Bistability analyses of a caspase activation model for receptor-induced apoptosis

Apoptosis is an important physiological process crucially involved in development and homeostasis of multicellular organisms. Although the major signaling pathways have been unraveled, a detailed mechanistic understanding of the complex underlying network remains elusive. We have translated here the current knowledge of the molecular mechanisms of the death-receptor-activated caspase cascade into a mathematical model. A reduction down to the apoptotic core machinery enables the application of analytical mathematical methods to evaluate the system behavior within a wide range of parameters. Using parameter values from the literature, the model reveals an unstable status of survival indicating the need for further control. Based on recent publications we tested one additional regulatory mechanism at the level of initiator caspase activation and demonstrated that the resulting system displays desired characteristics such as bistability. In addition, the results from our model studies allowed us to reconcile the fast kinetics of caspase 3 activation observed at the single cell level with the much slower kinetics found at the level of a cell population.     

Construction and analysis of a modular model of caspase activation in apoptosis

Background  

A key physiological mechanism employed by multicellular organisms is apoptosis, or programmed cell death. Apoptosis is triggered by the activation of caspases in response to both extracellular (extrinsic) and intracellular (intrinsic) signals. The extrinsic and intrinsic pathways are characterized by the formation of the death-inducing signaling complex (DISC) and the apoptosome, respectively; both the DISC and the apoptosome are oligomers with complex formation dynamics. Additionally, the extrinsic and intrinsic pathways are coupled through the mitochondrial apoptosis-induced channel via the Bcl-2 family of proteins.     

Fibrillogenic amylin evokes islet beta-cell apoptosis through linked activation of a caspase cascade and JNK1

Fibrillogenic human amylin elicits pancreatic beta-cell apoptosis that may contribute to development of type-2 diabetes. Here, we demonstrated that activation of a caspase cascade is necessary for induction of apoptosis by fibrillogenic amylin variants in two pancreatic beta-cell lines. Human amylin, as well as truncated 8-37human amylin, evoked sequential activation of caspases-8 and -3, and apoptosis, whereas non-beta-sheet forming and non-fibrillogenic homologs, such as [25,28,29triprolyl]human amylin, did not, implying that the beta-sheet conformer is required for human amylin-induced caspase activation. Significant inhibition of apoptosis was evoked by a selective caspase-1 inhibitor, indicating that caspase-1 is also essential for activation of the caspase cascade. Furthermore, we showed that specific jnk1 antisense oligonucleotides, which suppress phospho-JNK1 expression, effectively decreased human amylin-induced activation of c-Jun. Studies of the interplay between the caspase cascade and the JNK pathway showed that both apoptosis and caspase-3 activation were suppressed by treatment with a JNK inhibitor and by transfection of antisense jnk1 oligonucleotides or antisense-c-jun, whereas a selective inhibitor of caspases-1 and -3 prevented apoptosis but not c-Jun activation. Thus, the JNK1 activation preceded activation of caspases-1 and -3. However, selective JNK inhibition had no effect on caspase-8 activation, and selective caspase-8 inhibition only partially suppressed apoptosis and c-Jun activation, indicating that caspase-8 may partially act upstream of the JNK pathway. Our studies demonstrate a functional interaction of a caspase cascade and JNK1. Fibrillogenic amylin can evoke a JNK1-mediated apoptotic pathway, which is partially dependent and partially independent of caspase-8, and in which caspase-3 acts as a common downstream effector.     

Execution of macrophage apoptosis by Mycobacterium avium through apoptosis signal-regulating kinase 1/p38 mitogen-activated protein kinase signaling and caspase 8 activation

Macrophage apoptosis is an important component of the innate immune defense machinery (against pathogenic mycobacteria) responsible for limiting bacillary viability. However, little is known about the mechanism of how apoptosis is executed in mycobacteria-infected macrophages. Apoptosis signal-regulating kinase 1 (ASK1) was activated in Mycobacterium avium-treated macrophages and in turn activated p38 mitogen-activated protein (MAP) kinase. M. avium-induced macrophage cell death could be blocked in cells transfected with a catalytically inactive mutant of ASK1 or with dominant negative p38 MAP kinase arguing in favor of a central role of ASK1/p38 MAP kinase signaling in apoptosis of macrophages challenged with M. avium. ASK1/p38 MAP kinase signaling was linked to the activation of caspase 8. At the same time, M. avium triggered caspase 8 activation, and cell death occurred in a Fas-associated death domain (FADD)-dependent manner. The death signal induced upon caspase 8 activation linked to mitochondrial death signaling through the formation of truncated Bid (t-Bid), its translocation to the mitochondria and release of cytochrome c. Caspase 8 inhibitor (z-IETD-FMK) could block the release of cytochrome c as well as the activation of caspases 9 and 3. The final steps of apoptosis probably involved caspases 9 and 3, since inhibitors of both caspases could block cell death. Of foremost interest in the present study was the finding that ASK1/p38 signaling was essential for caspase 8 activation linked to M. avium-induced death signaling. This work provides the first elucidation of a signaling pathway in which ASK1 plays a central role in innate immunity.     

Lupeol induces p53 and cyclin-B-mediated G2/M arrest and targets apoptosis through activation of caspase in mouse skin

Lupeol, present in fruits and medicinal plants, is a biologically active compound that has been shown to have various pharmacological properties in experimental studies. In the present study, we demonstrated the modulatory effect of lupeol on 7,12-dimethylbenz[a]anthracene (DMBA)-induced alterations on cell proliferation in the skin of Swiss albino mice. Lupeol treatment showed significant (p < 0.05) preventive effects with marked inhibition at 48, 72, and 96 h against DMBA-mediated neoplastic events. Cell-cycle analysis showed that lupeol-induced G2/M-phase arrest (16-37%) until 72 h, and these inhibitory effects were mediated through inhibition of the cyclin-B-regulated signaling pathway involving p53, p21/WAF1, cdc25C, cdc2, and cyclin-B gene expression. Further lupeol-induced apoptosis was observed, as shown by an increased sub-G1 peak (28%) at 96 h, with upregulation of bax and caspase-3 genes and downregulation of anti-apoptotic bcl-2 and survivin genes. Thus, our results indicate that lupeol has novel anti-proliferative and apoptotic potential that may be helpful in designing strategies to fight skin cancer.     

Mechanisms of caspase activation and inhibition during apoptosis

Caspases are central components of the machinery responsible for apoptosis. Recent structural and biochemical studies on procaspases, IAPs, Smac/DIABLO, and apoptosome have revealed a conserved mechanism of caspase activation and inhibition. This article reviews these latest advances and presents our current understanding of caspase regulation during apoptosis.     

Mechanisms of caspase activation and inhibition during apoptosis

Apoptosis is primarily executed by active caspases, which are derived from the inactive procaspase zymogens through proteolytic cleavage. We determined the crystal structures of a caspase zymogen, procaspase-7, and an active caspase-7 without any bound inhibitors. Compared to the inhibitor-bound caspase-7, procaspase-7 zymogen exhibits significant structural differences surrounding the catalytic cleft, which precludes the formation of a productive conformation. Proteolytic cleavage in between the large and small subunits allows rearrangement of essential loops in the active site, priming active caspase-7 for inhibitor/substrate binding. Strikingly, binding by inhibitors causes a 180-degree-flipping of the N-terminus in the small subunit, which interacts with and stabilizes the catalytic cleft. These analyses reveal the structural mechanisms of caspase activation and demonstrate that the inhibitor/substrate binding is a process of     

E1A sensitizes cancer cells to TRAIL-induced apoptosis through enhancement of caspase activation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis of cancer cells. Sensitization of cancer cells to TRAIL, particularly TRAIL-resistant cancer cells, could improve the effectiveness of TRAIL as an anticancer agent. The adenovirus type 5 E1A that associates with anticancer activities including sensitization to apoptosis induced by tumor necrosis factor is currently being tested in clinical trials. In this study, we investigated the sensitivity to TRAIL in the E1A transfectants ip1-E1A2 and 231-E1A cells and the parental TRAIL-resistant human ovarian cancer SKOV3.ip1 and TRAIL-sensitive human breast cancer MDA-MB-231 cells. The results indicated that the percentage of TRAIL-induced apoptotic cells was significantly higher in the E1A transfectants of both cell lines than it was in the parental cell lines. To further investigate the cellular mechanism of this effect, we found that E1A enhances TRAIL-induced activation of caspase-8, caspase-9, and caspase-3. Inhibition of caspase-3 activity by a specific inhibitor, Z-DEVD-fmk, abolished TRAIL-induced apoptosis. In addition, E1A enhanced TRAIL expression in ip1-E1A2 cells, but not in 231-E1A cells, and the anti-TRAIL neutralizing antibody N2B2 blocked the E1A-mediated bystander effect in vitro. Taken together, these results suggest that E1A sensitizes both TRAIL-sensitive and TRAIL-resistant cancer cells to TRAIL-induced apoptosis, which occurs through the enhancement of caspase activation; activation of caspase-3 is required for TRAIL-induced apoptosis; and E1A-induced TRAIL expression is involved in the E1A-mediated bystander effect. Combination of E1A and TRAIL could be an effective treatment for cancer.     

Chrysin-induced apoptosis is mediated through caspase activation and Akt inactivation in U937 leukemia cells

Chrysin is a natural, biologically active compound extracted from many plants, honey, and propolis. It possesses potent anti-inflammation, anti-cancer, and anti-oxidation properties. The mechanism by which chrysin initiates apoptosis remains poorly understood. In the present report, we investigated the effect of chrysin on the apoptotic pathway in U937 human promonocytic cells. We show that chrysin induces apoptosis in association with the activation of caspase 3 and that Akt signal pathway plays a crucial role in chrysin-induced apoptosis in U937 cells. Furthermore, we have shown that inhibition of Akt phosphorylation in U937 cells by the specific PI3K inhibitor, LY294002 significantly, enhanced apoptosis. Overexpression of a constitutively active Akt (myr-Akt) in U937 cells inhibited the induction of apoptosis, activation of caspase 3, and PLC-gamma1 cleavage by chrysin. Together, these findings suggest that the Akt pathway plays a major role in regulating the apoptotic response of human leukemia cells to chrysin and raise the possibility that combined interruption of chrysin and PI3K/Akt-related pathways may represent a novel therapeutic strategy in hematological malignancies.     

MST, a physiological caspase substrate, highly sensitizes apoptosis both upstream and downstream of caspase activation

The human serine/threonine kinase, mammalian STE20-like kinase (MST), is considerably homologous to the budding yeast kinases, SPS1 and STE20, throughout their kinase domains. The cellular function and physiological activation mechanism of MST is unknown except for the proteolytic cleavage-induced activation in apoptosis. In this study, we show that MST1 and MST2 are direct substrates of caspase-3 both in vivo and in vitro. cDNA cloning of MST homologues in mouse and nematode shows that caspase-cleaved sequences are evolutionarily conserved. Human MST1 has two caspase-cleavable sites, which generate biochemically distinct catalytic fragments. Staurosporine activates MST either caspase-dependently or independently, whereas Fas ligation activates it only caspase-dependently. Immunohistochemical analysis reveals that MST is localized in the cytoplasm. During Fas-mediated apoptosis, cleaved MST translocates into the nucleus before nuclear fragmentation is initiated, suggesting it functions in the nucleus. Transiently expressed MST1 induces striking morphological changes characteristic of apoptosis in both nucleus and cytoplasm, which is independent of caspase activation. Furthermore, when stably expressed in HeLa cells, MST highly sensitizes the cells to death receptor-mediated apoptosis by accelerating caspase-3 activation. These findings suggest that MST1 and MST2 play a role in apoptosis both upstream and downstream of caspase activation.     

Molecular morphology of neuronal apoptosis: Analysis of caspase 3 activation during postnatal development of mouse cerebellar cortex

We have used the mammalian post-natal cerebellar cortex as a model to dissect out the molecular morphology of neuronal apoptosis in a well-defined population of central neurons: the cerebellar granule cells. By immunocytochemistry, in situ labeling of apoptotic cells, and analysis of cerebellar slices following particle-mediated gene transfer (biolistics), we have studied the relationship of cell death and cleavage of caspase 3, a key molecule in the execution of apoptosis, and monitored caspase 3 activation in living cells. Our results demonstrate the existence of caspase dependent and independent apoptotic pathways affecting the cerebellar granule cells at different stages of their life. Apoptosis of proliferating precursors and young pre-migratory cells occurs in the absence of caspase 3 cleavage, whereas cell death of post-mitotic post-migratory neurons is directly linked to caspase 3 activation. Data obtained from cerebellar cortex can be generalized to outline a more comprehensive picture of the cellular and molecular mechanisms of neuronal death not only in development, but also in a number of pathological conditions leading to neuronal loss.     

Adeno-associated virus type 2 Rep78 induces apoptosis through caspase activation independently of p53

Adeno-associated virus (AAV) type 2 Rep78 is a multifunctional protein required for AAV DNA replication, integration, and gene regulation. The biochemical activities of Rep78 have been described, but the effects of Rep proteins on the cell have not been characterized. We have analyzed Rep-mediated cytotoxicity. We demonstrated that Rep78 expression is sufficient to induce cell death and disruption of the cell cycle. Cell death was found to be mediated by apoptosis. Rep78 expression resulted in the activation of caspase-3, a terminal caspase directly involved in the execution of cell death. A peptidic inhibitor of caspase-3, Z-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK), abrogated Rep78-induced apoptosis, indicating that Rep78-mediated apoptosis is caspase-3 dependent. Rep78 induced apoptosis in wild-type p53-containing human embryonal carcinoma NT-2 cells and in p53-null promyelocytic human HL-60 cells, indicating that at least one pathway of Rep78-induced apoptosis is p53 independent. Apoptosis was shown to occur during the G(1) and early S phases of the cell cycle. By analyzing the effects of Rep78 mutations on cell viability, the cause of cell death was attributed in part to two biochemical activities of Rep78, DNA binding and ATPase/helicase activity. The endonuclease activity of Rep78 did not contribute to apoptosis induction.     

ExoS of Pseudomonas aeruginosa induces apoptosis through a Fas receptor/caspase 8-independent pathway in HeLa cells

Pseudomonas aeruginosa infection is a serious complication in immunocompromised individuals and in patients with cystic fibrosis. We have previously shown that the type III secreted effector ExoS triggers apoptosis in various cultured cell lines via its ADP-ribosyltransferase (ADPRT) activity. The apoptosis process was further shown to involve intrinsic signalling pathway requiring c-Jun N-terminal kinase (JNK)-initiated mitochondrial pathway. In the present study, we investigated the role of Fas pathway activation in P. aeruginosa-induced apoptosis. P. aeruginosa infection resulted in caspase 8 cleavage in HeLa cells, which was inhibited by overexpression of a dominant negative version of Fas-associated death domain (FADD), suggesting that Fas pathway was activated. In fact, confocal laser scanning microscopy showed that P. aeruginosa induced clustering of FasR. In addition, the ADPRT activity of the ExoS was required for the induction of FasR clustering and caspase 8 cleavage. However, blocking the FasR-FasL interaction by antagonistic antibodies to FasR or to FasL had no effect on P. aeruginosa-induced caspase 8 and caspase 3 activation, neither did the silencing of FasR by small interfering RNA (siRNA), suggesting that caspase 8 activation through the FADD bypasses FasR/FasL-mediated signalling. Thus, FADD-mediated caspase 8 activation involves intracellular ExoS in an ADPRT-dependent manner. Furthermore, silencing of caspase 8 by siRNA did not interfere with P. aeruginosa-induced apoptosis, whereas it rendered HeLa cells markedly increased resistance towards FasL-induced apoptosis. In conclusion, our findings indicate that ExoS of P. aeruginosa induces apoptosis through a mechanism that is independent of Fas receptor/caspase 8 pathway.     

TRAF6 regulates cell fate decisions by inducing caspase 8-dependent apoptosis and the activation of NF-kappaB

Tumor necrosis factor receptor-associated factor 6 (TRAF6) functions as an adaptor, positively regulating the NF-kappaB pathway. Here we report a new function of human TRAF6, the direct stimulation of apoptosis. The mechanism of apoptosis induction results from the capacity of human TRAF6 to interact and activate caspase 8. Both the C-terminal TRAF domain of human TRAF6, which directly interacts with the death effector domain of pro-caspase 8, and the N-terminal RING domain, which is required for activation of caspase 8, are necessary for the induction of apoptosis. The role of endogenous TRAF6 in regulating apoptosis was confirmed by extinguishing TRAF6 expression with specific small-hairpin RNA that resulted in diminished spontaneous apoptosis and resistance to induced apoptosis. In contrast to the human molecule, murine TRAF6 displayed less ability to induce apoptosis and a greater capacity to stimulate NF-kappaB activity. Human and murine TRAF6 are similar except in the region between zinc finger 5 and the TRAF domains. Reciprocal transfer of this connecting region completely exchanged the ability of human and murine TRAF6 to induce apoptosis and activate NF-kappaB. Unique regions of TRAF6 therefore play an important role in determining cell fate.     

ER stress-mediated apoptosis in a new mouse model of osteogenesis imperfecta

Osteogenesis imperfecta is an inherited disorder characterized by increased bone fragility, fractures, and osteoporosis, and most cases are caused by mutations affecting the type I collagen genes. Here, we describe a new mouse model for Osteogenesis imperfecta termed Aga2 (abnormal gait 2) that was isolated from the Munich N-ethyl-N-nitrosourea mutagenesis program and exhibited phenotypic variability, including reduced bone mass, multiple fractures, and early lethality. The causal gene was mapped to Chromosome 11 by linkage analysis, and a C-terminal frameshift mutation was identified in the Col1a1 (procollagen type I, alpha 1) gene as the cause of the disorder. Aga2 heterozygous animals had markedly increased bone turnover and a disrupted native collagen network. Further studies showed that abnormal proα1(I) chains accumulated intracellularly in Aga2/+ dermal fibroblasts and were poorly secreted extracellularly. This was associated with the induction of an endoplasmic reticulum stress-specific unfolded protein response involving upregulation of BiP, Hsp47, and Gadd153 with caspases-12 and −3 activation and apoptosis of osteoblasts both in vitro and in vivo. These studies resulted in the identification of a new model for Osteogenesis imperfecta, and identified a role for intracellular modulation of the endoplasmic reticulum stress-associated unfolded protein response machinery toward osteoblast apoptosis during the pathogenesis of disease.     

Effective caspase inhibition blocks neutrophil apoptosis and reveals Mcl-1 as both a regulator and a target of neutrophil caspase activation

Human tissue inflammation is terminated, at least in part, by the death of inflammatory neutrophils by apoptosis. The regulation of this process is therefore key to understanding and manipulating inflammation resolution. Previous data have suggested that the short-lived pro-survival Bcl-2 family protein, Mcl-1, is instrumental in determining neutrophil lifespan. However, Mcl-1 can be cleaved following caspase activity, and the possibility therefore remains that the observed fall in Mcl-1 levels is due to caspase activity downstream of caspase activation, rather than being a key event initiating apoptosis in human neutrophils.We demonstrate that apoptosis in highly purified neutrophils can be almost completely abrogated by caspase inhibition with the highly effective di-peptide caspase inhibitor, Q-VD.OPh, confirming the caspase dependence of neutrophil apoptosis. Effective caspase inhibition does not prevent the observed fall in Mcl-1 levels early in ultrapure neutrophil culture, suggesting that this fall in Mcl-1 levels is not a consequence of neutrophil apoptosis. However, at later timepoints, declines in Mcl-1 can be reversed with effective caspase inhibition, suggesting that Mcl-1 is both an upstream regulator and a downstream target of caspase activity in human neutrophils.     

Regulation of caspase activation in apoptosis: implications for transformation and drug resistance

Recent developments in the apoptosis field have uncovered a family of cysteine proteases, the Caspases, that act as signalling components as well as effectors of the cell death machinery. Caspases are constitutively present as inactive precursors within most cells and undergo proteolytic processing in response to diverse death-inducing stimuli to initiate the death programme. Active caspases can process other caspases of the same type as well as process caspases further downstream in the pathway that ultimately leads to collapse of the cell. This cellular collapse is thought to occur as a consequence of caspase-mediated cleavage of a diverse array of cellular substrates. Regulation of entry into the death programme is controlled at a number of levels by members of the Bcl-2 family, as well as by other cell death regulatory proteins. Recent data has shed light upon the mechanism of action of these regulatory molecules and suggests that the point of caspase activation is a major checkpoint in the cell death programme. Because many transformed cell populations possess derangements in cell death-regulatory genes, such as bcl-2, such cells frequently exhibit elevated resistance to cytotoxic chemotherapy. Thus, a deeper understanding of how apoptosis is normally regulated has therapeutic implications for disease states where the normal controls on the cell death machinery have been subverted. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cryopreservation and thawing of hematopoietic stem cell CD34-induced apoptosis through caspase pathway activation: Key role of granulocytes

IntroductionCell damage inescapably occurs during both the freezing and the thawing graft processes for autologous hematopoietic stem cell (HSC) transplantation. To estimate HSC injury, a quality control is performed including: (i) CD34⁺ quantification; (ii) percentage of CD34⁺ viability and (iii) evaluation of HSC functional ability to form colony forming unit–granulocyte macrophage (CFU-GM). Apoptosis involves complex pathways such as caspase enzymes. Here, we assess the extent of apoptosis that is caspase-dependent before and after cryoconservation of CD34⁺, using a Fluorescent Labeled Inhibitor of CAspases (FLICA).MethodsCaspase pathway activation status was evaluated in 46 patients (multiple myeloma [n = 24], lymphoma [n = 22]), by flow cytometry, using a 7-aminoactinomycin-D (7AAD)/FLICA staining test, in CD34⁺, CD3⁺, CD14⁺ and CD56⁺ cells. Viable 7AAD^?/FLICA⁺ cells were then correlated with various parameters.ResultsWe showed a significant caspase pathway activation, with 23% CD34⁺/7AAD^?/FLICA⁺ cells after thawing, compared with the 2% described in fresh CD34⁺ cells (P < 0.0001). Moreover, caspase pathway was significantly activated in thawing CD3⁺, CD56⁺ and CD14⁺ cells. We also report a significant correlation between the rate of CD34⁺/7AAD^?/FLICA⁺ cells and post-thawing granulocytes count (P = 0.042) and their potential to be differentiated into CFU-GM (P = 0.004).DiscussionOur results show substantial cell death, induced by the increase of caspase pathway activation, secondary to the thawing process, and across all study cell types. This observation may affect the immune response quality during recipient aplasia, without detecting a clinical impact. Moreover, caspase pathway activation through CD3⁺ and CD56⁺ subpopulations could modify the therapeutic result of donor lymphocytes infusion (DLI).