Induction of the intrinsic apoptosis pathway in insulin-secreting cells is dependent on oxidative damage of mitochondria but independent of caspase-12 activation (original) (raw)
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Apoptosis and non-inflammatory phagocytosis can be induced by mitochondrial damage without caspases
Cell Death and Differentiation, 2010
A central issue regarding vertebrate apoptosis is whether caspase activity is essential, particularly for its crucial biological outcome, non-inflammatory clearance of the dying cell. Caspase-9 is required for the proteolytic cascade unleashed by the mitochondrial outer membrane permeabilization (MOMP) regulated by the Bcl-2 protein family. However, despite the severely blunted apoptosis in cells from Casp9 −/− mice, some organs with copious apoptosis, such as the thymus, appear unaffected. To address this paradox, we investigated how caspase-9 loss affects apoptosis and clearance of mouse fibroblasts and thymocytes. Although Casp9 −/− cells were initially refractory to apoptotic insults, they eventually succumbed to slower caspase-independent cell death. Furthermore, in γ-irradiated mice, the dying Casp9 −/− thymocytes were efficiently cleared, without apparent inflammation. Notably, MOMP proceeded normally, and the impaired mitochondrial function, revealed by diminished mitochondrial membrane potential (Δψ m ), committed cells to die, as judged by loss of clonogenicity. Upon the eventual full collapse of Δψ m , presumably reflecting failure of respiration, intact dying Casp9 −/− cells unexpectedly exposed the prototypic "eat-me" signal phosphatidylserine, which allowed their recognition and engulfment by phagocytes without overt inflammation. Hence, caspase-9-induced proteolysis accelerates apoptosis, but impaired mitochondrial integrity apparently triggers a default caspase-independent program of cell death and non-inflammatory clearance. Thus, caspases appear dispensable for some essential biological functions of apoptosis.
Diabetes, 2004
Insulin-producing cells are known for their extremely low antioxidant equipment with hydrogen peroxide (H2O2)-inactivating enzymes. Therefore, catalase was stably overexpressed in mitochondria and for comparison in the cytoplasmic compartment of insulin-producing RINm5F cells and analyzed for its protective effect against toxicity of reactive oxygen species (ROS) and proinflammatory cytokines. Only mitochondrial overexpression of catalase provided protection against menadione toxicity, a chemical agent that preferentially generates superoxide radicals intramitochondrially. On the other hand, the cytoplasmic catalase overexpression provided better protection against H2O2 toxicity. Mitochondrial catalase overexpression also preferentially protected against the toxicity of interleukin-1β (IL-1β) and a proinflammatory cytokine mixture (IL-1β, tumor necrosis factor-α [TNF-α], and γ-interferon [IFN-γ]) that is more toxic than IL-1β alone. Thus, it can be concluded that targeted overexpres...
Promotion of Caspase Activation by Caspase-9-mediated Feedback Amplification of Mitochondrial Damage
Journal of clinical & cellular immunology, 2012
Mitochondrial disruption during apoptosis results in the activation of caspase-9 and a downstream caspase cascade. Triggering this caspase cascade leads to the cleavage of anti-apoptotic Bcl-2 family proteins, resulting in feedback amplification of mitochondrial disruption. However, whether such a feedback loop plays an important role in the promotion of caspase activation and execution of apoptosis has not been well established. We observed that mutated Bcl-2 or Bcl-xL that are resistant to cleavage by caspases inhibited caspase-9-induced caspase activation in human H9 T cells. The release of Smac after the activation of caspase-9 was also inhibited by cleavage-resistant Bcl-2 or Bcl-xL. Consistently, caspase-9-deficient cells were defective in the release of Smac after induction of apoptosis. Moreover, addition of a Smac mimetic overcame the inhibitory effects of cleavage-resistant Bcl-2/Bcl-xL, and restored caspase-9-mediated cell death. Our data suggest that caspase-9-induced fe...
Cell Biology and Toxicology, 2012
In this study, we have compared several features of cell death triggered by classical inducers of apoptotic pathways (etoposide and tumour necrosis factor (TNF)-α) versus exogenous reactive oxygen species (ROS; hydrogen peroxide (H 2 O 2 ), tert-butyl hydroperoxide (t-BHP)) or a ROS generator (paraquat). Our aim was to characterize relationships that exist between ROS, mitochondrial perturbations, Bcl-2 and caspases, depending on source and identity of ROS. First, we have found that these five inducers trigger oxidative stress, mitochondrial membrane permeabilization (MMP), cytochrome c (cyt c) release from mitochondria and cell death. In each case, cell death could be inhibited by several antioxidants, showing that it is primarily ROS dependent. Second, we have highlighted that during etoposide or TNF-α treatments, intracellular ROS level, MMP and cell death are all regulated by caspases and Bcl-2, with caspases acting early in the process. Third, we have demonstrated that H 2 O 2 -induced cell death shares many of these characteristics with etoposide and TNF-α, whereas t-BHP induces both caspasedependent and caspase-independent cell death. Surprisingly, paraquat-induced cell death, which harbours some characteristics of apoptosis such as cyt c release and caspase-3 activation, is not modulated by Bcl-2 and caspase inhibitors, suggesting that paraquat also triggers non-apoptotic cell death signals. On the one hand, these results show that endogenous or exogenous ROS can trigger multiple cell death pathways with Bcl-2 and caspases acting differentially. On the other hand, they suggest that H 2 O 2 could be an important mediator of etoposide and TNF-α-dependent cell death since these inducers trigger similar phenotypes.
Mitochondrial effectors in caspase-independent cell death
FEBS Letters, 2003
Activation of caspases is recognized as a key element in the apoptotic process. However, new evidence is drawing attention to the emergent role of cell death pathways where caspases are not involved. Recent advances in the molecular understanding of these new ways to die, called caspase-independent, have revealed that mitochondria play an important role via the release of proapoptotic proteins. The purpose of this review is to integrate, from a biological and structural point of view, the most recent advances in the knowledge of the main mitochondrial proapoptotic proteins involved in this cell death cascade. The origin of programmed cell death is discussed through these strongly conserved e¡ectors.
2021
Basic research on types 1 and 2 diabetes mellitus require early stage studies using beta cells or cell lines, ideally of human origin and with preserved insulin secretion in response to glucose. The 1.1E7 cells are a hybrid cell line resulting from the electrofusion of dispersed human islets and PANC-1 cells, capable of secreting insulin in response to glucose, but their survival and function under toxic conditions remains untested. This characterization is the purpose of the present study. We treated these cells with a cytokine mix, high glucose, palmitate, and the latter two combined. Under these conditions, we measured cell viability and apoptosis (MTT, Caspase Glo and TUNEL assays, as well as caspase-8 and -9 levels by Western blotting), endoplasmic reticulum stress markers (EIF2AK3, HSPA4, EIF2a, and HSPA5) by real-time PCR, and insulin secretion with a glucose challenge. All of these stimuli (i) induce apoptosis and ER stress markers expression, (ii) reduce mRNA amounts of 2-5...
Mitochondrial Release of Caspase-2 and -9 during the Apoptotic Process
Journal of Experimental Medicine, 1999
The barrier function of mitochondrial membranes is perturbed early during the apoptotic process. Here we show that the mitochondria contain a caspase-like enzymatic activity cleaving the caspase substrate Z-VAD.afc, in addition to three biological activities previously suggested to participate in the apoptotic process: (a) cytochrome c; (b) an apoptosis-inducing factor (AIF) which causes isolated nuclei to undergo apoptosis in vitro; and (c) a DNAse activity. All of these factors, which are biochemically distinct, are released upon opening of the permeability transition (PT) pore in a coordinate, Bcl-2–inhibitable fashion. Caspase inhibitors fully neutralize the Z-VAD.afc–cleaving activity, have a limited effect on the AIF activity, and have no effect at all on the DNase activities. Purification of proteins reacting with the biotinylated caspase substrate Z-VAD, immunodetection, and immunodepletion experiments reveal the presence of procaspase-2 and -9 in mitochondria. Upon inductio...
Diabetologia, 2005
Aims/hypothesis: Free radicals generated in mitochondria play a crucial role in the toxic effects of cytokines upon insulin-producing cells. This study therefore investigated the role of manganese superoxide dismutase (MnSOD) in cytokine-mediated toxicity in insulin-producing cells. Methods: MnSOD was either stably overexpressed (MnSODsense) or stably suppressed (MnSODantisense) in insulin-producing RINm5F cells. Cell viability was quantified after incubation with different chemical reactive oxygen species (ROS) generators and with cytokines (IL-1β alone or a mixture of IL-1β, TNF-α and IFN-γ). Additionally, cell proliferation and endogenous MnSOD protein expression were determined after exposure to cytokines. Results: After incubation with hydrogen peroxide (H 2 O 2 ) or hypoxanthine/xanthine oxidase no significant differences were observed in viability between control and MnSOD sense or MnSODantisense clones. MnSOD overexpression reduced the viability of MnSODsense cells after exposure to the intracellular ROS generator menadione compared with control and MnSODantisense cells. MnSODsense cells also showed the highest susceptibility to cytokine toxicity with more than 75% loss of viability and a significant reduction of the proliferation rate after 72 h of incubation with a cytokine mixture. In comparison with control cells (67% viability loss), the reduction of viability in MnSODantisense cells was lower (50%), indicating a sensitising role of MnSOD in the progression of cytokine toxicity. The cell proliferation rate decreased in parallel to the reduction of cell viability. The MnSOD expression level after exposure to cytokines was also significantly lower in Mn SODantisense cells than in control or MnSODsense cells. Conclusions/interpretation: The increase of the mitochondrial imbalance between the superoxide-and the H 2 O 2inactivating enzyme activities corresponds with a greater susceptibility to cytokines. Thus optimal antioxidative strategies to protect insulin-producing cells against cytokine toxicity may comprise a combined overexpression of H 2 O 2inactivating enzymes or suppression of MnSOD activity.