Fas activation of the p38 mitogen-activated protein kinase signalling pathway requires ICE/CED-3 family proteases.

The Fas receptor mediates a signalling cascade resulting in programmed cell death (apoptosis) within hours of receptor cross-linking. In this study Fas activated the stress-responsive mitogen-activated protein kinases, p38 and JNK, within 2 h in Jurkat T lymphocytes but not the mitogen-responsive kinase ERK1 or pp70S6k. Fas activation of p38 correlated temporally with the onset of apoptosis, and transfection of constitutively active MKK3 (glu), an upstream regulator of p38, potentiated Fas-induced cell death, suggesting a potential involvement of the MKK3/p38 activation pathway in Fas-mediated apoptosis. Fas has been shown to require ICE (interleukin-1 beta-converting enzyme) family proteases to induce apoptosis from studies utilizing the cowpox ICE inhibitor protein CrmA, the synthetic tetrapeptide ICE inhibitor YVAD-CMK, and the tripeptide pan-ICE inhibitor Z-VAD-FMK. In this study, crmA antagonized, and YVAD-CMK and Z-VAD-FMK completely inhibited, Fas activation of p38 kinase activity, demonstrating that Fas-dependent activation of p38 requires ICE/CED-3 family members and conversely that the MKK3/p38 activation cascade represents a downstream target for the ICE/CED-3 family proteases. Intriguingly, p38 activation by sorbitol and etoposide was resistant to YVAD-CMK and Z-VAD-FMK, suggesting the existence of an additional mechanism(s) of p38 regulation. The ICE/CED-3 family-p38 regulatory relationship described in the current work indicates that in addition to the previously described destructive cleavage of substrates such as poly(ADP ribose) polymerase, lamins, and topoisomerase, the apoptotic cysteine proteases also function to regulate stress kinase signalling cascades.     

Fas Induces Cytoplasmic Apoptotic Responses and Activation of the MKK7-JNK/SAPK and MKK6-p38 Pathways Independent of CPP32-like Proteases

IL-1β converting enzyme (ICE) family cysteine proteases are subdivided into three groups; ICE-, CPP32-, and Ich-1–like proteases. In Fas-induced apoptosis, activation of ICE-like proteases is followed by activation of CPP32-like proteases which is thought to be essential for execution of the cell death. It was recently reported that two subfamily members of the mitogen-activated protein kinase superfamily, JNK/SAPK and p38, are activated during Fas-induced apoptosis. Here, we have shown that MKK7, but not SEK1/ MKK4, is activated by Fas as an activator for JNK/ SAPK and that MKK6 is a major activator for p38 in Fas signaling. Then, to dissect various cellular responses induced by Fas, we used several peptide inhibitors for ICE family proteases in Fas-treated Jurkat cells and KB cells. While Z-VAD-FK which inhibited almost all the Fas-induced cellular responses blocked the activation of JNK/SAPK and p38, Ac-DEVD-CHO and Z-DEVD-FK, specific inhibitors for CPP32-like proteases, which inhibited the Fas-induced chromatin condensation and DNA fragmentation did not block the activation of JNK/SAPK and p38. Interestingly, these DEVD-type inhibitors did not block the Fas-induced morphological changes (cell shrinkage and surface blebbing), induction of Apo2.7 antigen, or the cell death (as assessed by the dye exclusion ability). These results suggest that the Fas-induced activation of the JNK/SAPK and p38 signaling pathways does not require CPP32-like proteases and that CPP32-like proteases, although essential for apoptotic nuclear events (such as chromatin condensation and DNA fragmentation), are not required for other apoptotic events in the cytoplasm or the cell death itself. Thus, the Fas signaling pathway diverges into multiple, separate processes, each of which may be responsible for part of the apoptotic cellular responses.     

Targeted expression of ced-3 and Ice induces programmed cell death in Drosophila

CED-3 is a cysteine protease required for programmed cell death in the nematode, Caenorhabditis elegans, and shares a sequence similarity with mammalian ICE (interleukin-1beta converting enzyme) family proteases. Both CED-3 and ICE family proteases can induce programmed cell death in mammalian cells. Structural and functional similarities between CED-3 and ICE family proteases indicate that the mechanism of cell death is evolutionarily conserved, suggesting the presence of a similar mechanism involving CED-3/ICE-like proteases in Drosophila. Here we determined whether CED-3 or ICE functions to induce programmed cell death in Drosophila. We have generated transformant lines in which ced-3 or Ice is ectopically expressed using the GAL4-UAS system. Expression of CED-3 and ICE can elicit cell death in Drosophila and the cell death was blocked by coexpressing the p35 gene which encodes a viral inhibitor of CED-3/ICE proteases. Results support the idea that the mechanism of programmed cell death controlled by CED-3/ICE is conserved among widely divergent animal species including Drosophila, and the system described provides a tool to dissect cell death mechanism downstream of CED-3/ICE proteases.     

The ICE family of cysteine proteases as effectors of cell death

Programmed cellular suicide follows a set of distinct morphological events involving profound cytoplasmic and nuclear changes. The recent discovery of a family of mammalian homologues of the Caenorhabditis elegans cell death protein CED-3 is now providing insight into how these events might be brought about. These mammalian proteins encode cysteine proteases with homology to the interleukin-1beta converting enzyme (ICE). CED-3 and seven of its currently known mammalian homologues cleave their substrates after an aspartate residue, a property shared only by the cytotoxic T cell (CTL) protease granzyme B which is necessary for the CTL-mediated killing of target cells. A number of proteins previously known to be cleaved in cells undergoing apoptosis have now been shown to be targeted by ICE-like proteases. Although many questions remain, it is becoming increasingly clear that this unique group of proteases play a central effector role in the process of physiological cell death. This article reviews various aspects of the ICE family of proteases.     

A functional role for death proteases in s-Myc- and c-Myc-mediated apoptosis.

Upon activation, cell surface death receptors, Fas/APO-1/CD95 and tumor necrosis factor receptor-1 (TNFR-1), are attached to cytosolic adaptor proteins, which in turn recruit caspase-8 (MACH/FLICE/Mch5) to activate the interleukin-1 beta-converting enzyme (ICE)/CED-3 family protease (caspase) cascade. However, it remains unknown whether these apoptotic proteases are generally involved in apoptosis triggered by other stimuli such as Myc and p53. In this study, we provide lines of evidence that a death protease cascade consisting of caspases and serine proteases plays an essential role in Myc-mediated apoptosis. When Rat-1 fibroblasts stably expressing either s-Myc or c-Myc were induced to undergo apoptosis by serum deprivation, a caspase-3 (CPP32)-like protease activity that cleaves a specific peptide substrate, Ac-DEVD-MCA, appeared in the cell lysates. Induction of s-Myc- and c-Myc-mediated apoptotic cell death was effectively prevented by caspase inhibitors such as Z-Asp-CH2-DCB and Ac-DEVD-CHO. Furthermore, exposing the cells to a serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), also significantly inhibited s-Myc- and c-Myc-mediated apoptosis and the appearance of the caspase-3-like protease activity in vivo. However, AEBSF did not directly inhibit caspase-3-like protease activity in the apoptotic cell lysates in vitro. Together, these results indicate that caspase-3-like proteases play a critical role in both s-Myc- and c-Myc-mediated apoptosis and that caspase-3-like proteases function downstream of the AEBSF-sensitive step in the signaling pathway of Myc-mediated apoptosis.     

Recruitment of apoptotic cysteine proteases (caspases) in influenza virus-induced cell death.

Influenza virus infection induces apoptosis in cultured cells with an augmented expression of Fas (APO-1/CD95). Caspases, a family of cysteine proteases structurally related to interleukin-1-beta-converting enzyme (ICE), play crucial roles in apoptosis induced by various stimuli, including Fas. However, activation of the caspase-cascade seems to be different in various pathways of apoptotic stimuli. We therefore examined the involvement of caspases in influenza virus-induced apoptosis using caspase inhibitors. We found that z-VAD-fmk and z-IETD-fmk effectively inhibited virus-induced apoptosis, whereas Ac-DEVD-CHO and Ac-YVAD-CHO showed partial and little effect on virus-induced cell death, respectively. Consistently, caspase-3-like activity, but not caspase-1-like activity, was increased in the virus-infected cells. The transfection of plasmids encoding viral inhibitors of caspase (v-FLIP or crmA) into HeLa cells inhibited apoptosis by virus infection. The peptide inhibitors of caspases used in this study did not inhibit viral replication. We conclude that influenza virus infection activates some caspases, and that this activation may be downstream of viral replication.     

ICE/CED-3 proteasesin apoptosis

An expanding family of cysteine proteases, of which interleukin-1beta-converting enzyme (ICE) is the prototype, has been shown to play a key role in mammalian cell apoptosis. ICE is both a structural and functional homologue of the nematode 'death gene' ced-3. Here, Moira Whyte discusses how functional characterization of these ICE-like proteases and identification of their substrates is helping to elucidate the biochemical processes underlying the stereotyped morphology of apoptosis and to identify potential targets for therapy.     

Ionizing radiation-induced, Bax-mediated cell death is dependent on activation of cysteine and serine proteases.

Bcl-2 family proteins and interleukin-1-beta converting enzyme/Caenorhabditis elegans cell death gene-3 (ICE/CED-3) family proteases (caspases) represent the basic regulators of apoptosis. However, the precise mechanism by which they interact is unclear. In this study, we found that gamma-radiation-induced apoptosis of leukemia cells was associated with activation of multiple caspases and bax up-regulation. Membrane changes and caspase activities were suppressed by specific caspase inhibitors. Similarly, the serine protease inhibitors z-Ala-Ala-Asp-cmk (AAD) and tosyl-lysine chloromethyl ketone (TLCK) also prevented caspase activation and poly(ADP-ribose) polymerase cleavage in vivo but had no effect on caspase activity in vitro. TLCK also prevented bax up-regulation as a result of its inhibitory effect on p53 function. Inhibitors of caspases and serine proteases partially prevented cell death, suggesting a caspase involvement in Bax-mediated cell death. We propose an ordering of signaling events in Bax-mediated cell death, including steps upstream and downstream of p53 and bax up-regulation.     

Bcl-2 prevents activation of CPP32 cysteine protease and cleavage of poly (ADP-ribose) polymerase and U1-70 kD proteins in staurosporine-mediated apoptosis

Members of the the Bcl-2 and ICE/ced-3 gene families have been implicated as essential components in the control of the cell death pathway. Bcl-2 overexpression can prevent programmed cell death (PCD) in different cell types. ICE/ced-3-like proteases are synthesized as pro-enzymes and are activated by limited proteolysis. When overexpressed in diverse cell types, they trigger PCD. Bcl-2 can inhibit PCD mediated by these proteases, although as yet it is not clear at what specific step in the cell death pathway the protein acts. Here, we demonstrate that CPP32/Yama/Apopain, a member of the ICE/Ced-3 gene family, is processed during staurosporine-induced apoptosis in HeLa cells and that concomitant with CPP32 activation, two other proteins, poly (ADP-ribose) polymerase (PARP) and the U1-70 K small ribonucleoprotein, also undergo proteolysis. Overexpression of Bcl-2 prevents cleavage of CPP32, PARP and U1-70 K and protects HeLa cells from PCD. These results demonstrate that Bcl-2 controls PCD, by acting upstream of CPP32/Yama/Apopain.     

Genetic and metabolic status of NGF-deprived sympathetic neurons saved by an inhibitor of ICE family proteases

Sympathetic neurons undergo programmed cell death (PCD) when deprived of NGF. We used an inhibitor to examine the function of interleukin-1 beta-converting enzyme (ICE) family proteases during sympathetic neuronal death and to assess the metabolic and genetic status of neurons saved by such inhibition. Bocaspartyl(OMe)-fluoromethylketone (BAF), a cell-permeable inhibitor of the ICE family of cysteine proteases, inhibited ICE and CPP32 (IC50 approximately 4 microM) in vitro and blocked Fas-mediated apoptosis in thymocytes (EC50 approximately 10 microM). At similar concentrations, BAF also blocked the NGF deprivation-induced death of rat sympathetic neurons in culture. Compared to NGF-maintained neurons, BAF-saved neurons had markedly smaller somas and maintained only basal levels of protein synthesis; readdition of NGF restored growth and metabolism. Although BAF blocked apoptosis in sympathetic neurons, it did not prevent the fall in protein synthesis or the increase in the expression of c-jun, c- fos, and other mRNAs that occur during neuronal PCD, implying that the ICE-family proteases function downstream of these events during PCD.NGF and BAF rescued sympathetic neurons with an identical time course, suggesting that NGF, in addition to inhibiting metabolic and genetic events associated with neuronal PCD, can act posttranslationally to abort apoptosis at a time point indistinguishable from the activation of cysteine proteases. Both poly-(ADP ribose) polymerase and pro-ICE and Ced-3 homolog-1 (ICH-1) appear to be cleaved in a BAF-inhibitable manner, although the majority of pro-CPP32 appears unchanged, suggesting that ICH-1 is activated during neuronal PCD. Potential implications of these findings for anti-apoptotic therapies are discussed.     

Fas stimulation induces RB dephosphorylation and proteolysis that is blocked by inhibitors of the ICE protease family

Fas antigen is a member of the tumor necrosis factor/nerve growth factor receptor family. Stimulation of Fas by Fas ligand or agonistic antibodies results in the activation of interleukin-1β converting enzyme-like (ICE-like) proteases, and proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). Ultimately, Fas activation leads to apoptotic cell death. The importance of PARP cleavage to the death process remains unclear. We have hypothesized that the cleavage of other cellular substrates may be important for Fas-mediated apoptosis. Here we show that stimulation of Fas results in significant alterations of retinoblastoma protein (RB). Treatment of Jurkat cells, a human leukemic T cell line, with anti-Fas induces dephosphorylation of RB, followed by proteolytic cleavage. These events precede internucleosomal DNA fragmentation. Dephosphorylation and cleavage of RB are inhibited by a specific tetrapeptide inhibitor of ICE-like proteases or by expression of cowpox virus CrmA protein or the Bcl-2 oncoprotein. Inhibition of these RB changes correlates with inhibition of apoptosis. We propose that cleavage of RB may represent an important step in the pathway of Fas-mediated apoptotic cell death. J. Cell. Biochem. 64:586–594. © 1997 Wiley-Liss, Inc.     

Upregulation of Fas-induced apoptosis by interferon-γ accompanied by increased ICE expression in renal cell cancer cells

The integration of Fas/Apo-1 (CD95) by Fas ligand or anti-Fas antibody induces apoptosis, and this system plays a pivotal role for the lysis of target cells by cytotoxic T lymphocytes. Fas-mediated apoptosis is also increased by a prior incubation of Fas-bearing cells with interferon(IFN)-. Interleukin-1- converting enzyme (ICE) and/or CPP32, or other members of ICE family act as direct cell death executors downstream of this mechanism, and a tetrapeptide inhibitor of these cysteine proteases blocks Fas-mediated apoptosis. In this study, we examined the effect of IFN- on Fas-mediated apoptosis in ACHN cells. IFN- augmented apoptosis in a dose dependent manner and reached a plateau at 400 U/ml when exposed for 48 h before the end of culture. The kinetics revealed a significant increase in apoptosis after 24 h. Exposing ACHN cells to IFN- increased pro-ICE expression accompanied with a decrease of pro-CPP32. These results suggest that direct enhancement of ICE expression and/or upregulation of conversion of pro-CPP32 to active form increases Fas-mediated apoptosis by IFN- in ACHN cells.     

The cytotoxic cell protease granzyme B initiates apoptosis in a cell-free system by proteolytic processing and activation of the ICE/CED-3 family protease, CPP32, via a novel two-step mechanism.

The major mechanism of cytotoxic lymphocyte killing involves the directed release of granules containing perforin and a number of proteases onto the target cell membrane. One of these proteases, granzyme B, has an unusual substrate site preference for Asp residues, a property that it shares with members of the emerging interleukin-1beta-converting enzyme (ICE)/CED-3 family of proteases. Here we show that granzyme B is sufficient to reproduce rapidly all of the key features of apoptosis, including the degradation of several protein substrates, when introduced into Jurkat cell-free extracts. Granzyme B-induced apoptosis was neutralized by a tetrapeptide inhibitor of the ICE/CED-3 family protease, CPP32, whereas a similar inhibitor of ICE had no effect. Granzyme B was found to convert CPP32, but not ICE, to its active form by cleaving between the large and small subunits of the CPP32 proenzyme, resulting in removal of the prodomain via an autocatalytic step. The cowpox virus protein CrmA, a known inhibitor of ICE family proteases as well as granzyme B, inhibited granzyme B-mediated CPP32 processing and apoptosis. These data demonstrate that CPP32 activation is a key event during apoptosis initiated by granzyme B.     

Caspase inhibition prevents staurosporine-induced apoptosis in CHO-K1 cells

Apoptosis is a distinct form of programmed cell death that plays an important role in many biological processes.Although the phenotypes of apoptotic cells are well documented, little is known of the central mechanismleading to programmed cell death. Over the past few years, a number of ICE/CED-3 family proteases(also termed caspases) have been discovered and implicated as the common effectors of apoptosis. Inthis report, we demonstrate that induction of apoptosis in CHO-K1 cells by staurosporine, a broad spectruminhibitor of protein kinases, results in an increase in DEVD-dependent protease activity. These events werefollowed by nuclear DNA fragmentation and cell death. Inhibition of the DEVD-cleaving activity by a synthetictetrapeptide inhibitor DEVD-CHO, blocked staurosporine-induced downstream apoptotic phenotypes, suchas morphological characteristics and DNA fragmentation. These results suggest that staurosporine-inducedapoptosis in CHO-K1 cells is mediated through the CPP32/caspase-3-like cysteine proteases.     

Activation of the c-Jun N-terminal kinase/stress-activated protein kinase pathway by overexpression of caspase-8 and its homologs.

Caspase-8 is the most proximal caspase in the caspase cascade and possesses a prodomain consisting of two homologous death effector domains (DEDs). We have discovered that caspase-8 and its homologs can physically interact with tumor necrosis factor receptor-associated factor family members and activate the c-Jun N-terminal kinase (JNK, or stress-activated protein kinase) pathway. This ability resides in the DED-containing prodomain of these proteins and is independent of their role as cell death proteases. A point mutant in the first DED of caspase-8 can block JNK activation induced by several death domain receptors. Inhibition of JNK activation blocks apoptosis mediated by caspase-10, Mach-related inducer of toxicity/cFLIP, and Fas/CD95, thereby suggesting a cooperative role of this pathway in the mediation of caspase-induced apoptosis.     

Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons.

Sympathetic neurons undergo programmed cell death (PCD) upon deprivation of nerve growth factor (NGF). PCD of neurons is blocked by inhibitors of the interleukin-1beta converting enzyme (ICE)/Ced-3-like cysteine protease, indicating involvement of this class of proteases in the cell death programme. Here we demonstrate that the proteolytic activities of the proteasome are also essential in PCD of neurons. Nanomolar concentrations of several proteasome inhibitors, including the highly selective inhibitor lactacystin, not only prolonged survival of NGF-deprived neurons but also prevented processing of poly(ADP-ribose) polymerase which is known to be cleaved by an ICE/Ced-3 family member during PCD. These results demonstrate that the proteasome is a key regulator of neuronal PCD and that, within this process, it is involved upstream of proteases of the ICE/Ced-3 family. This order of events was confirmed in macrophages where lactacystin inhibited the proteolytic activation of precursor ICE and the subsequent generation of active interleukin-1beta.     

Evidence for an interleukin-1β converting enzyme (ICE)-like activity distinct from ICE and responsible for ICE and CPP32 cleavage during apoptosis

IL-1 converting enzyme (ICE) and ICE-related proteases (IRPs) have been suggested to play a central role in apoptosis. We report the use of peptidic ICE inhibitors to reassess the role of this enzyme in the apoptosis induced by Fas or TNF receptor ligation in Jurkat cells, U937 cells or monocytes. Our results show that inhibition of IL-1 processing can be dissociated from inhibition of apoptosis. Indeed, two out of three com-pounds active on ICE are not inhibitory for apoptosis. This shows that ICE is not required for progression in the apoptotic pathway, but that one or several IRPs are necessary. In addition, Western blot analysis of cell lysates shows that both ICE and CPP32 precursors disappear rapidly after apoptosis induction, while ICH-1L precursor remains intact. Concomitant appearance of cleavage products can be visualized for CPP32, but not for ICE, suggesting that the former is proteolytically activated. In addition, this precursor cleavage can be blocked by an ICE inhibitor active on apoptosis. Altogether, our data support the hypothesis that one or several IRPs are necessary for apoptosis and are responsible for ICE and CPP32 cleavage during this process.     

Acute lung injury and cell death: how many ways can cells die?

Apoptosis has been considered as an underlying mechanism in acute lung injury/acute respiratory distress syndrome and multiorgan dysfunction syndrome. Recently, several alternative pathways for cell death (such as caspase-independent cell death, oncosis, and autophagy) have been discovered. Evidence of these pathways in the pathogenesis of acute lung injury has also come into light. In this article, we briefly introduce cell death pathways and then focus on studies related to lung injury. The different types of cell death that occur and the underlying mechanisms utilized depend on both experimental and clinical conditions. Lipopolysaccharide-induced acute lung injury is associated with apoptosis via Fas/Fas ligand mechanisms. Hyperoxia and ischemia-reperfusion injury generate reactive oxidative species, which induce complex cell death patterns composed of apoptosis, oncosis, and necrosis. Prolonged overexpression of inflammatory mediators results in increased production and activation of proteases, especially cathepsins. Activation and resistance to death of neutrophils also plays an important role in promoting parenchymal cell death. Knowledge of the coexisting multiple cell death pathways and awareness of the pharmacological inhibitors targeting different proteases critical to cell death may lead to the development of novel therapies for acute lung injury.     

Prevention of apoptosis in CNTF-dependent neurons by a mutant ICE and by viral protein CrmA but not by proto-oncogene product Bcl-2

The interleukin-1beta converting enzyme (ICE) gene family, (homologues of C. elegans cell death gene product Ced-3) plays an important role in controlling programmed cell death. Nerve growth factor (NGF) promotes survival of cultured embryonic chicken dorsal root ganglion neurons. Ciliary ganglion neurons depend exclusively on ciliary neurotrophic factor (CNTF) for survival. Complete depletion of NGF or CNTF from culture medium induces apoptosis in both types of neurons. We can prevent apoptosis, due either to NGF or CNTF withdrawal and in either type of neuron, by overexpression of a mutant inactive ICE and an ICE inhibitor, the product of cowpox virus gene crmA. Bcl-2 does not prevent apoptosis in CNTF-dependent ciliary neurons or DRG neurons as it does in NGF-dependent neurons. These results suggest that neuronal cell death is mediated through a common effector mechanism involving the Ice family of genes, whereas different suppression mechanisms are engaged depending upon the specific neurotrophic factors present.     

Caspase-3/CPP32-like activity is not sufficient to mediate apoptosis in an IL-2 dependent T cell line

CTLL cells undergo apoptosis when cultured in the absence of IL-2. The IL-1-converting-enzyme (ICE)/ caspase family has been implicated as an integral component of some forms of apoptosis. Numerous members of the caspase family have been identified, and it appears as if caspase-3/CPP32 plays a critical role. Previously we demonstrated that ICE/caspase-1 expression increases in CTLL cells during apoptosis; however, inhibition of ICE activity did not abrogate apoptotic death. The purpose of this report is to determine if other members of the caspase family are involved in T cell apoptosis induced by growth factor starvation. We show that cytosolic CPP32-like activity, as measured by the cleavage of DEVD-pNA and poly(ADP-ribose) polymerase (PARP), increases during apoptosis following growth factor deprivation. Cytosolic CPP32-like activity is inhibited in cells treated with the broad spectrum ICE family inhibitor boc-aspartyl(OMe)-fluoromethylketone (D-FMK) and by VAD-FMK and DEVD-FMK which have greater specificity for CPP32-like ICE homologs; however, only the broad spectrum ICE inhibitor D-FMK inhibited apoptosis. Our results suggest that apoptosis induced by growth factor deprivation involves the caspase family, but increased CPP32-like activity is not sufficient to mediate apoptosis induced by IL-2 starvation.