxy-terminus, requires caspase-3 and/or caspase-7, but not caspase-8. Blockage of the caspase cleavage site of p23 was associated with decreased cell death induced by ER stress. Immunodepletion of p23 or inhibition of p23 expression by siRNA resulted in enhancement of ER stress-induced cell death. While p23 co-immunoprecipitated with the BH3-only protein PUMA (p53-upregulated modulator of apoptosis) in untreated cells, prolonged ER stress disrupted this interaction. The results define a protective role for p23, and provide further support for a model in which ER stress is coupled to the mitochondrial intrinsic apoptotic pathway through the activities of BH3 family proteins.     

CHOP is involved in endoplasmic reticulum stress-induced apoptosis by enhancing DR5 expression in human carcinoma cells

It has been shown that excess stress to the endoplasmic reticulum (ER) triggers apoptosis, but the mechanisms underlying these processes remain unclear. We and others have reported previously that DR5 expression is up-regulated in thapsigargin (THG)-treated human cancer cells. Here, we provide evidence that CHOP is involved in THG up-regulation of DR5, which is a critical step for ER stress-induced apoptosis in human cancer cells. In human colon cancer HCT116 cells, knockdown of DR5 by siRNA blocked THG-induced Bax conformational change along with caspase-3 activation and cell death. Moreover, inhibition of CHOP expression attenuated DR5 up-regulation and apoptosis induced by THG, whereas ectopic expression of DR5 restored the sensitivity of CHOP siRNA-transfected cells to THG-induced apoptosis. In addition to HCT116 cells, inhibition of CHOP or DR5 induction also attenuated THG-induced cell death in other cancer cell lines including LNCaP, A2780S, and DU145, indicating that CHOP and DR5 are critical for ER stress-mediated apoptosis in human carcinoma cells. Furthermore, we identified a potential CHOP-binding site in the 5'-flanking region of the DR5 gene. Mutation of this site abrogated the enhanced reporter activity in response to THG treatment. Together, our findings suggest that CHOP regulates ER stress-induced apoptosis, at least in part, through enhancing DR5 expression in some types of human cancer cells.     

FTY720 induces apoptosis of chronic myelogenous leukemia cells via dual activation of BIM and BID and overcomes various types of resistance to tyrosine kinase inhibitors

PP2A activator FTY720 has been shown to possess the anti-leukemic activity for chronic myelogenous leukemia (CML), however, the cell killing mechanism underlying its anti-leukemic activity has remained to be verified. We investigated the precise mechanisms underlying the apoptosis induction by FTY720, especially focusing on the roles of BH3-only proteins, and the therapeutic potency of FTY720 for CML. Enforced expression of either BCL2 or the dominant-negative protein of FADD (FADD.DN) partly protected CML cells from apoptosis by FTY720, indicating the involvement of both cell extrinsic and intrinsic apoptosis pathways. FTY720 activates pro-apoptotic BH3-only proteins: BIM, which is essential for apoptosis by BCR-ABL1 tyrosine kinase inhibitors (TKIs), and BID, which accelerates the extrinsic apoptosis pathway. Gene knockdown of either BIM or BID partly protected K562 cells from apoptosis by FTY720, but the extent of cell protection was not as much as that by overexpression of either BCL2 or FADD.DN. Moreover, knockdown of both BIM and BID did not provide additional protection compared with knockdown of only BIM or BID, indicating that BIM and BID complement each other in apoptosis by FTY720, especially when either is functionally impaired. FTY720 can overcome TKI resistance caused by ABL kinase domain mutations, dysfunction of BIM resulting from gene deletion polymorphism, and galectin-3 overexpression. In addition, ABT-263, a BH3-mimetic, significantly augmented cell death induction by FTY720 both in TKI-sensitive and -resistant leukemic cells. These results provide the rationale that FTY720, with its unique effects on BIM and BID, could lead to new therapeutic strategies for CML.     

NELL2 Function in the Protection of Cells against Endoplasmic Reticulum Stress

Continuous intra- and extracellular stresses induce disorder of Ca²⁺ homeostasis and accumulation of unfolded protein in the endoplasmic reticulum (ER), which results in ER stress. Severe long-term ER stress triggers apoptosis signaling pathways, resulting in cell death. Neural epidermal growth factor-like like protein 2 (NELL2) has been reported to be important in protection of cells from cell death-inducing environments. In this study, we investigated the cytoprotective effect of NELL2 in the context of ER stress induced by thapsigargin, a strong ER stress inducer, in Cos7 cells. Overexpression of NELL2 prevented ER stress-mediated apoptosis by decreasing expression of ER stress-induced C/EBP homologous protein (CHOP) and increasing ER chaperones. In this context, expression of anti-apoptotic Bcl-xL was increased by NELL2, whereas NELL2 decreased expression of pro-apoptotic proteins, such as cleaved caspases 3 and 7. This anti-apoptotic effect of NELL2 is likely mediated by extracellular signal-regulated kinase (ERK) signaling, because its inhibitor, U0126, inhibited effects of NELL2 on the expression of anti- and pro-apoptotic proteins and on the protection from ER stress-induced cell death.     

PUMA mediates ER stress-induced apoptosis in portal hypertensive gastropathy

Mucosal apoptosis has been demonstrated to be an essential pathological feature in portal hypertensive gastropathy (PHG). p53-upregulated modulator of apoptosis (PUMA) was identified as a BH3-only Bcl-2 family protein that has an essential role in apoptosis induced by a variety of stimuli, including endoplasmic reticulum (ER) stress. However, whether PUMA is involved in mucosal apoptosis in PHG remains unclear, and whether PUMA induces PHG by mediating ER stress remains unknown. The aim of the study is to investigate whether PUMA is involved in PHG by mediating ER stress apoptotic signaling. To identify whether PUMA is involved in PHG by mediating ER stress, gastric mucosal injury and apoptosis were studied in both PHG patients and PHG animal models using PUMA knockout (PUMA-KO) and PUMA wild-type (PUMA-WT) mice. The induction of PUMA expression and ER stress signaling were investigated, and the mechanisms of PUMA-mediated apoptosis were analyzed. GES-1 and SGC7901 cell lines were used to further identify whether PUMA-mediated apoptosis was induced by ER stress in vitro. Epithelial apoptosis and PUMA were markedly induced in the gastric mucosa of PHG patients and mouse PHG models. ER stress had a potent role in the induction of PUMA and apoptosis in PHG models, and the apoptosis was obviously attenuated in PUMA-KO mice. Although the targeted deletion of PUMA did not affect ER stress, mitochondrial apoptotic signaling was downregulated in mice. Meanwhile, PUMA knockdown significantly ameliorated ER stress-induced mitochondria-dependent apoptosis in vitro. These results indicate that PUMA mediates ER stress-induced mucosal epithelial apoptosis through the mitochondrial apoptotic pathway in PHG, and that PUMA is a potentially therapeutic target for PHG.     

Mechanisms of lysophosphatidylcholine-induced hepatocyte lipoapoptosis

Isolated hepatocytes undergo lipoapoptosis, a feature of hepatic lipotoxicity, on treatment with saturated free fatty acids (FFA) such as palmitate (PA). However, it is unknown if palmitate is directly toxic to hepatocytes or if its toxicity is indirect via the generation of lipid metabolites such as lysophosphatidylcholine (LPC). PA-mediated hepatocyte lipoapoptosis is associated with endoplasmic reticulum (ER) stress, c-Jun NH(2)-terminal kinase (JNK) activation, and a JNK-dependent upregulation of the potent proapoptotic BH3-only protein PUMA (p53 upregulated modulator of apoptosis). Our aim was to determine which of these mechanisms of lipotoxicity are activated by PA-derived LPC. We employed Huh-7 cells and isolated murine and human primary hepatocytes. Intracellular LPC concentrations increase linearly as a function of the exogenous, extracellular PA, stearate, or LPC concentration. Incubation of Huh-7 cells or primary hepatocytes with LPC induced cell death by apoptosis in a concentration-dependent manner. Substituting LPC for PA resulted in caspase-dependent cell death that was accompanied by activating phosphorylation of JNK with c-Jun phosphorylation and an increase in PUMA expression. LPC also induced ER stress as manifest by eIF2α phosphorylation and CAAT/enhancer binding homologous protein (CHOP) induction. LPC cytotoxicity was attenuated by pharmacological inhibition of JNK or glycogen synthase kinase-3 (GSK-3). Similarly, short-hairpin RNA (shRNA)-targeted knockdown of CHOP protected Huh-7 cells against LPC-induced toxicity. The LPC-induced PUMA upregulation was prevented by JNK inhibition or shRNA-targeted knockdown of CHOP. Finally, genetic deficiency of PUMA rendered murine hepatocytes resistant to LPC-induced apoptosis. We concluded that LPC-induced lipoapoptosis is dependent on mechanisms largely indistinguishable from PA. These data suggest that FFA-mediated cytotoxicity is indirect via the generation of the toxic metabolite, LPC.     

PUMA dependent mitophagy by Abrus agglutinin contributes to apoptosis through ceramide generation

PUMA, a BH3-only pro-apoptotic Bcl2 family protein, is known to translocate from the cytosol into the mitochondria in order to induce apoptosis. Interestingly, the induction of PUMA by p53 plays a critical role in DNA damage-induced apoptosis. In this study, we reported mitophagy inducing potential of PUMA triggered by phytolectin Abrus agglutinin (AGG) in U87MG glioblastoma cells and established AGG-induced ceramide acts as the chief mediator of mitophagy dependent cell death through activation of both mitochondrial ROS as well as ER stress. Importantly, AGG upregulates PUMA expression in U87MG cells with the generation of dysfunctional mitochondria, with gain and loss of function of PUMA is shown to alter mitophagy induction. At the molecular level, our study identified that the LC3 interacting region (LIR) located at the C-terminal end of PUMA interacts with LC3 in order to stimulate mitophagy. In addition, AGG is also found to trigger ubiquitination of PUMA which in turn interacted with p62 for prompting mitophagy suggesting that AGG turns on PUMA-mediated mitophagy in U87MG cells in both p62-dependent as well as in p62-independent manner. Interestingly, AGG-triggered ceramide production through activation of ceramide synthase–1 leads to induction of ER stress and ROS accumulation to promote mitochondrial damage as well as mitophagy. Further, upon pre-treatment with Mdivi–1, DRP1 inhibitor, AGG exposure results in suppression of apoptosis in U87MG cells indicating AGG-induced mitophagy switches to apoptosis that can be exploited for better cancer therapeutics.