Caspase-3 deficiency reveals a physiologic role for Smac/DIABLO in regulating programmed cell death

Inhibitor of apoptosis protein (IAP)-binding proteins such as Grim, Reaper and HID have been shown to exert a critical role in regulating caspase activity in species such as D. Melanogaster. However, a comparable role for the mammalian homologue of second mitochondrial-derived activator of caspase/direct IAP-binding protein with low pI (Smac/DIABLO) has yet to be clearly established in vivo. Despite tremendous interest in recent years in the use of so-called Smac mimetics to enhance chemotherapeutic potency, our understanding of the true physiologic nature of Smac/DIABLO in regulating programmed cell death (PCD) remains elusive. In order to critically evaluate the role of Smac/DIABLO in regulating mammalian PCD, deficiency of caspase-3 was used as a sensitizing mutation in order to reduce aggregate levels of executioner caspase activity. We observe that combinatorial deletion of Diablo and Casp3, but neither alone, results in perinatal lethality in mice. Consistent with this, examination of both intrinsic and extrinsic forms of PCD in lines of murine embryonic fibroblasts demonstrate that loss of Smac/DIABLO alters both caspase-dependent and caspase-independent intrinsic PCD. Comparative small interfering RNA inhibition studies of X-linked inhibitor of apoptosis, cellular inhibitor of apoptosis (cIAP)-1, cIAP-2, caspase-6 and -7 in both wild-type and Casp3/Diablo DKO mouse embryonic fibroblast lineages, supports a model in which Smac/DIABLO acts to enhance the early phase executioner caspase activity through the modulation of inhibitory interactions between specific IAP family members and executioner caspases-3 and -7.     

The serine protease inhibitor TLCK attenuates intrinsic death pathways in neurons upstream of mitochondrial demise

It is well-established that activation of proteases, such as caspases, calpains and cathepsins are essential components in signaling pathways of programmed cell death (PCD). Although these proteases have also been linked to mechanisms of neuronal cell death, they are dispensable in paradigms of intrinsic death pathways, e.g. induced by oxidative stress. However, emerging evidence implicated a particular role for serine proteases in mechanisms of PCD in neurons. Here, we investigated the role of trypsin-like serine proteases in a model of glutamate toxicity in HT-22 cells. In these cells glutamate induces oxytosis, a form of caspase-independent cell death that involves activation of the pro-apoptotic protein BH3 interacting-domain death agonist (Bid), leading to mitochondrial demise and ensuing cell death. In this model system, the trypsin-like serine protease inhibitor Nα-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK) inhibited mitochondrial damage and cell death. Mitochondrial morphology alterations, the impairment of the mitochondrial membrane potential and ATP depletion were prevented and, moreover, lipid peroxidation induced by glutamate was completely abolished. Strikingly, truncated Bid-induced cell death was not affected by TLCK, suggesting a detrimental activity of serine proteases upstream of Bid activation and mitochondrial demise. In summary, this study demonstrates the protective effect of serine protease inhibition by TLCK against oxytosis-induced mitochondrial damage and cell death. These findings indicate that TLCK-sensitive serine proteases play a crucial role in cell death mechanisms upstream of mitochondrial demise and thus, may serve as therapeutic targets in diseases, where oxidative stress and intrinsic pathways of PCD mediate neuronal cell death.     

The execution phase of autophagy associated PCD during insect metamorphosis

During metamorphosis of Manduca sexta, involution of labial glands follows an autophagic pathway towards programmed cell death (PCD). We looked for evidence of both caspase dependent and independent pathways of PCD by assaying for caspases -1, -2, -3, and -6, proteasomal protease, and cathepsins B & L, using fluorogenic substrates and aldehyde and chloromethylketone inhibitors. The substrates FR-AMC and RR-AMC, preferentially degraded by cathepsins B and L, were the most rapidly degraded, increasing in rate as the gland involuted. Digestion of YVAD-AMC (preferential substrate for caspase-1) and DEVD-AMC (substrate for caspases-3 & -7) was barely detectable, less than 0.02% (on a per-unit-protein basis) of that seen in vertebrate embryos induced to undergo apoptosis. Cleavage of VDVAD-AFC (substrate for caspase -2) and VEID-AFC (substrate for caspase -6) was also assessed, but activity was negligible. Mitochondrial membrane permeabilization (MMP) and cytochrome c release were not detected. Exogenous caspase substrate, polyadenosyl ribose phosphorylase (PARP), is cleaved by labial gland extracts, but only at an acidic pH of 5.5–6.0, and into fragments different from those generated by caspases (confirmed by N-terminal sequencing). The cysteine protease inhibitor leupeptin inhibits PARP cleavage, but the caspase inhibitor DEVD-CHO does not. However, potential caspase-derived fragments of PARP are seen when cytochrome c and dATP are added to cytosolic extracts. Although apoptotic machinery is conserved and functional in this tissue, cell death occurs independently of caspases in metamorphosis. We also postulate that lysosomal proteases play the major proteolytic role similar to the caspase cascade seen in apoptosis.     

The murine TRAIL receptor signals caspase-independent cell death through ceramide

Death receptors such as the 55 kDa tumor necrosis factor (TNF) receptor (TNF-R55) or Fas can initiate both apoptotic (caspase-dependent) and caspase-independent routes to programmed cell death (PCD). Here, we demonstrate for the first time that the single murine receptor for (TNF)-related apoptosis-inducing ligand (mTRAIL-R2) can induce a caspase-independent form of PCD with necrosis-like features in addition to apoptosis. Analysis of morphological and cellular features of caspase-independent PCD in response to TRAIL and TNF suggests that mTRAIL-R2 and TNF-R55 elicit caspase-independent PCD through similar pathways, although without participation of cathepsins. Cells overexpressing acid ceramidase (AC), an enzyme that metabolizes the sphingolipid ceramide, show enhanced survival from TRAIL-induced caspase-independent PCD but not from apoptosis, implicating a function of ceramide as a key mediator in caspase-independent PCD (but not apoptosis) induced by mTRAIL-R2. In concert with the enhanced resistance of AC-overexpressing cells against caspase-independent PCD induced by TNF, our results suggest that ceramide acts as a common mediator of caspase-independent PCD caused by death receptors such as mTRAIL-R2 and TNF-R55.     

Control of mitochondrial integrity by Bcl-2 family members and caspase-independent cell death

Programmed cell death (PCD) is essential for normal development and maintenance of tissue homeostasis in multicellular organisms. While it is now evident that PCD can take many different forms, apoptosis is probably the most well-defined cell death programme. The characteristic morphological and biochemical features associated with this highly regulated form of cell death have until recently been exclusively attributed to the caspase family of cysteine proteases. As a result, many investigators affiliate apoptosis with its pivotal execution system, i.e. caspase activation. However, it is becoming increasingly clear that PCD or apoptosis can also proceed in a caspase-independent manner and maintain key characteristics of apoptosis. Mitochondrial integrity is central to both caspase-dependent and-independent cell death. The release of pro-apoptotic factors from the mitochondrial intermembrane space is a key event in a cell's commitment to die and is under the tight regulation of the Bcl-2 family. However, the underlying mechanisms governing the efflux of these pro-death molecules are largely unknown. This review will focus on the regulation of mitochondrial integrity by Bcl-2 family members with particular attention to the controlled release of factors involved in caspase-independent cell death.     

Stat3 controls lysosomal-mediated cell death in vivo

It is well established that lysosomes play an active role during the execution of cell death. A range of stimuli can lead to lysosomal membrane permeabilization (LMP), thus inducing programmed cell death without involvement of the classical apoptotic programme. However, these lysosomal pathways of cell death have mostly been described in vitro or under pathological conditions. Here we show that the physiological process of post-lactational regression of the mammary gland is accomplished through a non-classical, lysosomal-mediated pathway of cell death. We found that, during involution, lysosomes in the mammary epithelium undergo widespread LMP. Furthermore, although cell death through LMP is independent of executioner caspases 3, 6 and 7, it requires Stat3, which upregulates the expression of lysosomal proteases cathepsin B and L, while downregulating their endogenous inhibitor Spi2A (ref. 8). Our findings report a previously unknown, Stat3-regulated lysosomal-mediated pathway of cell death under physiological circumstances. We anticipate that these findings will be of major importance in the design of treatments for cancers such as breast, colon and liver, where cathepsins and Stat3 are commonly overexpressed and/or hyperactivated respectively.     

Caspase-like proteases regulate aluminum-induced programmed cell death in peanut

Recent evidence has proved that caspase protease activities are detected in both mammals and plants during programmed cell death (PCD). The characteristics and functions of caspase-like proteases play important roles in understanding the mechanisms of PCD in plants. In this work, we report firstly the involvement of caspase-like protease activities and effects in aluminum (Al) stress in two contrasting peanut genotypes. Caspase-like activities in the root tip cells of ‘Zhonghua 2’ (Al-sensitive) and ‘99-1507’ (Al-tolerant) were detected using synthetic caspase substrates during Al-triggered PCD. Caspase-1-, -2-, -3-, -4-, -5-, -6-, -8- and -9-like proteases were found in peanut root tip cells. VDQQDase (caspase-2-like) and WEHD (caspase-5-like) were the first detected in the plants, and almost all of the caspase-like proteases were activated during Al-induced PCD, especially caspase-3-like and caspase-1-like, which was higher in ‘Zhonghua 2’ than in ‘99-1507’. The highest activity levels of caspase-3- and caspase-1-like proteases occurred 8 and 4 h after 100 µM Al treatment, respectively. Compared with 100 µM AlCl₃ treatment alone, specific caspase-3 protease inhibitor Ac-DEVD-CHO inhibited the increase of caspase-3-like protease activity, Al content, Hsr203j expression, cell death and DNA fragmentation, and the decrease in root growth induced by 100 µM AlCl₃ treatment, but it was more obvious in ‘Zhonghua 2’ than in ‘99-1507’. In conclusion, there were different caspase-like proteases in root tips of peanut, and caspase-3-like protease was a crucial executioner in Al-induced PCD. Its effects in the ‘Zhonghua 2’ genotype were higher than in ‘99-1507’. An improved model of the mechanism of Al-induced PCD and Al tolerance differences in different genotypes is proposed.     

Epidermal growth factor triggers an original, caspase-independent pituitary cell death with heterogeneous phenotype

Programmed cell death (PCD) is physiologically involved in the regulation of cell division and differentiation. It encompasses caspase-dependent mitochondrial and nonmitochondrial pathways. Additional caspase-independent pathways have been characterized in mitochondrial PCDs but remain hypothetical in nonmitochondrial PCDs. Epidermal growth factor (EGF) has been shown to inhibit division of pituitary somato-lactotrope cells occurring in parallel with EGF-mediated differentiation of these precursors into lactotrope cells. We show here that in somato-lactotrope pituitary cell line GH4C1, EGF triggers a PCD characterized by an apoptosis-like DNA fragmentation, insensitivity to broad-range caspase inhibitors, and absence of either cytochrome c or apoptosis-inducing factor release from mitochondria. Dying cells display loose chromatin clustering and numerous cytoplasmic vacuoles, a fraction of which are autophagic, thus conferring a heterogeneous phenotype to this PCD. Moreover, overexpression of cell death inhibitor Bcl-2 prevented not only the EGF-induced PCD but also its prodifferentiation effects, thus pointing to a mechanistic relationship existing between these two phenomena. Overall, the characterized differentiation-linked cell death represents an original form of caspase-independent PCD. The mechanisms underlying this PCD involve combinatorial engagement of discrete death effectors leading to a heterogeneous death phenotype that might be evolutionary related to PCD seen during the differentiation of some unicellular organisms.     

A serine protease is involved in the initiation of DNA damage-induced apoptosis

Caspases are considered to be the key effector proteases of apoptosis. Initiator caspases cleave and activate downstream executioner caspases, which are responsible for the degradation of numerous cellular substrates. We studied the role of caspases in apoptotic cell death of a human melanoma cell line. Surprisingly, the pancaspase inhibitor zVAD-fmk was unable to block cleavage of poly(ADP-ribose) polymerase (PARP) after treatment with etoposide, while it did prevent DEVDase activity. It is highly unlikely that caspase-2, which is a relatively zVAD-fmk-resistant caspase, is mediating etoposide-induced PARP cleavage, as a preferred inhibitor of this caspase could not prevent cleavage. In contrast, caspase activation and PARP degradation were blocked by pretreatment of the cells with the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). We therefore conclude that a serine protease regulates an alternative initiation mechanism that leads to caspase activation and PARP cleavage. More importantly, while zVAD-fmk could not rescue melanoma cells from etoposide-induced death, the combination with AEBSF resulted in substantial protection. This indicates that this novel pathway fulfills a critical role in the execution of etoposide-induced programmed cell death.     

Bax/Bak-dependent release of DDP/TIMM8a promotes Drp1-mediated mitochondrial fission and mitoptosis during programmed cell death

Mitochondrial morphology within cells is controlled by precisely regulated rates of fusion and fission . During programmed cell death (PCD), mitochondria undergo extensive fragmentation and ultimately caspase-independent elimination through a process known as mitoptosis . Though this increased fragmentation is due to increased fission through the recruitment of the dynamin-like GTPase Drp1 to mitochondria , as well as to a block in mitochondrial fusion , cellular mechanisms underlying these processes remain unclear. Here, we describe a mechanism for the increased mitochondrial Drp1 levels and subsequent stimulation of mitochondrial fission seen during PCD. We observed Bax/Bak-mediated release of DDP/TIMM8a, a mitochondrial intermembrane space (IMS) protein , into the cytoplasm, where it binds to and promotes the mitochondrial redistribution of Drp1, a mediator of mitochondrial fission. Using both loss- and gain-of-function assays, we also demonstrate that the Drp1- and DDP/TIMM8a-dependent mitochondrial fragmentation observed during PCD is an important step in mitoptosis, which in turn is involved in caspase-independent cell death. Thus, following Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP), IMS proteins released comprise not only apoptogenic factors such as cytochrome c involved in caspase activation but also DDP/TIMM8a, which activates Drp1-mediated fission to promote mitochondrial fragmentation and subsequently elimination during PCD.     

The Bcr-Abl kinase inhibitor INNO-406 induces autophagy and different modes of cell death execution in Bcr-Abl-positive leukemias

Bcr-Abl tyrosine kinase (TK) inhibitors are promising therapeutic agents for Bcr-Abl-positive (Bcr-Abl(+)) leukemias. Although they are known to promote caspase-mediated apoptosis, it remains unclear whether caspase-independent cell death-inducing mechanisms are also triggered. Here we demonstrated that INNO-406, a second-generation Bcr-Abl TK inhibitor, induces programmed cell death (PCD) in chronic myelogenous leukemia (CML) cell lines through both caspase-mediated and caspase-independent pathways. The latter pathways include caspase-independent apoptosis (CIA) and necrosis-like cell death (CIND), and the cell lines varied regarding which mechanism was elicited upon INNO-406 treatment. We also observed that the propensity toward CIA or CIND in cells was strongly associated with cellular dependency on apoptosome-mediated caspase activity. Cells that undergo CIND have a high apoptosome activity potential whereas cells that undergo CIA tend to have a lower potential. Moreover, we found that INNO-406 promotes autophagy. When autophagy was inhibited with chloroquine or gene knockdown of beclin1 by shRNA, INNO-406-induced cell death was enhanced, which indicates that the autophagic response of the tumor cells is protective. These findings suggest new insights into the biology and therapy of Bcr-Abl(+) leukemias.     

Zinc induces caspase-dependent mitochondrial pathway of the programmed cell death in haemocytes of Drosophila melanogaster

Zinc is an essential trace element in cells. However, its high level in cytoplasm promotes activation of stress signaling pathways and may lead to cell death. In the present study we used Drosophila melanogaster blood cells (haemocytes), obtained from the third instar larvae, to study the effects of high concentrations of Zn(2+) on programmed cell death (PCD). We analyzed the activity of caspases, the level of caspase inhibitor protein DIAP1 and metallothioneins, as well as calcium concentrations and activity of mitochondria in haemocytes exposed to 0.35 and 1.7 mM concentrations of Zn. The obtained results showed that rapid increase of [Zn(2+)]( i ) in the cytoplasm up-regulates metallothionein MtnB but not MtnA gene expression in cells treated with Zn(2+) in both concentrations. Excess of Zn(2+) also induced activation of the initiator caspase Dronc, associated with the mitochondrial pathway of PCD, and the effector caspase DrICE. In turn, the activity of receptor-regulated Dredd caspase was not changed. The level of DIAP1 decreased significantly in haemocytes in the presence of high Zn(2+) concentration in comparison to untreated cells. Moreover, mitochondrial membrane potential was significantly decreased after exposure to Zn ions. These results indicate that high concentration of Zn(2+) in the cytoplasm of haemocytes induces PCD via a mitochondrial pathway and that caspases play a pivotal role in this process.     

Serine proteases mediate apoptosis-like cell death and phagocytosis under caspase-inhibiting conditions

Effective execution of apoptosis requires the activation of caspases. However, in many cases, broad-range caspase inhibitors such as Z-VAD.fmk do not inhibit cell death because death signaling continues via basal caspase activities or caspase-independent processes. Although death mediators acting under caspase-inhibiting conditions have been identified, it remains unknown whether they trigger a physiologically relevant cell death that shows typical signs of apoptosis, including phosphatidylserine (PS) exposure and the removal of apoptotic cells by phagocytosis. Here we show that cells treated with ER stress drugs or deprived of IL-3 still show hallmarks of apoptosis such as cell shrinkage, membrane blebbing, mitochondrial release of cytochrome c, PS exposure and phagocytosis in the presence of Z-VAD.fmk. Cotreatment of the stressed cells with Z-VAD.fmk and the serine protease inhibitor Pefabloc (AEBSF) inhibited all these events, indicating that serine proteases mediated the apoptosis-like cell death and phagocytosis under these conditions. The serine proteases were found to act upstream of an increase in mitochondrial membrane permeability as opposed to the serine protease Omi/HtrA2 which is released from mitochondria at a later stage. Thus, despite caspase inhibition or basal caspase activities, cells can still be phagocytosed and killed in an apoptosis-like fashion by a serine protease-mediated mechanism that damages the mitochondrial membrane.     

The apoptosis inhibitory domain of FE65-like protein 1 regulates both apoptotic and caspase-independent programmed cell death mediated by tumor necrosis factor

Tumor necrosis factor (TNF) can induce caspase-dependent (apoptotic) and caspase-independent pathways to programmed cell death (PCD). Here, we demonstrate that stable transfection of a cDNA encompassing the C-terminal apoptosis inhibitory domain (AID) of FE65-like protein 1 into mouse L929 fibrosarcoma cells protects from caspase-independent as well as from apoptotic PCD induced by TNF. We show that the AID does not protect from caspase-independent PCD elicited by 1-methyl-3-nitro-1-nitrosoguanidine, suggesting that the AID might prevent cell death by affecting assembly of the death inducing signaling complex of the 55 kDa TNF receptor or clustering of the receptor itself. Interference with caspase-independent PCD mediated by the sphingolipid ceramide further increases protection conferred by the AID, as does the antioxidant butylated hydroxyanisole, implicating ceramide and reactive oxygen species as potential factors interacting with caspase-independent PCD regulated by the AID.     

AIF-mediated caspase-independent necroptosis: a new chance for targeted therapeutics

Cell death has been initially divided into apoptosis, in which the cell plays an active role, and necrosis, which is considered a passive cell death program. Intense research performed in the last decades has concluded that "programmed" cell death (PCD) is a more complex physiological process than initially thought. Indeed, although in most cases the PCD process is achieved via a family of Cys proteases known as caspases, an important number of regulated PCD pathways do not involve this family of proteases. As a consequence, active forms of PCD are initially referred to as caspase-dependent and caspase-independent. More recent data has revealed that there are also active caspase-independent necrotic pathways defined as necroptosis (programmed necrosis). The existence of necroptotic forms of death was corroborated by the discovery of key executioners such as the kinase RIP1 or the mitochondrial protein apoptosis-inducing factor (AIF). AIF is a Janus protein with a redox activity in the mitochondria and a pro-apoptotic function in the nucleus. We have recently described a particular form of AIF-mediated caspase-independent necroptosis that also implicates other molecules such as PARP-1, calpains, Bax, Bcl-2, histone H2AX, and cyclophilin A. From a therapeutic point of view, the unraveling of this new form of PCD poses a question: is it possible to modulate this necroptotic pathway independently of the classical apoptotic paths? Because the answer is yes, a wider understanding of AIF-mediated necroptosis could theoretically pave the way for the development of new drugs that modulate PCD. To this end, we present here an overview of the current knowledge of AIF and AIF-mediated necroptosis. We also summarize the state of the art in some of the most interesting therapeutic strategies that could target AIF or the AIF-mediated necroptotic pathway.     

Calpain activates caspase-3 during UV-induced neuronal death but only calpain is necessary for death

While caspases have been strongly implicated in delayed neuronal death in a variety of experimental paradigms, other proteases such as calpain can also contribute to neuronal death. To evaluate the relative roles of caspase and calpain, we used a model system wherein UV treatment induced moderate or severe delayed cortical neuronal death, as quantified by propidium iodide and calcein AM. UV treatment led to increases in both caspase and calpain activation. Calpain inhibitor III (MDL-28170) reduced caspase activation, suggesting that caspase activation was mediated by calpain. Calpain contributed to neuronal death, as indicated by strong neuroprotection provided by calpain inhibitor III, calpeptin, or Ca2+-free medium. In contrast, caspase inhibitors were not neuroprotective. These results suggest that UV neurotoxicity is mediated by a loss of Ca2+ homeostasis which leads to a calpain-dependent, caspase-independent cell death. That calpain, but not caspase, may mediate death in instances involving the activation of both proteases may have relevance to other neuronal death models.     

Identification of early intermediates of caspase activation using selective inhibitors and activity-based probes

Caspases are cysteine proteases that are key effectors in apoptotic cell death. Currently, there is a lack of tools that can be used to monitor the regulation of specific caspases in the context of distinct apoptotic programs. We describe the development of highly selective inhibitors and active site probes and their applications to directly monitor executioner (caspase-3 and -7) and initiator (caspase-8 and -9) caspase activity. Specifically, these reagents were used to dissect the kinetics of caspase activation upon stimulation of apoptosis in cell-free extracts and intact cells. These studies identified a full-length caspase-7 intermediate that becomes catalytically activated early in the pathway and whose further processing is mediated by mature executioner caspases rather than initiator caspases. This form also shows distinct inhibitor sensitivity compared to processed caspase-7. Our data suggest that caspase-7 activation proceeds through a previously uncharacterized intermediate that is formed without cleavage of the intact zymogen.     

A Role for Caspases in Lens Fiber Differentiation

There is increasing evidence that programmed cell death (PCD) depends on a novel family of intracellular cysteine proteases, called caspases, that includes the Ced-3 protease in the nematode Caenorhabditis elegans and the interleukin-1β–converting enzyme (ICE)-like proteases in mammals. Some developing cells, including lens epithelial cells, erythroblasts, and keratinocytes, lose their nucleus and other organelles when they terminally differentiate, but it is not known whether the enzymatic machinery of PCD is involved in any of these normal differentiation events. We show here that at least one CPP32 (caspase-3)-like member of the caspase family becomes activated when rodent lens epithelial cells terminally differentiate into anucleate lens fibers in vivo, and that a peptide inhibitor of these proteases blocks the denucleation process in an in vitro model of lens fiber differentiation. These findings suggest that at least part of the machinery of PCD is involved in lens fiber differentiation.     

Serine protease inhibitor Spi2 mediated apoptosis of olfactory neurons

The olfactory epithelium of adult mouse, where primary sensory neurons are massively committed to apoptosis by removal of their synaptic target, was used as a model to determine in vivo mechanisms for neuronal cell death induction. A macro-array assay revealed that the death of olfactory neurons is accompanied with over-expression of the serine protease inhibitor Spi2. This over-expression is associated with decreased serine protease activity in the olfactory mucosa. Moreover, in vitro or in vivo inhibition of serine proteases induced apoptotic death of olfactory neuronal cells. Interestingly, Spi2 over-expression is not occurring in olfactory neurons but in cells of the lamina propria, suggesting that Spi2 may act extracellularly as a cell death inducer. In that sense, we present evidence that in vitro Spi2 overexpression generates a secreted signal for olfactory neuron death. Hence, taken together these results document a possible novel mechanism for apoptosis induction that might occur in response to neurodegenerative insults.