The mitochondrial permeability transition regulates cytochrome c release for apoptosis during endoplasmic reticulum stress by remodeling the cristae junction

The role of the mitochondrial permeability transition (MPT) in apoptosis and necrosis is controversial. Here we show that the MPT regulates the release of cytochrome c for apoptosis during endoplasmic reticulum (ER) stress by remodeling the cristae junction (CJ). CEM cells, HCT116 colon cancer cells, and murine embryo fibroblast cells were treated with the ER stressor thapsigargin (THG), which led to cyclophilin D-dependent mitochondrial release of the profusion GTPase optic atrophy 1 (OPA1), which controls CJ integrity, and cytochrome c, leading to apoptosis. Interference RNA knockdown of Bax blocked OPA1 and cytochrome c release after THG treatment but did not prevent the MPT, showing that Bax was essential for the release of cytochrome c by MPT. In isolated mitochondria, MPT led to OPA1 and cytochrome c release independently of voltage-dependent anion channel and the outer membrane, indicating that the MPT is an inner membrane phenomenon. Last, the MPT was regulated by the electron transport chain but not mitochondrial reactive oxygen species, since THG-induced cell death was not blocked by antioxidants and did not occur in cells lacking mitochondrial DNA. Our results show that the MPT regulates CJ remodeling for cytochrome c-dependent apoptosis induced by ER stress and that mitochondrial electron transport is indispensable for this process.     

Mitochondrial rhomboid PARL regulates cytochrome c release during apoptosis via OPA1-dependent cristae remodeling

Rhomboids, evolutionarily conserved integral membrane proteases, participate in crucial signaling pathways. Presenilin-associated rhomboid-like (PARL) is an inner mitochondrial membrane rhomboid of unknown function, whose yeast ortholog is involved in mitochondrial fusion. Parl-/- mice display normal intrauterine development but from the fourth postnatal week undergo progressive multisystemic atrophy leading to cachectic death. Atrophy is sustained by increased apoptosis, both in and ex vivo. Parl-/- cells display normal mitochondrial morphology and function but are no longer protected against intrinsic apoptotic death stimuli by the dynamin-related mitochondrial protein OPA1. Parl-/- mitochondria display reduced levels of a soluble, intermembrane space (IMS) form of OPA1, and OPA1 specifically targeted to IMS complements Parl-/- cells, substantiating the importance of PARL in OPA1 processing. Parl-/- mitochondria undergo faster apoptotic cristae remodeling and cytochrome c release. These findings implicate regulated intramembrane proteolysis in controlling apoptosis.     

Aspirin induces apoptosis through mitochondrial cytochrome c release

Aspirin and other non-steroidal anti-inflammatory drugs induce apoptosis in many cell types. Although the involvement of caspases has been demonstrated, the mechanism leading to caspase activation remains unknown. We have studied the role of the mitochondrial pathway in aspirin-induced apoptosis. The apoptotic effect of aspirin was analyzed in different cell lines (Jurkat, MOLT-4, Raji and HL-60) showing induction of mitochondrial cytochrome c release and caspases 9, 3 and 8 processing. Furthermore, early aspirin-induced cytochrome c release was not affected by the caspase inhibitor Z-VAD·fmk and preceded loss of mitochondrial membrane potential. Therefore, aspirin-induced apoptosis involves caspase activation through cytochrome c release.     

Nitrosylation of cytochrome c during apoptosis

Cytochrome c released from mitochondria into the cytoplasm plays a critical role in many forms of apoptosis by stimulating apoptosome formation and subsequent caspase activation. However, the mechanisms regulating cytochrome c apoptotic activity are not understood. Here we demonstrate that cytochrome c is nitrosylated on its heme iron during apoptosis. Nitrosylated cytochrome c is found predominantly in the cytoplasm in control cells. In contrast, when cytochrome c release from mitochondria is inhibited by overexpression of the anti-apoptotic proteins B cell lymphoma/leukemia (Bcl)-2 or Bcl-X(L), nitrosylated cytochrome c is found in the mitochondria. These data suggest that during apoptosis, cytochrome c is nitrosylated in mitochondria and then rapidly released into the cytoplasm in the absence of Bcl-2 or Bcl-X(L) overexpression. In vitro nitrosylation of cytochrome c increases caspase-3 activation in cell lysates. Moreover, the inhibition of intracellular cytochrome c nitrosylation is associated with a decrease in apoptosis, suggesting that cytochrome c nitrosylation is a proapoptotic modification. We conclude that nitrosylation of the heme iron of cytochrome c may be a novel mechanism of apoptosis regulation.     

Nuclear translocation of cytochrome c during apoptosis

Release of cytochrome c from mitochondria is a major event during apoptosis. Released cytochrome c has been shown to activate caspase-dependent apoptotic signals. In this report, we provide evidence for a novel role of cytochrome c in caspase-independent nuclear apoptosis. We showed that cytochrome c, released from mitochondria upon apoptosis induction, gradually accumulates in the nucleus as evidenced by both immunofluorescence and subcellular fractionation. Parallel to nuclear accumulation of cytochrome c, acetylated histone H2A, but not unmodified H2A, was released from the nucleus to the cytoplasm. Addition of purified cytochrome c to isolated nuclei recapitulated the preferential release of acetylated, but not deacetylated, histone H2A. Cytochrome c was also found to induce chromatin condensation. These results suggest that the nuclear accumulation of cytochrome c may be directly involved in the remodeling of chromatin. Our results provide evidence of a novel role for cytochrome c in inducing nuclear apoptosis.     

Lysosomal enzymes promote mitochondrial oxidant production, cytochrome c release and apoptosis.

Exposure of mammalian cells to oxidant stress causes early (iron catalysed) lysosomal rupture followed by apoptosis or necrosis. Enhanced intracellular production of reactive oxygen species (ROS), presumably of mitochondrial origin, is also observed when cells are exposed to nonoxidant pro-apoptotic agonists of cell death. We hypothesized that ROS generation in this latter case might promote the apoptotic cascade and could arise from effects of released lysosomal materials on mitochondria. Indeed, in intact cells (J774 macrophages, HeLa cells and AG1518 fibroblasts) the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH) causes lysosomal rupture, enhanced intracellular ROS production, and apoptosis. Furthermore, in mixtures of rat liver lysosomes and mitochondria, selective rupture of lysosomes by MSDH promotes mitochondrial ROS production and cytochrome c release, whereas MSDH has no direct effect on ROS generation by purifed mitochondria. Intracellular lysosomal rupture is associated with the release of (among other constituents) cathepsins and activation of phospholipase A2 (PLA2). We find that addition of purified cathepsins B or D, or of PLA2, causes substantial increases in ROS generation by purified mitochondria. Furthermore, PLA2 - but not cathepsins B or D - causes rupture of semipurified lysosomes, suggesting an amplification mechanism. Thus, initiation of the apoptotic cascade by nonoxidant agonists may involve early release of lysosomal constituents (such as cathepsins B and D) and activation of PLA2, leading to enhanced mitochondrial oxidant production, further lysosomal rupture and, finally, mitochondrial cytochrome c release. Nonoxidant agonists of apoptosis may, thus, act through oxidant mechanisms.     

Ultrastructural localization of cytochrome c in apoptosis demonstrates mitochondrial heterogeneity

Release of apoptogenic factors into the cytosol including cytochrome c is triggering the execution phase of apoptosis through activation of cytoplasmic effector caspases. How loss of function of the electron transport chain can be reconciled with an adequate energy supply necessary for executing the apoptotic program was studied in granulosa cell (GC) sheets cultured up to 72 h without gonadotrophic support. Cytochrome c was localized ultrastructurally by oxidation of diaminobenzidine tetrahydrochloride both in living and fixed cells. In uncultured GC sheets all cells show staining over their entire mitochondrial population. In 72 h cultured sheets in the absence of FSH pre-apoptotic GC's display two subsets of mitochondria: normal sized stained mitochondria and small orthodox mitochondria without demonstrable cytochrome function. Apoptotic cells contain several mitochondria with preservation of respiratory function besides unstained orthodox mitochondria. The cytochrome c containing mitochondria typically display dilated intracristal spaces, a mitochondrial conformation related to increased ATP production. Cytochrome c release was confirmed by Western blotting. In 72 h cultures supplemented with FSH, GC's displayed staining over their entire mitochondrial population. In cultures lacking FSH, but partially protected from apoptosis through caspase inhibition, the cytochrome c release was not inhibited. Thus in the present studied model dysfunction of only a subset of mitochondria is instrumental to initiate the apoptotic program while a functional electron transport chain is maintained until the degradation phase in a subset of respiring mitochondria.     

Bcr-Abl-mediated protection from apoptosis downstream of mitochondrial cytochrome c release

Bcr-Abl, activated in chronic myelogenous leukemias, is a potent cell death inhibitor. Previous reports have shown that Bcr-Abl prevents apoptosis through inhibition of mitochondrial cytochrome c release. We report here that Bcr-Abl also inhibits caspase activation after the release of cytochrome c. Bcr-Abl inhibited caspase activation by cytochrome c added to cell-free lysates and prevented apoptosis when cytochrome c was microinjected into intact cells. Bcr-Abl acted posttranslationally to prevent the cytochrome c-induced binding of Apaf-1 to procaspase 9. Although Bcr-Abl prevented interaction of endogenous Apaf-1 with the recombinant prodomain of caspase 9, it did not affect the association of endogenous caspase 9 with the isolated Apaf-1 caspase recruitment domain (CARD) or Apaf-1 lacking WD-40 repeats. These data suggest that Apaf-1 recruitment of caspase 9 is faulty in the presence of Bcr-Abl and that cytochrome c/dATP-induced exposure of the Apaf-1 CARD is likely defective. These data provide a novel locus of Bcr-Abl antiapoptotic action and suggest a distinct mechanism of apoptosomal inhibition.     

Endoplasmic reticulum BIK initiates DRP1-regulated remodelling of mitochondrial cristae during apoptosis

The endoplasmic reticulum (ER) can elicit proapoptotic signalling that results in transmission of Ca(2+) to the mitochondria, which in turn stimulates recruitment of the fission enzyme DRP1 to the surface of the organelle. Here, we show that BH3-only BIK activates this pathway at the ER in intact cells, resulting in mitochondrial fragmentation but little release of cytochrome c to the cytosol. The BIK-induced transformations in mitochondria are dynamic in nature and involve DRP1-dependent remodelling and opening of cristae, where the major stores of cytochrome c reside. This novel function for DRP1 is distinct from its recognized role in regulating mitochondrial fission. Selective permeabilization of the outer membrane with digitonin confirmed that BIK stimulation results in mobilization of intramitochondrial cytochrome c. Of note, BIK can cooperate with a weak BH3-only protein that targets mitochondria, such as NOXA, to activate BAX by a mechanism that is independent of DRP1 enzyme activity. When expressed together, BIK and NOXA cause rapid release of mobilized cytochrome c and activation of caspases.     

Nitric oxide inhibits execution of apoptosis at two distinct ATP-dependent steps upstream and downstream of mitochondrial cytochrome c release

The endogenous mediator nitric oxide (NO) blocked apoptosis of Jurkat cells elicited by staurosporine, anti-CD95 or chemotherapeutics, and switched death to necrosis. The switch in the mode of cell death was dependent on the ATP loss elicited by NO. This affected two distinct steps of the apoptotic cascade. First, the release of cytochrome c from mitochondria was delayed by NO. Second, processing of procaspases-3/7 to the active proteases was prevented even after cytochrome c had been released. Thus, NO interferes with execution steps of apoptosis both upstream and downstream of cytochrome c release.     

Increased mitochondrial cytochrome c levels and mitochondrial hyperpolarization precede camptothecin-induced apoptosis in Jurkat cells

Mitochondria play a central role in apoptosis through release of cytochrome c and activation of caspases. In the present study, we showed that, in Jurkat human T cells, camptothecin-induced apoptosis is preceded by (i) an increase in cytochrome c and subunit IV of cytochrome c oxidase (COX IV) levels in mitochondria; and (ii) an elevation of the mitochondrial membrane potential (Delta(Psi)m). These events are followed by cytochrome c release into the cytosol, cytochrome c and COX IV depletion from mitochondria, externalization of phosphatidylserine (PS), disruption of Delta(Psi)m, caspase activation, poly(ADP-ribose)polymerase cleavage and DNA fragmentation. The pan-caspase inhibitor z-VAD.fmk blocked camptothecin-induced PS externalization, disruption of Delta(Psi)m and DNA fragmentation, suggesting that these events are mediated by caspase activation. In contrast, z-VAD did not prevent cytochrome c release, despite preventing cytochrome c and COX IV depletion from mitochondria. Together, these data suggest that mitochondrial cytochrome c and COX IV enrichment are early events preceding the onset of apoptosis and that cytochrome c release is upstream of caspase activation and loss of Delta(Psi)m. Furthermore, prevention by z-VAD of cytochrome c and COX IV depletion in mitochondria suggests the possibility that a caspase-like activity in mitochondria is involved in the proteolytic depletion of respiratory chain proteins. Activation of this activity may play an important role in drug-induced apoptosis.     

Phosphatidyl serine exposure during apoptosis precedes release of cytochrome c and decrease in mitochondrial transmembrane potential

Time kinetics of phosphatidyl serine (PS) exposure were compared to other apoptotic parameters following different apoptotic stimuli. Our data indicate that anti-Fas treatment of L929sAhFas cells results in rapid exposure of PS, which precedes decrease in mitochondrial transmembrane potential (DeltaPsi(m)) and release of cytochrome c, indicating that PS exposure occurs independently of these mitochondrial events. Also during TNF-, etoposide- or staurosporine-mediated apoptosis in PC60 RI/RII cells, PS-positive cells were observed before they had a decreased DeltaPsi(m). However, during growth factor depletion-induced death of 32D cells, both phenomena seemed to occur at the same time.     

Inhibiting Drp1-mediated mitochondrial fission selectively prevents the release of cytochrome c during apoptosis

Most cell death stimuli trigger the mitochondrial release of cytochrome c and other cofactors that induce caspase activation and ensuing apoptosis. Apoptosis is also associated with massive mitochondrial fragmentation and cristae remodeling. Dynamin-related protein 1 (Drp1), a protein of the mitochondrial fission machinery, has been reported to participate in apoptotic mitochondrial fragmentation. Several theories explaining the mechanisms of cytochrome c release have been proposed. One suggests that it relies on the activation of Drp1-mediated mitochondrial fission. Here, we report that downregulation of Drp1 inhibits fragmentation of the mitochondrial network and partially prevents the release of cytochrome c but fails to prevent the release of other mitochondrial factors such as second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI, Omi/HtrA2, adenylate kinase 2 and deafness dystonia peptide/TIMM8a. An explanation for the prevention of cytochrome c release is provided by our observation that inhibiting Drp1-mediated mitochondrial fission prevents the mitochondrial release of soluble OPA1 that was proposed to regulate cristae remodeling and complete cytochrome c release during apoptosis. Finally, we observed that downregulation of Drp1 delays but does not inhibit apoptosis, suggesting that mitochondrial fragmentation is not a prerequisite for apoptosis.     

Bid-cardiolipin interaction at mitochondrial contact site contributes to mitochondrial cristae reorganization and cytochrome C release

Release of cytochrome c from the mitochondrial intermembrane space is critical to apoptosis induced by a variety of death stimuli. Bid is a BH3-only prodeath Bcl-2 family protein that can potently activate this efflux. In the current study, we investigated the mitochondrial localization of Bid and its interactions with mitochondrial phospholipids, focusing on their relationships with Bid-induced cytochrome c release. We found that Bid binding to the mitochondria required only three of its eight helical structures (alpha4-alpha6), but not the BH3 domain, and the binding could not be inhibited by the antideath molecule Bcl-x(L). Membrane fractionations indicated that tBid bound to mitochondrial outer membranes at both contact and noncontact sites. Bid could interact with specific cardiolipin species on intact mitochondria as identified by mass spectrometry. Like the binding to the mitochondria, this interaction could not be blocked by the mutation in the BH3 domain or by Bcl-x(L.) However, a cardiolipin-specific dye, 10-N-nonyl acridine orange, could preferentially suppress Bid binding to the mitochondrial contact site and inhibit Bid-induced mitochondrial cristae reorganization and cytochrome c release. These findings thus suggest that interactions of Bid with mitochondrial cardiolipin at the contact site can contribute significantly to its functions.     

Bax and the mitochondrial permeability transition cooperate in the release of cytochrome c during endoplasmic reticulum-stress-induced apoptosis

Endoplasmic reticulum (ER) stress induces apoptosis by mechanisms that are not fully clear. Here we show that ER stress induced by the Ca(2+)-ATPase inhibitor thapsigargin (THG) activates cytochrome c-dependent apoptosis through cooperation between Bax and the mitochondrial permeability transition (MPT) in human leukemic CEM cells. Pharmacological inhibition of the MPT as well as small interfering RNA (siRNA) knockdown of the MPT core component cyclophilin D blocked cytochrome c release and caspase-dependent apoptosis but did not prevent Bax activation, translocation or N-terminal exposure in mitochondria. siRNA knockdown of Bax also blocked THG-mediated cytochrome c release and apoptosis, but did not prevent MPT activation and resulted in caspase-independent cell death. Our results show that ER-stress-induced cell death involves a caspase and Bax-dependent pathway as well as a caspase-independent MPT-directed pathway.     

ATP binding to cytochrome c diminishes electron flow in the mitochondrial respiratory pathway.

Eukaryotic cytochrome c possesses an ATP-binding site of substantial specificity and high affinity that is conserved between highly divergent species and which includes the invariant residue arginine91. Such evolutionary conservatism strongly suggests a physiological role for ATP binding that demands further investigation. We report the preparation of adducts of the protein and the affinity labels 8-azido adenosine 5'-triphosphate, adenosine 5'-triphosphate-2',3'-dialdehyde, and 5'-p-fluorosulfonylbenzoyladenosine. The two former reagents were seen to react at the arginine91-containing site, yet the reaction of the latter, although specific, occurred elsewhere, suggesting caution is necessary in its use. None of the adducts displayed significant modification of global structure, stability, or physicochemical properties, leading us to believe that the 8-N3-ATP and oATP adducts are good stabilized models of the noncovalent interaction; yet modification led to significant, and sometimes pronounced, effects on biological activity. We therefore propose that the role of ATP binding to this site, which we have shown to occur when the phosphorylation potential of the system is high under the equivalent of physiological conditions, is to cause a decrease in electron flow through the mitochondrial electron transport chain. Differences in the degree of inhibition produced by differences in adduct chemistry suggest that this putative regulatory role is mediated primarily by electrostatic effects.     

A distinct pathway of cell-mediated apoptosis initiated by granulysin.

Granulysin is an antimicrobial and tumoricidal molecule expressed in granules of CTL and NK cells. In this study, we show that granulysin damages cell membranes based upon negative charge, disrupts the transmembrane potential (Deltapsi) in mitochondria, and causes release of cytochrome c. Granulysin-induced apoptosis is blocked in cells overexpressing Bcl-2. Despite the release of cytochrome c, procaspase 9 is not processed. Nevertheless, activation of caspase 3 is observed in granulysin-treated cells, suggesting that granulysin activates a novel pathway of CTL- and NK cell-mediated death distinct from granzyme- and death receptor-induced apoptosis.     

Gelsolin inhibits apoptosis by blocking mitochondrial membrane potential loss and cytochrome c release

Apoptotic cell death, characterized by chromatin condensation, nuclear fragmentation, cell membrane blebbing, and apoptotic body formation, is also accompanied by typical mitochondrial changes. The latter includes enhanced membrane permeability, fall in mitochondrial membrane potential (Deltapsi(m)) and release of cytochrome c into the cytosol. Gelsolin, an actin regulatory protein, has been shown to inhibit apoptosis, but when cleaved by caspase-3, a fragment that is implicated as an effector of apoptosis is generated. The mechanism by which the full-length form of gelsolin inhibits apoptosis is unclear. Here we show that the overexpression of gelsolin inhibits the loss of Deltapsi(m) and cytochrome c release from mitochondria resulting in the lack of activation of caspase-3, -8, and -9 in Jurkat cells treated with staurosporine, thapsigargin, and protoporphyrin IX. These effects were corroborated in vitro using recombinant gelsolin protein on isolated rat mitochondria stimulated with Ca(2+), atractyloside, or Bax. This protective function of gelsolin, which was not due to simple Ca(2+) sequestration, was inhibited by polyphosphoinositide binding. In addition we confirmed that gelsolin, besides its localization in the cytosol, is also present in the mitochondrial fraction of cells. Gelsolin thus acts on an early step in the apoptotic signaling at the level of mitochondria.