Effect of Hypoxia on Caspase-3, -8, and -9 Activity and Expression in the Cerebral Cortex of Newborn Piglets

Caspases play an important role in programmed cell death. Caspase-3 is a key executioner of apoptosis, whose activation is mediated by the initiator caspases, caspase-8 and caspase-9. The present study tested the hypothesis that cerebral hypoxia results in increased activation and expression of caspases-3, -8, and -9 in the cytosolic fraction of the cerebral cortex of newborn piglets. To test this hypothesis the activity and expression of caspases-3, -8, and -9 were determined in newborn piglets divided into normoxic and hypoxic groups. Caspase activity was determined spectrofluorometrically using enzyme specific substrates. The expression of caspase protein was assessed by Western blot analysis using enzyme specific antibody. Caspases-3, -8, and -9 activity and expression was significantly higher in the hypoxic group than in the normoxic group. These results demonstrate that hypoxia induces activation and increased expression of both the initiator caspases and the executioner caspase in the cerebral cortex of newborn piglets. We conclude that hypoxia results in stimulation of both the pathways of caspase-3 activation.     

Apoptosis triggered redistribution of caspase-9 from cytoplasm to mitochondria

Caspase-9 is an apoptosis initiator protease activated as a response to the mitochondrial damage in the cytoplasmic complex apoptosome. By fluorescence labelling of proteins, confocal microscopy and subcellular fractionations we demonstrate that caspase-9 is in the cytoplasm of non-apoptotic pituitary cells. The activation of apoptosis with rotenone triggers the redistribution of caspase-9 to mitochondria. Experiments using the general caspase inhibitor z-VAD.fmk and the specific caspase-9 inhibitor z-LEHD.fmk show that the caspase-9 redistribution is a regulated process and requires the activity of a caspase other than the caspase-9. We propose that this spatial regulation is required to control the activity of caspase-9.     

Inhibition of caspase-9 through phosphorylation at Thr 125 by ERK MAPK

Many pro-apoptotic signals activate caspase-9, an initiator protease that activates caspase-3 and downstream caspases to initiate cellular destruction. However, survival signals can impinge on this pathway and suppress apoptosis. Activation of the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) pathway is associated with protection of cells from apoptosis and inhibition of caspase-3 activation, although the targets are unknown. Here, we show that the ERK MAPK pathway inhibits caspase-9 activity by direct phosphorylation. In mammalian cell extracts, cytochrome c-induced activation of caspases-9 and -3 requires okadaic-acid-sensitive protein phosphatase activity. The opposing protein kinase activity is overcome by treatment with the broad-specificity kinase inhibitor staurosporine or with inhibitors of MEK1/2. Caspase-9 is phosphorylated at Thr 125, a conserved MAPK consensus site targeted by ERK2 in vitro, in a MEK-dependent manner in cells stimulated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Phosphorylation at Thr 125 is sufficient to block caspase-9 processing and subsequent caspase-3 activation. We suggest that phosphorylation and inhibition of caspase-9 by ERK promotes cell survival during development and tissue homeostasis. This mechanism may also contribute to tumorigenesis when the ERK MAPK pathway is constitutively activated.     

Heat shock induces apoptosis independently of any known initiator caspase-activating complex

Adaptive responses to mild heat shock are among the most widely conserved and studied in nature. More intense heat shock, however, induces apoptosis through mechanisms that remain largely unknown. Herein, we present evidence that heat shock activates an apical protease that stimulates mitochondrial outer membrane permeabilization and processing of the effector caspase-3 in a benzyloxycarbonyl-VAD-fluoromethyl ketone (polycaspase inhibitor)- and Bcl-2-inhibitable manner. Surprisingly, however, neither FADD.caspase-8 nor RAIDD.caspase-2 PIDDosome (p53-induced protein with a death domain) complexes were detected in dying cells, and neither of these initiator caspases nor the endoplasmic reticulum stress-activated caspases-4/12 were required for mitochondrial outer membrane permeabilization. Similarly, although cytochrome c was released from mitochondria following heat shock, functional Apaf-1.caspase-9 apoptosome complexes were not formed, and caspase-9 was not essential for the activation of caspase-3 or the induction of apoptosis. Thus, heat shock does not require any of the known initiator caspases or their activating complexes to promote apoptotic cell death but instead relies upon the activation of an apparently novel apical protease with caspase-like activity.     

Oligomerization is a general mechanism for the activation of apoptosis initiator and inflammatory procaspases

Proteolytic activation of initiator procaspases is a crucial step in the cellular commitment to apoptosis. Alternative models have been postulated for the activation mechanism, namely the oligomerization or induced proximity model and the allosteric regulation model. While the former holds that procaspases become activated upon proper oligomerization by an adaptor protein, the latter states that the adaptor is an allosteric regulator for procaspases. The allosteric regulation model has been applied for the activation of procaspase-9 by apoptotic protease-activating factor (Apaf-1) in an oligomeric complex known as the apoptosome. Using approaches that allow for controlled oligomerization, we show here that aggregation of multiple procaspase-9 molecules can induce their activation independent of the apoptosome. Oligomerization-induced procaspase-9 activation, both within the apoptosome and in artificial systems, requires stable homophilic association of the protease domains, raising the possibility that the function of Apaf-1 is not only to oligomerize procaspase-9 but also to maintain the interaction of the caspase-9 protease domain after processing. In addition, we provide biochemical evidence that other apoptosis initiator caspases (caspase-2 and -10) as well as a procaspase involved in inflammation (murine caspase-11) are also activated by oligomerization. Thus, oligomerization of precursor molecules appears to be a general mechanism for the activation of both apoptosis initiator and inflammatory procaspases.     

Baculovirus P35 protein does not inhibit caspase-9 in a cell-free system of apoptosis

The P35 protein derived from the baculovirus Autographa californica NPV has been characterized as an inhibitor of apoptotic cell death in a great number of organisms and situations. This potential has been further mapped to the capacity of P35 to inhibit all caspases investigated. Here we show that P35 does not inhibit caspase-9 activity in a cell-free system of mammalian caspase activation. In cell extracts, cytochrome c addition led to the activation of caspase-9, -3 and -7. When cytosolic extract from cells expressing P35 was added, caspase-9-mediated maturation of caspase-3 proceeded normally but caspase-3-mediated further events were prevented, such as complete processing of caspase-3, processing of caspase-7 and the appearance of DEVD-cleaving activity. The P35 protein from Bombyx mori NPV, which has been reported to have a much weaker anti-apoptosis activity in vivo, was found also to have significant caspase-3-inhibiting activity. These data suggest that P35 evolved specifically to inhibit effector rather than initiator caspases.     

Protease Activity of Procaspase-8 Is Essential for Cell Survival by Inhibiting Both Apoptotic and Nonapoptotic Cell Death Dependent on Receptor-interacting Protein Kinase 1 (RIP1) and RIP3

Caspase-8 has an important role as an initiator caspase during death receptor-mediated apoptosis. Moreover, it has been reported to contribute to the regulation of cell fate in various types of cells including T-cells. In this report, we show that caspase-8 has an essential role in cell survival in mouse T-lymphoma-derived L5178Y cells. The knockdown of caspase-8 expression decreased the growth rate and increased cell death, both of which were induced by the absence of protease activity of procaspase-8. The cell death was associated with reactive oxygen species (ROS) accumulation, caspase activation, and autophagosome formation. The cell death was inhibited completely by treatment with ROS scavengers, but only partly by treatment with caspase inhibitors, expression of Bcl-xL, and knockdown of caspase-3 or Atg-7 which completely inhibits apoptosis or autophagosome formation, respectively, indicating that apoptosis and autophagy-associated cell death are induced simultaneously by the knockdown of caspase-8 expression. Further analysis indicated that RIP1 and RIP3 regulate this multiple cell death, because the cell death as well as ROS production was completely inhibited by not only treatment with the RIP1 inhibitor necrostatin-1, but also by knockdown of RIP3. Thus, in the absence of protease activity of procaspase-8, RIP1 and RIP3 simultaneously induce not only nonapoptotic cell death conceivably including autophagic cell death and necroptosis but also apoptosis through ROS production in mouse T-lymphoma cells.     

Vesicular stomatitis viruses expressing wild-type or mutant M proteins activate apoptosis through distinct pathways

Vesicular stomatitis virus (VSV) induces apoptosis by at least two mechanisms. The viral matrix (M) protein induces apoptosis via the mitochondrial pathway due to the inhibition of host gene expression. However, in some cell types, the inhibition of host gene expression by VSV expressing wild-type (wt) M protein delays VSV-induced apoptosis, indicating that another mechanism is involved. In support of this, the recombinant M51R-M (rM51R-M) virus, expressing a mutant M protein that is defective in its ability to inhibit host gene expression, induces apoptosis much more rapidly in L929 cells than do viruses expressing wt M protein. Here, we determine the caspase pathways by which the rM51R-M virus induces apoptosis. An analysis of caspase activity, using fluorometric caspase assays and Western blots, indicated that each of the main initiator caspases, caspase-8, caspase-9, and caspase-12, were activated during infection with the rM51R-M virus. The overexpression of Bcl-2, an inhibitor of the mitochondrial pathway, or MAGE-3, an inhibitor of caspase-12 activation, did not delay apoptosis induction in rM51R-M virus-infected L929 cells. However, an inhibitor of caspase-8 activity significantly delayed apoptosis induction. Furthermore, the inhibition of caspase-8 activity prevented the activation of caspase-9, suggesting that caspase-9 is activated by cross talk with caspase-8. These data indicate that VSV expressing the mutant M protein induces apoptosis via the death receptor apoptotic pathway, a mechanism distinct from that induced by VSV expressing the wt M protein.     

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis is dependent on activation of cysteine and serine proteases

We examined the role of caspases and serine protease(s) in cell death induced by tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). After incubation of adenocarcinoma cells with TRAIL, caspase-3, -8 were activated and the cleavage of Bid induced the release of cytochrome c, from the mitochondria to the cytosol. Tetrapeptide inhibitors of caspase-1, -2, -3, and -8 suppressed DNA fragmentation and attenuated the release of cytochrome c, whereas inhibitors of caspase-5 did not. Interestingly, the general serine protease(s) inhibitor 4-(2-aminoethyl)benzylsulfonyl fluoride (AEBSF) resulted in the arrest of apoptosis. However, the AEBSF did not prevent the release of mitochondrial cytochrome c during TRAIL-induced apoptosis. From these results, we postulate that serine protease(s) may be involved in post-mitochondrial apoptotic events, that lead to the activation of the initiator, caspase-9.     

Sequential caspase-2 and caspase-8 activation upstream of mitochondria during ceramideand etoposide-induced apoptosis

Recently, caspase-2 was shown to act upstream of mitochondria in stress-induced apoptosis. Activation of caspase-8, a key event in death receptor-mediated apoptosis, also has been demonstrated in death receptor-independent apoptosis. The regulation of these initiator caspases, which trigger the mitochondrial apoptotic pathway, is unclear. Here we report a potential regulatory role of caspase-2 on caspase-8 during ceramide-induced apoptosis. Our results demonstrate the sequential events of initiator caspase-2 and caspase-8 activation, Bid cleavage and translocation, and mitochondrial damage followed by downstream caspase-9 and -3 activation and cell apoptosis after ceramide induction in T cell lines. The expression of truncated Bid (tBid) and the reduction in mitochondrial transmembrane potential were blocked by caspase-2 or caspase-8, but not caspase-3, knockdown using an RNA interference technique. Ceramide-induced caspase-8 activation, mitochondrial damage, and apoptosis were blocked in caspase-2 short interfering RNA-expressing cells. Therefore, caspase-2 acts upstream of caspase-8 during ceramide-induced mitochondrial apoptosis. Similarly, sequential caspase-2 and caspase-8 activation upstream of mitochondria was also observed in etoposide-induced apoptosis. These data suggest sequential initiator caspase-2 and caspase-8 activation in the mitochondrial apoptotic pathway induced by ceramide or etoposide.     

Proteolytic Processing of the Caspase-9 Zymogen Is Required for Apoptosome-mediated Activation of Caspase-9

Maturation of the single-chain caspase-9 zymogen through autoproteolytic processing is mediated by the Apaf-1 apoptosome at the onset of apoptosis. Processed caspase-9 and the apoptosome form a holoenzyme with robust proteolytic activity that is 2–3 orders of magnitude higher than that of free processed caspase-9. An unresolved important question is the role of caspase-9 processing, with some experimental data suggesting its dispensability. In this study, we demonstrate that, in contrast to wild-type caspase-9, the unprocessed single-chain caspase-9 triple mutant E306A/D315A/D330A (Casp9-TM) could no longer be adequately activated by the apoptosome. Compared with the protease activity of wild-type caspase-9, that of Casp9-TM in the presence of the apoptosome was drastically reduced. The crippled protease activity of Casp9-TM in the presence of the apoptosome is likely attributable to a markedly reduced ability of Casp9-TM to form homodimers. These data identify an essential role for the autoproteolytic processing of caspase-9 in its activation.     

羊栖菜多糖通过激活Caspase途径诱导Lovo细胞凋亡

研究了羊栖菜多糖（Sargassum Fusiforme Polysaccharides,SFPS）诱导人大肠癌lovo细胞凋亡及凋亡过程中caspase-3、caspase-8、caspase-9的活性变化。MTT法检测SFPS对lovo细胞增殖的抑制率;通过电镜、琼脂糖凝胶电泳、流式细胞术鉴定细胞凋亡;应用Western印迹法测定caspase-3酶原和caspase-9的变化;RToPCR检测caspase-3 mRNA表达;caspase-3,caspase-8、caspase-9活性检测试剂盒观察caspase-3、caspase-8、caspase-9的活性改变。结果显示,SFPS对lovo细胞增殖有显著抑制作用,经形态变化、DNA条带和流式细胞分析,可见明显的细胞凋亡特征。SFPS处理lovo细胞后,发现caspase-3酶原蛋白表达降低,caspase-3 mRNA高表达,并具有剂量和时间的依赖性。而在检测蛋白中,也发现caspase-9被激活进而形成具有活性的片段。另外,caspase的活性检测也进一步发现caspase-3、caspase-9的活性逐步增高。实验结果提示SFPS在体外诱导lovo胞凋亡,这可能是SFPS抑制肿瘤增殖的机制之一,并且是通过激活启动caspase-9,进而激活下游效应caspase-3的级联反应来实现的。     

Loss of caspase-9 reveals its essential role for caspase-2 activation and mitochondrial membrane depolarization

Caspase-9 plays an important role in apoptosis induced by genotoxic stress. Irradiation and anticancer drugs trigger mitochondrial outer membrane permeabilization, resulting in cytochrome c release and caspase-9 activation. Two highly contentious issues, however, remain: It is unclear whether the loss of the mitochondrial membrane potential DeltaPsi(M) contributes to cytochrome c release and whether caspases are involved. Moreover, an unresolved question is whether caspase-2 functions as an initiator in genotoxic stress-induced apoptosis. In the present study, we have identified a mutant Jurkat T-cell line that is deficient in caspase-9 and resistant to apoptosis. Anticancer drugs, however, could activate proapoptotic Bcl-2 proteins and cytochrome c release, similarly as in caspase-9-proficient cells. Interestingly, despite these alterations, the cells retained DeltaPsi(M). Furthermore, processing and enzyme activity of caspase-2 were not observed in the absence of caspase-9. Reconstitution of caspase-9 expression restored not only apoptosis but also the loss of DeltaPsi(M) and caspase-2 activity. Thus, we provide genetic evidence that caspase-9 is indispensable for drug-induced apoptosis in cancer cells. Moreover, loss of DeltaPsi(M) can be functionally separated from cytochrome c release. Caspase-9 is not only required for DeltaPsi(M) loss but also for caspase-2 activation, suggesting that these two events are downstream of the apoptosome.     

Caspase-4 is required for activation of inflammasomes

IL-1β and IL-18 are crucial regulators of inflammation and immunity. Both cytokines are initially expressed as inactive precursors, which require processing by the protease caspase-1 for biological activity. Caspase-1 itself is activated in different innate immune complexes called inflammasomes. In addition, caspase-1 activity regulates unconventional protein secretion of many other proteins involved in inflammation and repair. Human caspase-4 is a poorly characterized member of the caspase family, which is supposed to be involved in endoplasmic reticulum stress-induced apoptosis. However, its gene is located on the same locus as the caspase-1 gene, which raises the possibility that caspase-4 plays a role in inflammation. In this study, we show that caspase-4 expression is required for UVB-induced activation of proIL-1β and for unconventional protein secretion by skin-derived keratinocytes. These processes require expression of the nucleotide-binding domain leucine-rich repeat containing, Pyrin domain containing-3 inflammasome, and caspase-4 physically interacts with its central molecule caspase-1. As the active site of caspase-4 is required for activation of caspase-1, the latter most likely represents a substrate of caspase-4. Caspase-4 expression is also essential for efficient nucleotide-binding domain leucine-rich repeat containing, Pyrin domain containing-3 and for absent in melanoma 2 inflammasome-dependent proIL-1β activation in macrophages. These results demonstrate an important role of caspase-4 in inflammation and innate immunity through activation of caspase-1. Therefore, caspase-4 represents a novel target for the treatment of (auto)inflammatory diseases.     

Engineering a dimeric caspase-9: a re-evaluation of the induced proximity model for caspase activation

Caspases are responsible for the execution of programmed cell death (apoptosis) and must undergo proteolytic activation, in response to apoptotic stimuli, to function. The mechanism of initiator caspase activation has been generalized by the induced proximity model, which is thought to drive dimerization-mediated activation of caspases. The initiator caspase, caspase-9, exists predominantly as a monomer in solution. To examine the induced proximity model, we engineered a constitutively dimeric caspase-9 by relieving steric hindrance at the dimer interface. Crystal structure of the engineered caspase-9 closely resembles that of the wild-type (WT) caspase-9, including all relevant structural details and the asymmetric nature of two monomers. Compared to the WT caspase-9, this engineered dimer exhibits a higher level of catalytic activity in vitro and induces more efficient cell death when expressed. However, the catalytic activity of the dimeric caspase-9 is only a small fraction of that for the Apaf-1-activated caspase-9. Furthermore, in contrast to the WT caspase-9, the activity of the dimeric caspase-9 can no longer be significantly enhanced in an Apaf-1-dependent manner. These findings suggest that dimerization of caspase-9 may be qualitatively different from its activation by Apaf-1, and in conjunction with other evidence, posit an induced conformation model for the activation of initiator caspases.     

The non-apoptotic role of caspase-9 promotes differentiation in leukemic cells

Caspase family contains cysteine proteases involving in the key cellular processes, such as apoptosis, inflammation, and autophagy. There is a growing body of evidence that caspase family also plays a role in cellular differentiation. Evidence suggests that caspase-9 is among the most important members with non-apoptotic roles in the execution of differentiation. Since drug-induced differentiation in some types of cancer cells is a promising treatment, we have investigated caspase-9 activity during differentiation of a cancer cell; leukemia. We demonstrate that caspase-9 has increased activity during differentiation and also the inhibition of caspase-9 will prevent the granulocytic differentiation of leukemic cells. In addition, we studied the differentiation induction mediated by caspase-9 using an inducible variant of caspase-9. Results indicate the caspase-9 mediated differentiation accompanied by a reduction in the expression of CD33 and an increase in CD15. Notably, all of the events occur when cell viability remains constant. Owing to the evidence, caspase-9 activity is considered as a central factor in the execution of differentiation in leukemic cells.     

Mitochondrial pro-apoptotic indices do not precede the transient caspase activation associated with myogenesis

Skeletal muscle differentiation requires activity of the apoptotic protease caspase-3. We attempted to identify the source of caspase activation in differentiating C2C12 skeletal myoblasts. In addition to caspase-3, caspase-2 was transiently activated during differentiation; however, no changes were observed in caspase-8 or -9 activity. Although mitochondrial Bax increased, this was matched by Bcl-2, resulting in no change to the mitochondrial Bax:Bcl-2 ratio early during differentiation. Interestingly, mitochondrial membrane potential increased on a timeline similar to caspase activation and was accompanied by an immediate, temporary reduction in cytosolic Smac and cytochrome c. Since XIAP protein expression dramatically declined during myogenesis, we investigated whether this contributes to caspase-3 activation. Despite reducing caspase-3 activity by up to 57%, differentiation was unaffected in cells overexpressing normal or E3-mutant XIAP. Furthermore, a XIAP mutant which can inhibit caspase-9 but not caspase-3 did not reduce caspase-3 activity or affect differentiation. Administering a chemical caspase-3 inhibitor demonstrated that complete enzyme inhibition was required to impair myogenesis. These results suggest that neither mitochondrial apoptotic signaling nor XIAP degradation is responsible for transient caspase-3 activation during C2C12 differentiation.