p38 mitogen-activated protein kinase regulates a novel, caspase-independent pathway for the mitochondrial cytochrome c release in ultraviolet B radiation-induced apoptosis

The mechanisms of UVB-induced apoptosis and the role of p38 mitogen-activated protein kinase (MAPK) were investigated in HaCaT cells. UVB doses that induced apoptosis also produced a sustained activation of p38 MAPK and mitochondrial cytochrome c release, leading to pro-caspase-3 activation. Late into the apoptotic process, UVB also induced a caspase-mediated cleavage of Bid. Caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone substantially blocked the UVB-induced apoptosis without preventing the release of mitochondrial cytochrome c and the p38 MAPK activation. The inhibition of p38 MAPK counteracted both apoptosis and cytochrome c release as well as the DEVD-amino-4-methylcoumarin cleavage activity without affecting the processing of pro-caspase-8. These results indicate that UVB induces multiple and independent apoptotic pathways, which culminate in pro-caspase-3 activation, and that the initial cytochrome c release is independent of caspase activity. Importantly, we show that a sustained p38 MAPK activation contributes to the UVB-induced apoptosis by mediating the release of mitochondrial cytochrome c into the cytosol.     

Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition

We report here the identification of a novel protein, Smac, which promotes caspase activation in the cytochrome c/Apaf-1/caspase-9 pathway. Smac promotes caspase-9 activation by binding to inhibitor of apoptosis proteins, IAPs, and removing their inhibitory activity. Smac is normally a mitochondrial protein but is released into the cytosol when cells undergo apoptosis. Mitochondrial import and cleavage of its signal peptide are required for Smac to gain its apoptotic activity. Overexpression of Smac increases cells' sensitivity to apoptotic stimuli. Smac is the second mitochondrial protein, along with cytochrome c, that promotes apoptosis by activating caspases.     

Mitochondria from TRAIL-resistant prostate cancer cells are capable of responding to apoptotic stimuli

TNFalpha-related apoptosis inducing ligand (TRAIL) has been shown to induce apoptosis in prostate cancer cells. However, some prostate cancer cells, such as LNCaP are resistant to TRAIL. In addition to the involvement of several pathways in the TRAIL-resistance of LNCaP, it has been shown that mitochondrial response to TRIAL is low in these cells. Therefore, in this study, using in vitro cell free and reconstitution models, we have demonstrated that mitochondria from these cells are capable of responding to apoptotic stimuli. Furthermore, experiments to determine the influence of cytochrome c on apoptotic response noted that incubation of cytosol with exogenous cytochrome c induced truncation of Bid. We have demonstrated that truncation of Bid by exogenous cytochrome c is mediated through the activation of caspases-9 and -3. Incubation of cytosol with recombinant caspases-9 and -3 in the absence or presence of inhibitors showed that activation of caspase-9, leading to the activation of caspase-3 was necessary for the truncation of Bid. Published results indicate that in apoptotic cells cytochrome c is released from the mitochondria in two installments, an early small amount and a late larger amount. Our results suggest that the initial release of cytochrome generates tBid that is capable of translocation into the mitochondria causing further release of cytochrome c. Thus, in addition to providing functional explanation for the biphasic release of cytochrome c from mitochondria, we demonstrate the presence of a feedback amplification of mitochondrial apoptotic signal.     

Mitochondrial cytochrome c release and caspase-3-like protease activation during indomethacin-induced apoptosis in rat gastric mucosal cells

Indomethacin (IND), a nonsteroidal anti-inflammatory drug, has been known to cause gastric mucosal injury as a side effect. Using a rat gastric mucosal cell line, RGM1, we determined whether apoptosis is involved in IND-mediated gastropathy, and whether caspase activation and mitochondrial cytochrome c release play an important role in producing apoptosis of IND-treated RGM1 cells in the presence of serum. IND caused caspase-3-like protease activation followed by apoptosis in a dose- and time-dependent manner. Caspase-1-like protease activity did not change during IND-induced apoptosis. IND also increased mitochondrial cytochrome c release in a time-dependent fashion. Mitochondrial cytochrome c efflux occurred just before or at the same time as caspase-3-like protease activation, and preceded the increase in apoptotic cell numbers. Z-VAD-FMK, a caspase inhibitor, inhibited both the increase in caspase-3-like protease activity and apoptosis in IND-treated RGM1 cells but did not affect caspase-1-like protease activity or mitochondrial cytochrome c release. These observations suggest that the apoptosis of gastric mucosal cells could be involved in IND-induced gastropathy, that cytochrome c is released from mitochondria into the cytosol during the early phase of IND-mediated apoptosis, and that subsequent activation of caspase-3-like protease, but not caspase-1-like protease, is required for the execution of apoptosis.     

Cytochrome c acts as a cardiolipin oxygenase required for release of proapoptotic factors

Programmed death (apoptosis) is turned on in damaged or unwanted cells to secure their clean and safe self-elimination. The initial apoptotic events are coordinated in mitochondria, whereby several proapoptotic factors, including cytochrome c, are released into the cytosol to trigger caspase cascades. The release mechanisms include interactions of B-cell/lymphoma 2 family proteins with a mitochondria-specific phospholipid, cardiolipin, to cause permeabilization of the outer mitochondrial membrane. Using oxidative lipidomics, we showed that cardiolipin is the only phospholipid in mitochondria that undergoes early oxidation during apoptosis. The oxidation is catalyzed by a cardiolipin-specific peroxidase activity of cardiolipin-bound cytochrome c. In a previously undescribed step in apoptosis, we showed that oxidized cardiolipin is required for the release of proapoptotic factors. These results provide insight into the role of reactive oxygen species in triggering the cell-death pathway and describe an early role for cytochrome c before caspase activation.     

Apoptosis induces efflux of the mitochondrial matrix enzyme deoxyguanosine kinase

Deoxyguanosine kinase (dGK) initiates the salvage of purine deoxynucleosides in mitochondria and is a key enzyme in mitochondrial DNA precursor synthesis. The active form of the enzyme is a 60-kDa protein normally located in the mitochondrial matrix. Here we describe the subcellular distribution of dGK during apoptosis in human epithelial kidney 293 cells and human lymphoblast Molt-4 cells. Immunological methods were used to monitor dGK as well as other mitochondrial proteins. Surprisingly, dGK was found to relocate to the cytosolic compartment at a similar rate as cytochrome c, a mitochondrial intermembraneous enzyme known to enter the cytosol early in apoptosis. The redistribution of dGK from the mitochondria to the cytosol may be of importance for the activation of apoptotic purine nucleoside cofactors such as dATP and demonstrates that mitochondrial matrix proteins may selectively leak out during apoptosis.     

Mitochondrially localized active caspase-9 and caspase-3 result mostly from translocation from the cytosol and partly from caspase-mediated activation in the organelle. Lack of evidence for Apaf-1-mediated procaspase-9 activation in the mitochondria

Active caspase-9 and caspase-3 have been observed in the mitochondria, but their origins are unclear. Theoretically, procaspase-9 might be activated in the mitochondria in a cytochrome c/Apaf-1-dependent manner, or activated caspase-9 and -3 may translocate to the mitochondria, or the mitochondrially localized procaspases may be activated by the translocated active caspases. Here we present evidence that the mitochondrially localized active caspase-9 and -3 result mostly from translocation from the cytosol (into the intermembrane space) and partly from caspase-mediated activation in the organelle rather than from the Apaf-1-mediated activation. Apaf-1 localizes exclusively in the cytosol and, upon apoptotic stimulation, translocates to the perinuclear area but not to the mitochondria. In most cases, the mitochondrially localized procaspase-9 and -3 are released early during apoptosis and translocate to the cytosol and/or perinuclear area. Cytochrome c and the mitochondrial matrix protein Hsp60 are also rapidly released to the cytosol early during apoptosis. Both the early release of proteins like cytochrome c and Hsp60 from the mitochondria as well as the later translocation of the active caspase-9/-3 are partially inhibited by cyclosporin A, an inhibitor of mitochondrial membrane permeabilization. The mitochondrial active caspases may function as a positive feedback mechanism to further activate other or residual mitochondrial procaspases, degrade mitochondrial constituents, and disintegrate mitochondrial functions.     

腺苷酸激酶与细胞凋亡

腺苷酸激酶(AK)在维持细胞能量平衡中起着重要作用,新近又发现它与细胞凋亡有着密切的关系.在凋亡过程中,AK2从线粒体膜间释放到胞浆是一种非常普遍的现象,但其在细胞凋亡中作用仍很不清楚.综述了近年对腺苷酸激酶的研究进展,探讨了腺苷酸激酶在细胞凋亡程序中所扮演的角色.     

Laser micro-irradiation of mitochondria: is there an amplified mitochondrial death signal in neural cells?

Several mitochondrial proteins, such as cytochrome c, are directly involved in the pathway for caspase activation following induction of apoptosis. Release of mitochondrial cytochrome c early in apoptosis is rapid and almost complete. Microinjection of cytochrome c into resting cells induces apoptosis, but the amount needed approaches the total cellular content. These observations suggest that mitochondrial protein release is an all-or-nothing process inside the cell and not an amplifiable apoptotic signal. To test this hypothesis, laser micro-irradiation was used to rupture membranes of individual mitochondria within living rat neural cells. Laser micro-irradiation caused swelling, fragmentation, depolarization, and cytochrome c depletion in targeted mitochondria. These effects were explained by correlative electron microscopic analysis showing local rupture of outer and inner membranes at the site of irradiation. In all cases, there were no detectable changes in the structure, membrane potential, or cytochrome c content of neighboring, non-irradiated organelles. Furthermore, irradiation of up to 15% of the mitochondria in a cell did not induce apoptosis. The results from these laser micro-irradiation experiments prove that local release of mitochondrial proteins does not constitute an amplifiable apoptotic signal in resting neural cells.     

Modulation of mitochondrial apoptosis by PI3K inhibitors

Most anticancer therapies exert their action by triggering programmed cell death (apoptosis) in cancer cells. The mitochondrial pathway of apoptosis is initiated by mitochondrial outer membrane permeabilization, leading to the release of apoptogenic factors such as cytochrome c or Smac from the mitochondrial intermembrane space into the cytosol. Mitochondrial outer membrane permeabilization is tightly controlled, for example by pro- and anti-apoptotic proteins of the Bcl-2 family. Recent evidence indicates that inhibition of the PI3K/Akt/mTOR pathway by small-molecule PI3K inhibitors primes cancer cells to mitochondrial apoptosis by tipping the balance towards pro-apoptotic Bcl-2 proteins, resulting in increased mitochondrial outer membrane permeabilization. Thus, mitochondrial apoptotic events play an important role in PI3K inhibitor-mediated sensitization for apoptosis.     

Heat stress prevents mitochondrial injury in ATP-depleted renal epithelial cells

The events that precipitate cell death and the stress proteins responsible for cytoprotection during ATP depletion remain elusive. We hypothesize that exposure to metabolic inhibitors damages mitochondria, allowing proapoptotic proteins to leak into the cytosol, and suggest that heat stress-induced hsp72 accumulation prevents mitochondrial membrane injury. To test these hypotheses, renal epithelial cells were transiently ATP depleted with sodium cyanide and 2-deoxy-D-glucose in the absence of medium dextrose. Recovery from ATP depletion was associated with the release into the cytosol of cytochrome c and apoptosis-inducing factor (AIF), proapoptotic proteins that localize to the intermitochondrial membrane space. Concomitant with mitochondrial cytochrome c leak, a seven- to eightfold increase in caspase 3 activity was observed. In controls, state III mitochondrial respiration was reduced by 30% after transient exposure to metabolic inhibitors. Prior heat stress preserved mitochondrial ATP production and significantly reduced both cytochrome c release and caspase 3 activation. Despite less cytochrome c release, prior heat stress increased binding between cytochrome c and hsp72. The present study demonstrates that mitochondrial injury accompanies exposure to metabolic inhibitors. By reducing outer mitochondrial membrane injury and by complexing with cytochrome c, hsp72 could inhibit caspase activation and subsequent apoptosis.     

c-Myc induces cytochrome c release in Rat1 fibroblasts by increasing outer mitochondrial membrane permeability in a Bid-dependent manner

Ectopic expression of c-myc sensitises cells to a wide range of apoptotic stimuli by inducing the release of cytochrome c from the mitochondrial intermembrane space into the cytosol. To elucidate the molecular mechanisms of mitochondrial permeabilisation in response to c-Myc activation, we carried out a biochemical fractionation analysis of Rat1 fibroblasts expressing an inducible c-Myc protein. We find that cytoplasmic extracts from cells in which c-Myc has been activated contain a soluble factor capable of inducing cytochrome c release from isolated mouse liver mitochondria. This factor is present only under growth factor deprivation conditions and its activity is inhibited by addition of Bcl-X(L). The c-Myc-induced factor copurifies with full-length Bid, a "BH3-only" proapoptotic member of the Bcl-2 family, and antibodies raised against the BH3 domain of Bid inhibit c-Myc-induced cytochrome c releasing activity. These results are consistent with a model in which the activation of c-Myc regulates factors capable of enhancing the mitochondrial membrane destabilisation function of "BH3-only" proteins.     

A novel ubiquitin fusion system bypasses the mitochondria and generates biologically active Smac/DIABLO

Smac/DIABLO is a mitochondrial protein that is proteolytically processed and released during apoptosis along with cytochrome c and other proapoptotic factors. Once in the cytosol, Smac protein binds to inhibitors of apoptosis (IAP) proteins and disrupts the ability of the IAPs to inhibit caspases 3, 7, and 9. The requirement for mitochondrial processing and release has complicated efforts to delineate the effect of Smac overexpression and IAP inhibition on cell death processes. In this report, we document a novel expression system using ubiquitin fusions to express mature, biologically active Smac in the cytosol of transfected cells. Processing of the ubiquitin-Smac fusions is rapid and complete and generates mature Smac protein initiating correctly with the Ala-Val-Pro-Ile tetrapeptide sequence that is required for proper function. The biological activity of this exogenous protein was demonstrated by its interaction with X-linked IAP, one of the most potent of the IAPs. The presence of mature Smac was not sufficient to trigger apoptosis of healthy cells. However, cells with excess Smac protein were greatly sensitized to apoptotic triggers such as etoposide exposure. Cancer cells typically display deregulated apoptotic pathways, including Bcl2 overexpression, thereby suppressing the release of cytochrome c and Smac. The ability to circumvent the requirement for mitochondrial processing and release is critical to developing Smac as a possible gene therapy payload in cancer chemosensitization.     

Influence of cytochrome c on apoptosis induced by Anagrapha (Syngrapha) falcifera multiple nuclear polyhedrosis virus (AfMNPV) in insect Spodoptera litura cells

We investigated the influence of cytochrome c on apoptosis induced by Anagrapha (Syngrapha) falcifera multiple nuclear polyhedrosis virus (AfMNPV). Microscopic observation revealed that infection of SL-1 cells with AfMNPV resulted in apoptosis, displaying apoptotic bodies in fluorescent-stained nuclei of AfMNPV-infected SL-1cells. Western blot analysis demonstrated that AfMNPV-induced apoptosis in insect SL-1 cells was significantly inhibited by cyclosporin A which blocked a translocation of cytochrome c from the mitochondria to the cytosol. As determined by using AC-DEVD-AFC as substrate, the activity of caspase-3 in AfMNPV-induced cells was detected as early as 4h post infection, gradually increased with time extension, and reached a highest level after 16h of infection. However, activity of caspase-3 in apoptotic cells decreased in the presence of cyclosporin A (30microM), indicating that activation of caspase-3 in SfaMNPV-induced cells was dependent on the release of cytochrome c from the mitochondria. In addition, cyclosporin A could markedly inhibit mitochondrial transmembrane potential (DeltaPsim) disruption in undergoing apoptotic cells. These data indicate that cytochrome c plays a key role in AfMNPV-induced apoptosis in S. litura cells and may be required for caspase activation during the induction of apoptosis.     

Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system.

In a cell-free system based on Xenopus egg extracts, Bcl-2 blocks apoptotic activity by preventing cytochrome c release from mitochondria. We now describe in detail the crucial role of cytochrome c in this system. The mitochondrial fraction, when incubated with cytosol, releases cytochrome c. Cytochrome c in turn induces the activation of protease(s) resembling caspase-3 (CPP32), leading to downstream apoptotic events, including the cleavage of fodrin and lamin B1. CPP32-like protease activity plays an essential role in this system, as the caspase inhibitor, Ac-DEVD-CHO, strongly inhibited fodrin and lamin B1 cleavage, as well as nuclear morphology changes. Cytochrome c preparations from various vertebrate species, but not from Saccharomyces cerevisiae, were able to initiate all signs of apoptosis. Cytochrome c by itself was unable to process the precursor form of CPP32; the presence of cytosol was required. The electron transport activity of cytochrome c is not required for its pro-apoptotic function, as Cu- and Zn-substituted cytochrome c had strong pro-apoptotic activity, despite being redox-inactive. However, certain structural features of the molecule were required for this activity. Thus, in the Xenopus cell-free system, cytosol-dependent mitochondrial release of cytochrome c induces apoptosis by activating CPP32-like caspases, via unknown cytosolic factors.     

Involvement of endoplasmic reticulum in paclitaxel-induced apoptosis

The participation of the mitochondrial pathway in paclitaxel-induced apoptosis has been well documented. After addition of paclitaxel to U937 cells, however, we observed an early expression of five endoplasmic reticulum (ER) stress response genes that preceded the release of cytochrome c from the mitochondria and the cleavage of the caspases. Involvement of the ER was supported by the following evidence. Paclitaxel treatment not only activated calpain and caspase-4, but also induced a gradual increase in the cytosolic Ca(2+) concentration at 3-6 h. Paclitaxel-induced apoptosis can be inhibited by the calpain inhibitor calpeptin and IP(3) receptor inhibitors. Either buffering of the cytosolic Ca(2+) or inhibition of mitochondrial calcium uptake reduced BiP expression. These inhibitors also reduced mitochondrial apoptotic signals, such as mitochondrion membrane potential disruption, cytochrome c release and eventually reduced the death of U937 cells. Paclitaxel-induced Bax/Bak translocation to the ER and Bax dimerization on the ER membrane occurred within 3 h, which led to a Ca(2+) efflux into cytosol. Moreover, we found that cytochrome c translocated to the ER after releasing from mitochondria and then interacted with the IP(3) receptor at 12-15 h. This phenomenon has been known to amplify apoptotic signaling. Taken together, ER would seem to contribute to paclitaxel-induced apoptosis via both the early release of Ca(2+) and the late amplification of mitochondria-mediated apoptotic signals.     

Photodynamic therapy: a mitochondrial inducer of apoptosis

Photodamage to the mitochondria of murine leukemia P388 cells resulted in immediate loss of the mitochondrial membrane potential together with the release of cytochrome c into the cytosol. This was followed by a rapid activation of caspase 3-like proteases, as indicated by a marked rise in DEVDase activity. There was no significant effect on WEHDase or VEIDase activities, suggesting that only the late-stage caspases had been effected. The apoptotic response to mitochondrial photodamage was abolished by the broad-spectrum caspase inhibitor zVAD-fmk, but this did not prevent loss of viability after mitochondrial photodamage. These studies indicate that the release of cytochrome c from photodamaged mitochondria is sufficient to directly initiate a caspase-dependent apoptotic response.     

Bcl-2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells

The pro-apoptotic protein, Bax, has been reported to translocate from cytosol to mitochondria following exposure of cells to apoptotic stresses including cytokine withdrawal and treatment with glucocorticoids and cytotoxic drugs. These observations, coupled with reports showing that Bax causes the release of mitochondrial cytochrome c, implicate Bax as a central mediator of the apoptotic process. In this report we demonstrate by subcellular fractionation a significant shift in Bax localization from cytosol to cellular membranes in two human tumor cell lines exposed to staurosporine or etoposide. Immunofluorescence studies confirmed that Bax specifically relocalized to the mitochondria. This redistribution of Bax occurred in concert with, or just prior to, proteolytic processing of procaspase-3, activation of DEVD-specific cleavage activity and degradation of poly(ADP-ribose) polymerase. However, Bax membrane translocation was independent of caspase activity as determined using the broad-range caspase inhibitor z-VAD-fmk. High level overexpression of the anti-apoptotic protein Bcl-2 prevented Bax redistribution to the mitochondria, caspase activation and apoptosis following exposure to staurosporine or etoposide. These data confirm the role of Bax in mitochondrial cytochrome c release, and indicate that prevention of Bax translocation to the mitochondrial membrane represents a novel mechanism by which Bcl-2 inhibits drug-induced apoptosis.     

Apoptosis-associated release of Smac/DIABLO from mitochondria requires active caspases and is blocked by Bcl-2.

Smac/DIABLO is a mitochondrial protein that potentiates some forms of apoptosis, possibly by neutralizing one or more members of the IAP family of apoptosis inhibitory proteins. Smac has been shown to exit mitochondria and enter the cytosol during apoptosis triggered by UV- or gamma-irradiation. Here, we report that Smac/DIABLO export from mitochondria into the cytosol is provoked by cytotoxic drugs and DNA damage, as well as by ligation of the CD95 death receptor. Mitochondrial efflux of Smac/DIABLO, in response to a variety of pro-apoptotic agents, was profoundly inhibited in Bcl-2-overexpressing cells. Thus, in addition to modulating apoptosis-associated mitochondrial cytochrome c release, Bcl-2 also regulates Smac release, suggesting that both molecules may escape via the same route. However, whereas cell stress-associated mitochondrial cytochrome c release was largely caspase independent, release of Smac/DIABLO in response to the same stimuli was blocked by a broad-spectrum caspase inhibitor. This suggests that apoptosis-associated cytochrome c and Smac/DIABLO release from mitochondria do not occur via the same mechanism. Rather, Smac/DIABLO efflux from mitochondria is a caspase-catalysed event that occurs downstream of cytochrome c release.     

Bax and other pro-apoptotic Bcl-2 family "killer-proteins" and their victim the mitochondrion

Two major intracellular apoptosis signaling cascades have been characterized, the mitochondrial pathway and the death receptor pathway. The mitochondrial pathway is regulated by members of the Bcl-2 protein family. The members of this family can be subdivided into anti- and pro-apoptotic proteins. The pro-apoptotic members are further divided into two groups, the multidomain and the 'BH3 domain only' proteins. When cells are exposed to apoptotic stimulation, pro-apoptotic proteins are activated through post-translational modifications or changes in their conformation. The main site of action of the multidomain proteins are the mitochondria, where these proteins induce permeabilization of the outer membrane resulting in the release of proteins, including cytochrome c, from the intermembrane space. In the cytosol cytochrome c activates caspase cascades ultimately leading to cell death. Mounting evidence indicates that apoptosis is involved in a wide range of pathological conditions. Recent studies suggest that the mitochondrial signaling pathway is involved in several diseases. Although, so far, with the exception of C. elegans, most studies on apoptosis have been performed in mammalian systems, recently homologues to the Bcl-2 family members, including pro-apoptotic members, have been identified in Drosophila and zebrafish. Here the structure and function of the various pro-apoptotic Bcl-2 family members, their effects on mitochondria, and their involvement in diseases are discussed.