Search tips
Search criteria

Results 1-25 (1193453)

Clipboard (0)

Related Articles

1.  Both the Caspase CSP-1 and a Caspase-Independent Pathway Promote Programmed Cell Death in Parallel to the Canonical Pathway for Apoptosis in Caenorhabditis elegans 
PLoS Genetics  2013;9(3):e1003341.
Caspases are cysteine proteases that can drive apoptosis in metazoans and have critical functions in the elimination of cells during development, the maintenance of tissue homeostasis, and responses to cellular damage. Although a growing body of research suggests that programmed cell death can occur in the absence of caspases, mammalian studies of caspase-independent apoptosis are confounded by the existence of at least seven caspase homologs that can function redundantly to promote cell death. Caspase-independent programmed cell death is also thought to occur in the invertebrate nematode Caenorhabditis elegans. The C. elegans genome contains four caspase genes (ced-3, csp-1, csp-2, and csp-3), of which only ced-3 has been demonstrated to promote apoptosis. Here, we show that CSP-1 is a pro-apoptotic caspase that promotes programmed cell death in a subset of cells fated to die during C. elegans embryogenesis. csp-1 is expressed robustly in late pachytene nuclei of the germline and is required maternally for its role in embryonic programmed cell deaths. Unlike CED-3, CSP-1 is not regulated by the APAF-1 homolog CED-4 or the BCL-2 homolog CED-9, revealing that csp-1 functions independently of the canonical genetic pathway for apoptosis. Previously we demonstrated that embryos lacking all four caspases can eliminate cells through an extrusion mechanism and that these cells are apoptotic. Extruded cells differ from cells that normally undergo programmed cell death not only by being extruded but also by not being engulfed by neighboring cells. In this study, we identify in csp-3; csp-1; csp-2 ced-3 quadruple mutants apoptotic cell corpses that fully resemble wild-type cell corpses: these caspase-deficient cell corpses are morphologically apoptotic, are not extruded, and are internalized by engulfing cells. We conclude that both caspase-dependent and caspase-independent pathways promote apoptotic programmed cell death and the phagocytosis of cell corpses in parallel to the canonical apoptosis pathway involving CED-3 activation.
Author Summary
Caspases are cysteine proteases that in many cases drive apoptosis, an evolutionarily conserved and highly stereotyped form of cellular suicide with functions in animal development and tissue maintenance. The dysregulation of apoptosis can contribute to diseases as diverse as cancer, autoimmunity, and neurodegeneration. Caspases are often thought to be required for, or even to define, apoptosis. Although there is evidence that apoptosis can occur in the absence of caspase activity, caspase-independence can be difficult to prove, as most animals have multiple caspases. The nematode Caenorhabditis elegans has four caspases, CED-3, CSP-1, CSP-2, and CSP-3. CED-3 has a well-established role in apoptosis, but less is known about the functions of the CSP caspases. In this study, we show that CSP-1 promotes apoptosis in the developing C. elegans embryo and that CSP-1 is regulated differently than its homolog CED-3. Furthermore, we show that apoptosis and the engulfment of dying cells can occur in mutants lacking all four caspases, proving that neither apoptosis nor cell-corpse engulfment require caspase function and that caspase-independent activities can contribute to apoptosis of some cells during animal development.
PMCID: PMC3591282  PMID: 23505386
2.  Caspase-2 Maintains Bone Homeostasis by Inducing Apoptosis of Oxidatively-Damaged Osteoclasts 
PLoS ONE  2014;9(4):e93696.
Osteoporosis is a silent disease, characterized by a porous bone micro-structure that enhances risk for fractures and associated disabilities. Senile, or age-related osteoporosis (SO), affects both men and women, resulting in increased morbidity and mortality. However, cellular and molecular mechanisms underlying senile osteoporosis are not fully known. Recent studies implicate the accumulation of reactive oxygen species (ROS) and increased oxidative stress as key factors in SO. Herein, we show that loss of caspase-2, a cysteine aspartate protease involved in oxidative stress-induced apoptosis, results in total body and femoral bone loss in aged mice (20% decrease in bone mineral density), and an increase in bone fragility (30% decrease in fracture strength). Importantly, we demonstrate that genetic ablation or selective inhibition of caspase-2 using zVDVAD-fmk results in increased numbers of bone-resorbing osteoclasts and enhanced tartrate-resistant acid phosphatase (TRAP) activity. Conversely, transfection of osteoclast precursors with wild type caspase-2 but not an enzymatic mutant, results in a decrease in TRAP activity. We demonstrate that caspase-2 expression is induced in osteoclasts treated with oxidants such as hydrogen peroxide and that loss of caspase-2 enhances resistance to oxidants, as measured by TRAP activity, and decreases oxidative stress-induced apoptosis of osteoclasts. Moreover, oxidative stress, quantified by assessment of the lipid peroxidation marker, 4-HNE, is increased in Casp2-/- bone, perhaps due to a decrease in antioxidant enzymes such as SOD2. Taken together, our data point to a critical and novel role for caspase-2 in maintaining bone homeostasis by modulating ROS levels and osteoclast apoptosis during conditions of enhanced oxidative stress that occur during aging.
PMCID: PMC3972236  PMID: 24691516
3.  The Enigmatic Roles of Caspases in Tumor Development 
Cancers  2010;2(4):1952-1979.
One function ascribed to apoptosis is the suicidal destruction of potentially harmful cells, such as cancerous cells. Hence, their growth depends on evasion of apoptosis, which is considered as one of the hallmarks of cancer. Apoptosis is ultimately carried out by the sequential activation of initiator and executioner caspases, which constitute a family of intracellular proteases involved in dismantling the cell in an ordered fashion. In cancer, therefore, one would anticipate caspases to be frequently rendered inactive, either by gene silencing or by somatic mutations. From clinical data, however, there is little evidence that caspase genes are impaired in cancer. Executioner caspases have only rarely been found mutated or silenced, and also initiator caspases are only affected in particular types of cancer. There is experimental evidence from transgenic mice that certain initiator caspases, such as caspase-8 and -2, might act as tumor suppressors. Loss of the initiator caspase of the intrinsic apoptotic pathway, caspase-9, however, did not promote cellular transformation. These data seem to question a general tumor-suppressive role of caspases. We discuss several possible ways how tumor cells might evade the need for alterations of caspase genes. First, alternative splicing in tumor cells might generate caspase variants that counteract apoptosis. Second, in tumor cells caspases might be kept in check by cellular caspase inhibitors such as c-FLIP or XIAP. Third, pathways upstream of caspase activation might be disrupted in tumor cells. Finally, caspase-independent cell death mechanisms might abrogate the selection pressure for caspase inactivation during tumor development. These scenarios, however, are hardly compatible with the considerable frequency of spontaneous apoptosis occurring in several cancer types. Therefore, alternative concepts might come into play, such as compensatory proliferation. Herein, apoptosis and/or non-apoptotic functions of caspases may even promote tumor development. Moreover, experimental evidence suggests that caspases might play non-apoptotic roles in processes that are crucial for tumorigenesis, such as cell proliferation, migration, or invasion. We thus propose a model wherein caspases are preserved in tumor cells due to their functional contributions to development and progression of tumors.
PMCID: PMC3840446  PMID: 24281211
caspase; cancer; apoptosis; tumor suppressor; non-apoptotic functions; mutations; LOH
4.  Inflammatory arthritis in caspase-1 gene deficient mice: Contribution of proteinase 3 for caspase-1-independent production of bioactive IL-1β 
Arthritis and rheumatism  2009;60(12):3651-3662.
Caspase-1 is a known cysteine proteases and is a critical component of the inflammasome. Caspase-1 and neutrophil serine proteases, such as proteinase 3 (PR3) can process pro-IL-1β a crucial cytokine linked to the pathogenesis of rheumatoid arthritis, but their relative importance is unknown.
To this end we induced acute and chronic arthritis in caspase-1−/− mice and investigated the lack of caspase-1 on joint swelling, cartilage metabolism and joint pathology. In addition, caspase-1 activity was inhibited in mice lacking active cysteine proteases and evaluated the effect of dual blockade of caspase-1 and serine proteinase on arthritis severity and joint pathology.
Surprisingly, caspase-1−/− mice developed joint swelling similar to wild-type mice in models of neutrophil-dominated arthritis. Joint fluid concentrations of bioactive IL-1β were comparable in caspase-1−/− mice and controls. In contrast, induction of chronic arthritis with minimal numbers of neutrophils in caspase-1−/− mice lead to reduced joint inflammation and cartilage damage, implying caspase-1 dependence. In mice lacking neutrophil serine PR3, inhibition caspase-1 activity results in decreased bioactive IL-1β concentrations in synovial tissue and less suppression of chondrocyte anabolic function. In addition, dual blockade of both PR-3 and caspase-1 lead to protection against cartilage and bone destruction.
We conclude that caspase-1 deficiency does not affect neutrophil-dominated joint inflammation, whereas in chronic arthritis the lack of caspase-1 results in reduced joint pathology. This study implies that caspase-1 inhibitors are not able to interfere with the whole spectrum of IL-1β production and hence may be of therapeutic value only in inflammatory conditions where limited numbers of neutrophils are present.
PMCID: PMC2993325  PMID: 19950280
5.  Caspase-1-Dependent and -Independent Cell Death Pathways in Burkholderia pseudomallei Infection of Macrophages 
PLoS Pathogens  2014;10(3):e1003986.
The cytosolic pathogen Burkholderia pseudomallei and causative agent of melioidosis has been shown to regulate IL-1β and IL-18 production through NOD-like receptor NLRP3 and pyroptosis via NLRC4. Downstream signalling pathways of those receptors and other cell death mechanisms induced during B. pseudomallei infection have not been addressed so far in detail. Furthermore, the role of B. pseudomallei factors in inflammasome activation is still ill defined. In the present study we show that caspase-1 processing and pyroptosis is exclusively dependent on NLRC4, but not on NLRP3 in the early phase of macrophage infection, whereas at later time points caspase-1 activation and cell death is NLRC4- independent. In the early phase we identified an activation pathway involving caspases-9, -7 and PARP downstream of NLRC4 and caspase-1. Analyses of caspase-1/11-deficient infected macrophages revealed a strong induction of apoptosis, which is dependent on activation of apoptotic initiator and effector caspases. The early activation pathway of caspase-1 in macrophages was markedly reduced or completely abolished after infection with a B. pseudomallei flagellin FliC or a T3SS3 BsaU mutant. Studies using cells transfected with the wild-type and mutated T3SS3 effector protein BopE indicated also a role of this protein in caspase-1 processing. A T3SS3 inner rod protein BsaK mutant failed to activate caspase-1, revealed higher intracellular counts, reduced cell death and IL-1β secretion during early but not during late macrophage infection compared to the wild-type. Intranasal infection of BALB/c mice with the BsaK mutant displayed a strongly decreased mortality, lower bacterial loads in organs, and reduced levels of IL-1β, myeloperoxidase and neutrophils in bronchoalveolar lavage fluid. In conclusion, our results indicate a major role for a functional T3SS3 in early NLRC4-mediated caspase-1 activation and pyroptosis and a contribution of late caspase-1-dependent and -independent cell death mechanisms in the pathogenesis of B. pseudomallei infection.
Author Summary
Inflammasome activation is important for host defence against bacterial infection. Many gram-negative pathogens use secretion systems to inject bacterial proteins such as flagellin or structural components of the secretion machinery itself into the host cytosol leading to caspase-1 activation and pyroptotic cell death. However, little is known about the B. pseudomallei factors that trigger caspase-1 activation as well as downstream signalling pathways and effector mechanisms of caspase-1. Here, we identified the B. pseudomallei T3SS3 inner rod protein BsaK as an early activator of caspase-1-dependent cell death and IL-1β secretion in primary macrophages and as a virulence factor in murine melioidosis. We could show that upon infection of macrophages, caspase-7 is activated downstream of the NLRC4/caspase-1 inflammasome and requires caspase-9 processing. Although caspase-7 was essential for cleavage of the DNA damage sensor PARP during pyroptosis, it did neither contribute to cytokine production nor B. pseudomallei growth restriction by promoting early macrophage death. In addition to a rapid NLRC4/caspase-1- dependent induction of pyroptosis in wild-type macrophages, we observed a delayed activation of classical apoptosis in macrophages lacking caspase-1/11. Thus, initiation of different cell death pathways seems to be an effective strategy to limit intracellular B. pseudomallei infection.
PMCID: PMC3953413  PMID: 24626296
The long-lived growth hormone (GH) receptor knockout (GHRKO; KO) mice are GH resistant due to targeted disruption of the GH receptor (Ghr) gene. Apoptosis is a physiological process in which cells play an active role in their own death and is a normal component of the development and health of multicellular organisms. Aging is associated with the progressive loss of strength of skeletal and heart muscles. Calorie restriction (CR) is a well known experimental model to delay aging and increase lifespan. The aim of the study was to examine the expression of the following apoptosis-related genes: caspase-3, caspase-9, caspase-8, bax, bcl-2, Smac/DIABLO, p53 and cytochrome c1 (cyc1) in the skeletal muscles and hearts of female normal and GHRKO mice, fed ad libitum or subjected to 40% CR for 6 months, starting at 2 months of age. Moreover, skeletal muscle caspase-3, caspase-9, caspase-8, bax, bcl-2, Smac/DIABLO, Apaf-1, bad, phospho-bad (pbad), phospho-p53 (pp53) and cytochrome c (cyc) protein expression levels were assessed.
Expression of caspase-3, caspase-9, bax and Smac/DIABLO genes and proteins was decreased in GHRKO’s skeletal muscles. The Apaf-1 protein expression also was diminished in this tissue. In contrast, bcl-2 and pbad protein levels were increased in skeletal muscles in knockouts. No changes were demonstrated for the examined genes expression in GHRKO’s hearts except for the increased level of cyc1 mRNA. CR did not alter the expression of the examined genes and proteins in skeletal muscles of knockouts vs. normal (N) mice. In heart homogenates, CR increased caspase-3 mRNA level as compared to ad libitum (AL) mice.
decreased expression of certain pro-apoptotic genes and/or proteins may constitute the potential mechanism of prolonged longevity in GHRKO mice, protecting these animals from aging; this potential beneficial mechanism is not affected by calorie restriction.
PMCID: PMC3703836  PMID: 21321312
7.  Comparison of activated caspase detection methods in the gentamicin-treated chick cochlea 
Hearing research  2008;240(1-2):1-11.
Aminoglycoside antibiotics induce caspase-dependent apoptotic death in cochlear hair cells. Apoptosis, a regulated form of cell death, can be induced by many stressors, which activate signaling pathways that result in the controlled dismantling of the affected cell. The caspase family of proteases is activated in the apoptotic signaling pathway and is responsible for cellular destruction. The initiator caspase-9 and the effector caspase-3 are both activated in chick cochlear hair cells following aminoglycoside exposure. We have analyzed caspase activation in the avian cochlea during gentamicin-induced hair cell death to compare two different methods of caspase detection: caspase antibodies and CaspaTag kits. Caspase antibodies bind to the cleaved activated form of caspase-9 or caspase-3 in specific locations in fixed tissue. CaspaTag is a fluorescent inhibitor that binds to a reactive cysteine residue on the large subunit of the caspase heterodimer in unfixed tissue.
To induce cochlear hair cell loss, 1-2 week-old chickens received a single injection of gentamicin (300 mg/kg). Chicks were sacrificed 24, 30, 42, 48, 72, or 96 h after injection. Cochleae were dissected and labeled for activated caspase-9 or caspase-3 using either caspase-directed antibodies or CaspaTag kits. Ears were co-labeled with either phalloidin or myosin VI to visualize hair cells and to determine the progression of cochlear damage. The timing of caspase activation was similar for both assays; however, caspase-9 and caspase-3 antibodies labeled only those cells currently undergoing apoptotic cell death. Conversely, CaspaTag-labeled all the cells that have undergone apoptotic cell death and ejection from the sensory epithelium, in addition to those that are currently in the cell death process. This makes CaspaTag ideal for showing an overall pattern or level of cell death over a period of time, while caspase antibodies provide a snapshot of cell death at a specific time point.
PMCID: PMC2430666  PMID: 18487027
Apoptosis; Caspase; Avian; Aminoglycoside; Cochlea; Gentamicin; Hair cells
8.  Leptin attenuates cardiac apoptosis after chronic ischaemic injury 
Cardiovascular Research  2009;83(2):313-324.
We have previously shown that activation of leptin signalling in the heart reduces cardiac morbidity and mortality after myocardial infarction (MI). In the present study, we tested the hypothesis that leptin signalling limits cardiac apoptosis after MI through activation of signal transducer and activator of transcription (STAT)-3 responsive anti-apoptotic genes, including B-cell lymphoma (bcl)-2 and survivin, that serve to downregulate the activity of caspase-3.
Methods and results
Hearts from C57BL/6J and three groups of leptin-deficient Ob/Ob mice (food-restricted, ad libitum, and leptin-repleted) were examined 4 weeks after permanent left coronary artery ligation or sham operation. Inflammatory and apoptotic cell number was determined in cardiac sections by immunostaining. Expression of cardiac bcl-2, survivin, and pro and active caspase-3 was determined and correlated with in vitro caspase-3 activity. In the absence of MI, both lean and obese leptin-deficient mice exhibited increased cardiac apoptosis compared with wild-type mice. After MI, the highest rates of apoptosis were seen in the infarcted tissue of lean and obese Ob/Ob mice. Further, leptin-deficient hearts, as well as hearts from wild-type mice treated with the STAT-3 inhibitor WP1066, exhibited blunted anti-apoptotic bcl-2 and survivin gene expression, and increased caspase-3 protein expression and activity. The increased caspase-3 activity and apoptosis in hearts of leptin-deficient mice after MI was significantly attenuated in Ob/Ob mice replete with leptin, reducing apoptosis to levels comparable to that observed in wild-type mice after MI.
These results demonstrate that intact leptin signalling post-MI acts through STAT-3 to increase anti-apoptotic bcl-2 and survivin gene expression and reduces caspase-3 activity, consistent with a cardioprotective role of leptin in the setting of chronic ischaemic injury.
PMCID: PMC2701718  PMID: 19233863
Apoptosis; Leptin; Survivin; bcl-2; Heart failure
9.  Prostaglandin F2alpha- and FAS-activating antibody-induced regression of the corpus luteum involves caspase-8 and is defective in caspase-3 deficient mice 
We recently demonstrated that caspase-3 is important for apoptosis during spontaneous involution of the corpus luteum (CL). These studies tested if prostaglandin F2α (PGF2α) or FAS regulated luteal regression, utilize a caspase-3 dependent pathway to execute luteal cell apoptosis, and if the two receptors work via independent or potentially shared intracellular signaling components/pathways to activate caspase-3. Wild-type (WT) or caspase-3 deficient female mice, 25–26 days old, were given 10 IU equine chorionic gonadotropin (eCG) intraperitoneally (IP) followed by 10 IU human chorionic gonadotropin (hCG) IP 46 h later to synchronize ovulation. The animals were then injected with IgG (2 micrograms, i.v.), the FAS-activating antibody Jo2 (2 micrograms, i.v.), or PGF2α (10 micrograms, i.p.) at 24 or 48 h post-ovulation. Ovaries from each group were collected 8 h later for assessment of active caspase-3 enzyme and apoptosis (measured by the TUNEL assay) in the CL. Regardless of genotype or treatment, CL in ovaries collected from mice injected 24 h after ovulation showed no evidence of active caspase-3 or apoptosis. However, PGF2α or Jo2 at 48 h post-ovulation and collected 8 h later induced caspase-3 activation in 13.2 ± 1.8% and 13.7 ± 2.2 % of the cells, respectively and resulted in 16.35 ± 0.7% (PGF2α) and 14.3 ± 2.5% TUNEL-positive cells when compared to 1.48 ± 0.8% of cells CL in IgG treated controls. In contrast, CL in ovaries collected from caspase-3 deficient mice whether treated with PGF2α , Jo2, or control IgG at 48 h post-ovulation showed little evidence of active caspase-3 or apoptosis. CL of WT mice treated with Jo2 at 48 h post-ovulation had an 8-fold increase in the activity of caspase-8, an activator of caspase-3 that is coupled to the FAS death receptor. Somewhat unexpectedly, however, treatment of WT mice with PGF2α at 48 h post-ovulation resulted in a 22-fold increase in caspase-8 activity in the CL, despite the fact that the receptor for PGF2α has not been shown to be directly coupled to caspase-8 recruitment and activation. We hypothesize that PGF2α initiates luteolysis in vivo, at least in part, by increasing the bioactivity or bioavailability of cytokines, such as FasL and that multiple endocrine factors work in concert to activate caspase-3-driven apoptosis during luteolysis.
PMCID: PMC152637  PMID: 12657159
10.  The Pro-Apoptotic BH3-only, Bcl-2 Family Member Puma Is Critical for Acute Ethanol-Induced Neuronal Apoptosis 
Synaptogenesis in humans occurs in the last trimester of gestation and in the first few years of life whereas it occurs in the postnatal period in rodents. A single exposure of neonatal rodents to ethanol during this period evokes extensive neuronal apoptosis. Previous studies indicate that ethanol triggers the intrinsic apoptotic pathway in neurons and that this requires the multi-BH domain, pro-apoptotic Bcl-2 family member Bax. To define the upstream regulators of this apoptotic pathway, we examined the possible roles of p53 and a subclass of pro-apoptotic Bcl-2 family members (i.e. the BH3 domain-only proteins) in neonatal wild-type and gene-targeted mice that lack these cell death inducers. Acute ethanol exposure produced greater caspase-3 activation and neuronal apoptosis in wild type mice than in saline-treated littermate controls. Loss of Puma resulted in marked protection from ethanol-induced caspase-3 activation and apoptosis. Although Puma expression has been reported to be regulated by p53, p53-deficient mice exhibited a similar extent of ethanol-induced caspase-3 activation and neuronal apoptosis as wild-type mice. Mice deficient in other pro-apoptotic BH3-only proteins, including Noxa, Bim or Hrk, showed no significant protection from ethanol-induced neuronal apoptosis. Collectively, these studies indicate a p53-independent, Bax- and Puma-dependent mechanism of neuronal apoptosis and identify Puma as a possible molecular target for inhibiting the effects of intrauterine ethanol exposure in humans.
PMCID: PMC2745204  PMID: 19535997
Apoptosis; Bax; BH3-only; Caspase-3; Ethanol; Noxa; p53; Puma
11.  Lyapunov exponents and phase diagrams reveal multi-factorial control over TRAIL-induced apoptosis 
Kinetic modeling, phase diagrams analysis, and quantitative single-cell experiments are combined to investigate how multiple factors, including the XIAP:caspase-3 ratio and ligand concentration, regulate receptor-mediated apoptosis.
Based on protein expression levels, Lyapunov-based phase diagrams predict which pathways are required for a cell to undergo receptor-mediated cell death.Multiple inter-dependent factors, including the XIAP:caspase-3 ratio and ligand concentration, regulate the requirement for mitochondrial outer membrane permeabilization during receptor-mediated apoptosis.The E3 ubiquitin ligase activity of XIAP is essential for maintaining the ‘snap-action' regulation of effector caspase activity.Cell-to-cell variability in protein expression gives rise to mixed phenotypes in cell lines that map close to boundaries (separatrices) identified by Lyapunov exponent analysis.
In mammalian cells, extrinsic (receptor-mediated) apoptosis is triggered by binding of extracellular death ligands such as tumor necrosis factor (TNF) and TRAIL (TNF-related apoptosis-inducing ligand) to cognate receptors. When death receptors are activated, death inducing signaling complexes (DISCs) assemble causing activation and cleavage of initiator pro-caspases-8 and -10, which then cleave effector pro-caspases-3 and -7 in a multi-enzyme cascade (Riedl and Shi, 2004). Active effector caspases digest essential cellular proteins and activate the CAD nucleases that cleave genomic DNA, thereby killing cells. This cascade of DISC assembly followed by initiator and then effector caspase activation is sufficient to kill so-called type I cells (e.g. B lymphocytes), but most cell types exhibit a type II behavior in which mitochondrial outer membrane permeabilization (MOMP) is an essential step in the march to death (Scaffidi et al, 1998; Barnhart et al, 2003; Letai, 2008). Identifying factors that determine whether cells are type I or II is of practical and theoretical interest. From a practical perspective, whether a cell requires MOMP for apoptosis determines the potency of Bcl2 and similar oncogenes, the efficacy of anti-Bcl2 drugs such as navitoclax (ABT-263), and the sensitivity of cells to TRAIL and anti-TRAIL receptor antibodies, which are also investigational anti-cancer drugs (Newsom-Davis et al, 2009). From a theoretical perspective, the type I versus II choice exemplifies a common situation in mammalian cells in which overlapping signaling pathways play a greater or lesser role in controlling cell fate depending on cell type: it is remarkable that a simple three-step (receptor→initiator caspase→effector caspase) process is sufficient to trigger apoptosis in some cell types but that a much more complex route involving MOMP is required in others.
Attempts to understand why some cells require MOMP for cell death and others do not have identified differences in the oligomeric state of death ligand receptors and the efficiency of DISC formation as important variables. In cells in which DISCs form efficiently, initiator caspases are cleaved rapidly and sufficient effector pro-caspases are processed into their active forms to kill cells (type I cells; Scaffidi et al, 1999b). In type II cells, DISC formation seems to be less efficient, and it has been proposed that MOMP is required to amplify weak initiator caspase signals and thereby generate lethal effector caspase levels (Barnhart et al, 2003). However, it has recently become apparent that XIAP also plays a role in type I versus II choice: in XIAP knockout mice, liver cells switch from a type II to a type I phenotype (Jost et al, 2009) and XIAP is observed to be involved in the survival of type I cells treated with death ligands in culture (Maas et al, 2010).
In this paper, we attempt to place these observations in a quantitative context by analyzing a computational model of extrinsic cell death using a method drawn from dynamical system analysis, direct finite-time Lyapunov exponent (DLE) analysis. Our implementation of DLE analysis relates changes in the concentrations of protein in a model to an outcome several hours later. We computed DLEs for six regulators of apoptosis over a range of concentrations determined experimentally to represent a natural range of variation in parental or genetically modified tumor cell lines. This generated a phase space onto which individual cell lines could be mapped using quantitative immunoblotting data. Cell-to-cell variation was estimated by flow cytometry and also mapped onto the phase space. The most interesting regions of the space were those in which a small change in one or more initial protein concentration resulted in a dramatic change in phenotype. Such a boundary or separatrix was observed in slices of phase space corresponding XIAP versus pro-caspase-3 concentration (the [XIAP]:[caspase-3] ratio). In cells in which the ratio is low, a type I phenotype is predicted to occur; when the ratio is high, a type II phenotype is favored; and in cell lines that lie close to the separatrix, cell-to-cell variability is expected, with some cells exhibiting a type I phenotype and others a type II behavior. DLE analysis shows that the [XIAP]:[caspase-3] ratio is not the only controlling factor in type I versus II control: as receptor activity or ligand concentration increase, the position of the separatrix changes so as to expand the region in which the type I phenotype is favored.
We tested these predictions by manipulating XIAP and ligand levels in multiple cell lines and then followed cell death by imaging, flow cytometry, or clonogenic assays. We observed that when XIAP was knocked out (by homologous recombination) in the HCT116 colorectal cancer line, cells shifted from a pure type II to a type I phenotype, as predicted from the DLE phase diagram. SKW6.4 B-cell lymphoma cells were predicted to lie at a position in phase space that is insensitive to XIAP levels (within the range achievable by over-expression) and we confirmed this experimentally. Finally, T47D breast cancer cells were predicted—and observed—to straddle the separatrix and to exhibit cell-to-cell variability in fate, with some cells showing a type I and others a type II phenotype. As the concentration of TRAIL was increased, the ratio of type I to type II T47D cells increased, confirming the prediction that this ratio is controlled in a multi-factorial manner.
To extend our approach to mutations that change protein activity rather than protein level, we simulated the effects of changing rate constants that control ubiquitylation of caspase-3 following its binding to XIAP. We generated cells carrying a truncated form of XIAP that lacks the RING domain (XIAPΔRING) and cannot mediate the ubiquitylation of caspase-3 (this truncation leaves the affinity of XIAP for caspase-3 unchanged). We predicted and demonstrated experimentally that expression of XIAPΔRING disrupts normal snap-action control over caspase-3 activation. Our findings not only advance understanding of extrinsic apoptosis but also constitute a proof of principle for an approach to quantitative modeling of dynamic regulatory processes in diverse cell types.
Receptor-mediated apoptosis proceeds via two pathways: one requiring only a cascade of initiator and effector caspases (type I behavior) and the second requiring an initiator–effector caspase cascade and mitochondrial outer membrane permeabilization (type II behavior). Here, we investigate factors controlling type I versus II phenotypes by performing Lyapunov exponent analysis of an ODE-based model of cell death. The resulting phase diagrams predict that the ratio of XIAP to pro-caspase-3 concentrations plays a key regulatory role: type I behavior predominates when the ratio is low and type II behavior when the ratio is high. Cell-to-cell variability in phenotype is observed when the ratio is close to the type I versus II boundary. By positioning multiple tumor cell lines on the phase diagram we confirm these predictions. We also extend phase space analysis to mutations affecting the rate of caspase-3 ubiquitylation by XIAP, predicting and showing that such mutations abolish all-or-none control over activation of effector caspases. Thus, phase diagrams derived from Lyapunov exponent analysis represent a means to study multi-factorial control over a complex biochemical pathway.
PMCID: PMC3261706  PMID: 22108795
apoptosis; caspases; dynamical systems analysis; kinetic modeling; XIAP
12.  Caspase-Dependent Inhibition of Mousepox Replication by gzmB 
PLoS ONE  2009;4(10):e7512.
Ectromelia virus is a natural mouse pathogen, causing mousepox. The cytotoxic T (Tc) cell granule serine-protease, granzyme B, is important for its control, but the underlying mechanism is unknown. Using ex vivo virus immune Tc cells, we have previously shown that granzyme B is able to activate several independent pro-apoptotic pathways, including those mediated by Bid/Bak/Bax and caspases-3/-7, in target cells pulsed with Tc cell determinants.
Methods and Findings
Here we analysed the physiological relevance of those pro-apoptotic pathways in ectromelia infection, by incubating ectromelia-immune ex vivo Tc cells from granzyme A deficient (GzmB+ Tc cells) or granzyme A and granzyme B deficient (GzmA×B−/− Tc cell) mice with ectromelia-infected target cells. We found that gzmB-induced apoptosis was totally blocked in ectromelia infected or peptide pulsed cells lacking caspases-3/-7. However ectromelia inhibited only partially apoptosis in cells deficient for Bid/Bak/Bax and not at all when both pathways were operative suggesting that the virus is able to interfere with apoptosis induced by gzmB in case not all pathways are activated. Importantly, inhibition of viral replication in vitro, as seen with wild type cells, was not affected by the lack of Bid/Bak/Bax but was significantly reduced in caspase-3/-7-deficient cells. Both caspase dependent processes were strictly dependent on gzmB, since Tc cells, lacking both gzms, neither induced apoptosis nor reduced viral titers.
Out findings present the first evidence on the biological importance of the independent gzmB-inducible pro-apoptotic pathways in a physiological relevant virus infection model.
PMCID: PMC2759507  PMID: 19838298
13.  The Inflammasome-Mediated Caspase-1 Activation Controls Adipocyte Differentiation and Insulin Sensitivity 
Cell metabolism  2010;12(6):593-605.
Obesity-induced inflammation originating from expanding adipose tissue interferes with insulin sensitivity. Important metabolic effects have been recently attributed to IL-1β and IL-18, two members of the IL-1 family of cytokines. Processing of IL-1β and IL-18 requires cleavage by caspase-1, a cysteine protease regulated by a protein complex called the inflammasome. We demonstrate that the inflamma-some/caspase-1 governs adipocyte differentiation and insulin sensitivity. Caspase-1 is upregulated during adipocyte differentiation and directs adipocytes toward a more insulin-resistant phenotype. Treatment of differentiating adipocytes with recombinant IL-1β and IL-18, or blocking their effects by inhibitors, reveals that the effects of caspase-1 on adipocyte differentiation are largely conveyed by IL-1β. Caspase-1 and IL-1β activity in adipose tissue is increased both in diet-induced and genetically induced obese animal models. Conversely, mice deficient in caspase-1 are more insulin sensitive as compared to wild-type animals. In addition, differentiation of preadipocytes isolated from caspase-1−/− or NLRP3−/− mice resulted in more metabolically active fat cells. In vivo, treatment of obese mice with a caspase-1 inhibitor significantly increases their insulin sensitivity. Indirect calorimetry analysis revealed higher fat oxidation rates in caspase-1−/− animals. In conclusion, the inflammasome is an important regulator of adipocyte function and insulin sensitivity, and caspase-1 inhibition may represent a novel therapeutic target in clinical conditions associated with obesity and insulin resistance.
PMCID: PMC3683568  PMID: 21109192
14.  TNF-α Mediates Diabetes-Enhanced Chondrocyte Apoptosis During Fracture Healing and Stimulates Chondrocyte Apoptosis Through FOXO1 
Journal of Bone and Mineral Research  2010;25(7):1604-1615.
To gain insight into the effect of diabetes on fracture healing, experiments were carried out focusing on chondrocyte apoptosis during the transition from cartilage to bone. Type 1 diabetes was induced in mice by multiple low-dose streptozotocin injections, and simple transverse fractures of the tibia or femur was carried out. Large-scale transcriptional profiling and gene set enrichment analysis were performed to examine apoptotic pathways on total RNA isolated from fracture calluses on days 12, 16, and 22, a period of endochondral bone formation when cartilage is resorbed and chondrocyte numbers decrease. Tumor necrosis factor α (TNF-α) protein levels were assessed by ELISA and caspase-3 by bioactivity assay. The role of TNF was examined by treating mice with the TNF-specific inhibitor pegsunercept. In vitro studies investigated the proapoptotic transcription factor FOXO1 in regulating TNF-induced apoptosis of chondrogenic ATDC5 and C3H10T1/2 cells as representative of differentiated chondrocytes, which are important during endochondral ossification. mRNA profiling revealed an upregulation of gene sets related to apoptosis in the diabetic group on day 16 when cartilage resorption is active but not day 12 or day 22. This coincided with elevated TNF-α protein levels, chondrocyte apoptosis, enhanced caspase-3 activity, and increased FOXO1 nuclear translocation (p < .05). Inhibition of TNF significantly reduced these parameters in the diabetic mice but not in normoglycemic control mice (p < .05). Silencing FOXO1 using siRNA in vitro significantly reduced TNF-induced apoptosis and caspase activity in differentiated chondrocytes. The mRNA levels of the proapoptotic genes caspase-3, caspase-8, caspase-9, and TRAIL were significantly reduced with silencing of FOXO1 in chondrocytic cells. Inhibiting caspase-8 and caspase-9 significantly reduced TNF-induced apoptosis in chondrogenic cells. These results suggest that diabetes causes an upregulation of proapoptotic genes during the transition from cartilage to bone in fracture healing. Diabetes increased chondrocyte apoptosis through a mechanism that involved enhanced production of TNF-α, which stimulates chondrocyte apoptosis and upregulates mRNA levels of apoptotic genes through FOXO1 activation. © 2010 American Society for Bone and Mineral Research.
PMCID: PMC3154002  PMID: 20200974
apoptosis; bone; cell death; chondrocyte; cartilage; cytokine; fracture; forkhead; nuclear localization; transcription factor
15.  Glutamate-induced apoptosis in primary cortical neurons is inhibited by equine estrogens via down-regulation of caspase-3 and prevention of mitochondrial cytochrome c release 
BMC Neuroscience  2005;6:13.
Apoptosis plays a key role in cell death observed in neurodegenerative diseases marked by a progressive loss of neurons as seen in Alzheimer's disease. Although the exact cause of apoptosis is not known, a number of factors such as free radicals, insufficient levels of nerve growth factors and excessive levels of glutamate have been implicated. We and others, have previously reported that in a stable HT22 neuronal cell line, glutamate induces apoptosis as indicated by DNA fragmentation and up- and down-regulation of Bax (pro-apoptotic), and Bcl-2 (anti-apoptotic) genes respectively. Furthermore, these changes were reversed/inhibited by estrogens. Several lines of evidence also indicate that a family of cysteine proteases (caspases) appear to play a critical role in neuronal apoptosis. The purpose of the present study is to determine in primary cultures of cortical cells, if glutamate-induced neuronal apoptosis and its inhibition by estrogens involve changes in caspase-3 protease and whether this process is mediated by Fas receptor and/or mitochondrial signal transduction pathways involving release of cytochrome c.
In primary cultures of rat cortical cells, glutamate induced apoptosis that was associated with enhanced DNA fragmentation, morphological changes, and up-regulation of pro-caspase-3. Exposure of cortical cells to glutamate resulted in a time-dependent cell death and an increase in caspase-3 protein levels. Although the increase in caspase-3 levels was evident after 3 h, cell death was only significantly increased after 6 h. Treatment of cells for 6 h with 1 to 20 mM glutamate resulted in a 35 to 45% cell death that was associated with a 45 to 65% increase in the expression of caspase-3 protein. Pretreatment with caspase-3-protease inhibitor z-DEVD or pan-caspase inhibitor z-VAD significantly decreased glutamate-induced cell death of cortical cells. Exposure of cells to glutamate for 6 h in the presence or absence of 17β-estradiol or Δ8, 17β-estradiol (10 nM-10 μM) resulted in the prevention of cell death and was associated with a significant dose-dependent decrease in caspase-3 protein levels, with Δ8, 17β-E2 being more potent than 17β-E2. Protein levels of Fas receptor remained unchanged in the presence of glutamate. In contrast, treatment with glutamate induced, in a time-dependent manner, the release of cytochrome c into the cytosol. Cytosolic cytochrome c increased as early as 1.5 h after glutamate treatment and these levels were 5 fold higher after 6 h, compared to levels in the untreated cells. Concomitant with these changes, the levels of cytochrome c in mitochondria decreased significantly. Both 17β-E2 and Δ8, 17β-E2 reduced the release of cytochrome c from mitochondria into the cytosol and this decrease in cytosolic cytochrome c was associated with inhibition of glutamate-induced cell death.
In the primary cortical cells, glutamate-induced apoptosis is accompanied by up-regulation of caspase-3 and its activity is blocked by caspase protease inhibitors. These effects of glutamate on caspase-3 appear to be independent of changes in Fas receptor, but are associated with the rapid release of mitochondrial cytochrome c, which precedes changes in caspase-3 protein levels leading to apoptotic cell death. This process was differentially inhibited by estrogens with the novel equine estrogen Δ8, 17β-E2 being more potent than 17β-E2. To our knowledge, this is the first study to demonstrate that equine estrogens can prevent glutamate-induced translocation of cytochrome c from mitochondria to cytosol in rat primary cortical cells.
PMCID: PMC555946  PMID: 15730564
16.  Impaired antioxidant defence and accumulation of oxidative stress in caspase-2-deficient mice 
Cell Death and Differentiation  2012;19(8):1370-1380.
Caspase-2 has been implicated in apoptosis and in non-apoptotic processes such as cell cycle regulation, tumor suppression and ageing. Using caspase-2 knockout (casp2−/−) mice, we show here that the putative anti-ageing role of this caspase is due in part to its involvement in the stress response pathway. The old casp2−/− mice show increased cellular levels of oxidized proteins, lipid peroxides and DNA damage, suggesting enhanced oxidative stress. Furthermore, murine embryonic fibroblasts from casp2−/− mice showed increased reactive oxygen species generation when challenged with pro-oxidants. Reduced activities of antioxidant enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were observed in the old casp2−/− mice. Interestingly, in the old casp2−/− animals expression of FoxO1 and FoxO3a was significantly reduced, whereas p21 levels and the number of senescent hepatocytes were elevated. In contrast to young wild-type mice, the casp2−/− animals fed an on ethanol-based diet failed to show enhanced GSH-Px and SOD activities. Thus, caspase-2, most likely via FoxO transcription factors, regulates the oxidative stress response in vivo.
PMCID: PMC3392626  PMID: 22343713
oxidative stress; ROS; antioxidant; caspases; ageing
17.  Caspase-10-Dependent Cell Death in Fas/CD95 Signalling Is Not Abrogated by Caspase Inhibitor zVAD-fmk 
PLoS ONE  2010;5(10):e13638.
Upon CD95/Fas ligation, the initiator caspase-8 is known to activate effector caspases leading to apoptosis. In the presence of zVAD-fmk, a broad-spectrum caspase inhibitor, Fas engagement can also trigger an alternative, non-apoptotic caspase-independent form of cell death, which is initiated by RIP1. Controversy exists as to the ability of caspase-10 to mediate cell death in response to FasL (CD95L or CD178). Herein, the role of caspase-10 in FasL-induced cell death has been re-evaluated.
Methodology and Principal Findings
The present study shows that FasL-induced cell death was completely impaired in caspase-8- and caspase-10-doubly deficient (I9-2e) Jurkat leukaemia T-cell lines. Over-expressing of either caspase-8 or caspase-10 in I9-2e cells triggered cell death and restored sensitivity to FasL, further arguing for a role of both initiator caspases in Fas apoptotic signalling. In the presence of zVAD-fmk, FasL triggered an alternative form of cell death similarly in wild-type (A3) and in caspase-8-deficient Jurkat cells expressing endogenous caspase-10 (clone I9-2d). Cell death initiated by Fas stimulation in the presence of zVAD-fmk was abrogated in I9-2e cells as well as in HeLa cells, which did not express endogenous caspase-10, indicating that caspase-10 somewhat participates in this alternative form of cell death. Noteworthy, ectopic expression of caspase-10 in I9-2e and HeLa cells restored the ability of FasL to trigger cell death in the presence of zVAD-fmk. As a matter of fact, FasL-triggered caspase-10 processing still occurred in the presence of zVAD-fmk.
Conclusions and Significance
Altogether, these data provide genetic evidence for the involvement of initiator caspase-10 in FasL-induced cell death and indicate that zVAD-fmk does not abrogate caspase-10 processing and cytotoxicity in Fas signalling. Our study also questions the existence of an alternative caspase-independent cell death pathway in Fas signalling.
PMCID: PMC2964310  PMID: 21049020
18.  The role of aquaporin 4 in apoptosis after intracerebral hemorrhage 
We previously reported that aquaporin-4 deletion (AQP4−/−) in mice increased edema and altered blood-brain barrier integrity following intracerebral hemorrhage (ICH). To date, little is known about the role of AQP4 in apoptosis after ICH. The purpose of this study was to examine the role of AQP4 in apoptosis and its mechanisms after ICH using AQP4−/− mice.
We compared the survival rate and neurological deficits in wild-type (AQP4+/+) mice with those in AQP4−/− mice following ICH. Histological changes were detected with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and Hoechst staining. The cell types involved were determined by immunocytochemical studies. We also measured activated caspase-3, caspase-9, caspase-8, Bax, and Bcl-2 with Western blotting at 1, 3, and 7 days after ICH. A cytokine protein assay was used to detect cytokines in AQP4+/+ and AQP4−/− mice following ICH, and the results were verified by ELISA.
We found more apoptotic cells in AQP4−/− mice following ICH; the cell types involved were predominantly neurons and astrocytes. Western blotting showed that the expression of activated caspase-3 and caspase-8 was significantly increased (P <0.05). Moreover, we demonstrated a greater enhancement in the release of TNF-α and IL-1β, as well as their receptors, in AQP4−/− mice following ICH than in AQP4+/+ mice by cytokine protein assay and Western blotting (P <0.05). The inhibitors of TNF-α and IL-1β reduced apoptotic cells after ICH in AQP4−/− mice compared with wild-type mice (P <0.05).
AQP4 deletion increases apoptosis following ICH, and the underlying mechanism may be through cytokines, especially TNF-α and IL-1β, initiating the apoptotic cascade, as well as activation of caspase-3 and caspase-8.
PMCID: PMC4243419  PMID: 25359421
apoptosis; aquaporin-4; cytokine; intracerebral hemorrhage
19.  Combined inhibition of cell death induced by apoptosis inducing factor and caspases provides additive neuroprotection in experimental traumatic brain injury 
Neurobiology of Disease  2012;46(3):745-758.
Neuronal programmed cell death (PCD) contributes to delayed tissue damage after traumatic brain injury (TBI). Both caspase-dependent and caspase-independent mechanisms have been implicated, with the latter including apoptosis inducing factor (AIF). The peptidyl-proplyl isomerase Cyclophilin A (CypA) transports AIF from the cytosol to the nucleus, a key step for AIF-dependent cell death. We compared the effects of single versus combined inhibition of caspase and AIF pathways in a mouse controlled cortical impact (CCI) model, by examining the effects of CypA gene knockout (CypA−/−), caspase inhibition with a pan-caspase inhibitor (boc-aspartyl(OMe)-fluoromethylketone, BAF), or combined modulation. TBI caused caspase activation as well as translocation of AIF to the nucleus. Markers of caspase activation including caspase-specific fodrin cleavage fragments and number of FLIVO positive cells were reduced in BAF-treated CypA+/+ mice, whereas markers of AIF activation including AIF/H2AX interaction and AIF translocation to the nucleus were attenuated in CypA−/− mice. Each single intervention, (CypA−/− or BAF-treated CypA+/+) reduced the number of apoptotic cells (TUNEL-positive) in the cortex and improved long-term sensorimotor function; CypA−/− also attenuated microglial activation after injury. Importantly, BAF-treated CypA−/− mice, showed greater effects than either intervention alone on multiple outcomes including: reduction in TUNEL-positive cells, decrease in neuroinflammation, improved motor and cognitive recovery, and attenuation of lesion volume and neuronal loss in the hippocampus. Using two in vitro neuronal cell death models known to induce AIF-mediated PCD, we also showed that neurons from CypA−/− animals were protected and that effects were unrelated to caspase activation. These data indicate that AIF-mediated and caspase-dependent pathways contribute independently and in parallel to secondary injury after TBI, and suggest that combined therapeutic strategies directed at multiple PCD pathways may provide superior neuroprotection than those directed at single mechanisms.
PMCID: PMC3352990  PMID: 22426396
Traumatic Brain Injury; Apoptosis Inducing Factor; Caspase; Cyclophilin A
20.  Caspase-3 deficiency reveals a physiologic role for Smac/DIABLO in regulating programmed cell death 
Cell Death and Differentiation  2011;18(11):1780-1790.
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.
PMCID: PMC3190114  PMID: 21597464
apoptosis; caspases; Smac/DIABLO; IAPs; knockout
21.  Enhanced HtrA2/Omi Expression in Oxidative Injury to Retinal Pigment Epithelial Cells and Murine Models of Neurodegeneration 
To investigate the role of HtrA2/Omi, a nuclear-encoded mitochondrial serine protease with a proapoptosis function, under H2O2-induced oxidative stress in human RPE, in the Ccl2−/− Cx3cr1−/− double-knockout (DKO) mouse retina, and the HtrA2/Omi-deficient mice.
Oxidative stress was induced in ARPE-19 cells by 1 mM H2O2 for 2 hours. HtrA2/Omi and caspase-3 expression was evaluated using RQ-PCR, immunohistochemistry, or Western blot. Cell viability was detected by MTT assay. HtrA2/Omi expression in the subcellular components and activated caspase-3 were measured. These processes were also evaluated in cells treated with UCF-101, an HtrA2/Omi inhibitor or in cells subjected to RNAi against HtrA2/Omi. Oxidative stress was assayed and compared in retinas of DKO and wild-type (WT) mice by determining serum NADPH oxidase subunits and nitrite levels. Transmission electron microscopy was used to view the retinal ultrastructure of the HtrA2/Omi-deficient mice.
H2O2-induced oxidative damage resulted in HtrA2/Omi translocation from mitochondria to cytosol, leading to RPE cell apoptosis via a caspase-mediated pathway. Treatment of RPE cells with UCF-101 reduced the cytosolic translocation of HtrA2/Omi, attenuated caspase-3 activation, and decreased apoptosis. After specific HtrA2 downregulation, increased cell viability was measured in H2O2-treated ARPE-19 cells. Retina of DKO mice exhibit increased oxidative stress and upregulation of HtrA2/Omi. Fewer and abnormal mitochondria were found in HtrA2/Omi−/− photoreceptors and RPE.
These findings suggest that HtrA2/Omi is related to RPE apoptosis due to oxidative stress, which may play an important role in the integrity of mitochondria and the pathogenesis of AMD.
PMCID: PMC2903625  PMID: 19443712
22.  Caspase-1 as a Central Regulator of High Fat Diet-Induced Non-Alcoholic Steatohepatitis 
PLoS ONE  2013;8(2):e56100.
Nonalcoholic steatohepatitis (NASH) is associated with caspase activation. However, a role for pro-inflammatory caspases or inflammasomes has not been explored in diet-induced liver injury. Our aims were to examine the role of caspase-1 in high fat-induced NASH. C57BL/6 wild-type and caspase 1-knockout (Casp1-/-) mice were placed on a 12-week high fat diet. Wild-type mice on the high fat diet increased hepatic expression of pro-caspase-1 and IL-1β. Both wild-type and Casp1-/- mice on the high fat diet gained more weight than mice on a control diet. Hepatic steatosis and TG levels were increased in wild-type mice on high fat diet, but were attenuated in the absence of caspase-1. Plasma cholesterol and free fatty acids were elevated in wild-type, but not Casp1-/- mice, on high fat diet. ALT levels were elevated in both wild-type and Casp1-/- mice on high fat diet compared to control. Hepatic mRNA expression for genes associated with lipogenesis was lower in Casp1-/- mice on high fat diet compared to wild-type mice on high fat diet, while genes associated with fatty acid oxidation were not affected by diet or genotype. Hepatic Tnfα and Mcp-1 mRNA expression was increased in wild-type mice on high fat diet, but not in Casp1-/- mice on high fat diet. αSMA positive cells, Sirius red staining, and Col1α1 mRNA were increased in wild-type mice on high fat diet compared to control. Deficiency of caspase-1 prevented those increases. In summary, the absence of caspase-1 ameliorates the injurious effects of high fat diet-induced obesity on the liver. Specifically, mice deficient in caspase-1 are protected from high fat-induced hepatic steatosis, inflammation and early fibrogenesis. These data point to the inflammasome as an important therapeutic target for NASH.
PMCID: PMC3567081  PMID: 23409132
23.  Caspase-12 mediates carbon tetrachloride-induced hepatocyte apoptosis in mice 
World Journal of Gastroenterology : WJG  2014;20(48):18189-18198.
AIM: To investigate the role of caspase-12 and its downstream targets in carbon tetrachloride (CCl4)-induced hepatocyte apoptosis.
METHODS: The role of caspase-12 was determined by using caspase-12 knock-out (-/-) mice. CCl4 (300 μL/kg body weight) or vehicle (corn oil) was administered to caspase-12+/+ or caspase-12-/- mice as a single intraperitoneal injection. The animals were sacrificed 24 h after the CCl4 treatment. Blood was collected to evaluate liver function by the measurement of the activity of alanine aminotransferase. Liver samples were used for the measurements of reactive oxygen species using plasma malondialdehyde as biomarker, hepatocyte apoptosis was evaluated via terminal transferase-mediated dUTP nick-end labeling and controlled by morphologic study, and cytochrome C release and caspase activations were measured by Western blotting.
RESULTS: Administration of a low dose of CCl4 resulted in hepatocyte apoptosis and acute liver injury in wild-type mice. CCl4 also induced the generation of reactive oxygen species and induction of endoplasmic reticulum stress in the liver followed by activations of caspase-12, -9 and -3 as well as release of small amounts of cytochrome C. However, in the CCl4-treated caspase-12-/- mice, activation of caspase-9 and -3 were significantly attenuated (P < 0.05); no effect was seen in cytochrome C release. CCl4-induced apoptosis and liver damage was markedly reduced in caspase-12-/- mice compared to caspase-12+/+ mice (P < 0.05). The active form of caspase-8 was not detected in either caspase-12+/+ or caspase-12-/- mice. There was no significant different in the formation of reactive oxygen species in the livers of caspase-12+/+ and caspase-12-/- mice treated with CCl4.
CONCLUSION: Caspase-12 plays a pivotal role in CCl4-induced hepatic apoptosis through the activation of the downstream effector caspase-3 directly and/or indirectly via capase-9 activation.
PMCID: PMC4277956  PMID: 25561786
Apoptosis; Carbon tetrachloride; Caspases; Endoplasmic reticulum; Hepatocyte; Reactive oxygen species
24.  ICAD Deficiency in Human Colon Cancer and Predisposition to Colon Tumorigenesis: Linkage to Apoptosis Resistance and Genomic Instability 
PLoS ONE  2013;8(2):e57871.
We previously showed that DNA fragmentation factor, which comprises a caspase-3-activated DNase (CAD) and its inhibitor (ICAD), may influence the rate of cell death by generating PARP-1-activating DNA breaks. Here we tested the hypothesis that ICAD-deficient colon epithelial cells exhibiting resistance to death stimuli may accumulate additional genetic modifications, leading to a tumorigenic phenotype. We show that ICAD deficiency may be associated with colon malignancy in humans. Indeed, an examination of ICAD expression using immunohistochemistry in an array of both colon cancer and normal tissues revealed that ICAD expression levels were severely compromised in the cancerous tissues. Upon DNA damage caused by a low dose of irradiation, ICAD cells acquire a tumorigenic phenotype. Colon epithelial cells derived from ICAD mice showed a significant resistance to death induced by the colon carcinogen dimethylhydrazine in vitro and in mice. Such resistance was associated with a decrease in PARP-1 activation. In an animal model of dimethylhydrazine-induced colon tumorigenesis, ICAD−/− mice developed significantly higher numbers of tumors with markedly larger sizes than the wild-type counterparts. Interestingly, the phenotype of the ICAD−/− mice was not associated with a significant increase in the precancerous aberrant crypt foci suggesting a potential link to tumor progression rather than initiation. More importantly, ICAD deficiency was associated with severe genomic instability as assessed by array comparative genomic hybridization. Such genomic instability consisted most prominently of amplifications but with sizable deletions as compared to the wild-type counterparts affecting several cancer-related genes including RAF-1, GSN, LMO3, and Fzd6 independently of p53. Altogether, our results present a viable case for the involvement of ICAD deficiency in colon carcinogenesis and show that apoptosis and genomic instability may comprise the means by which such deficiency may contribute to the process of increasing susceptibility to carcinogen-induced tumorigenesis.
PMCID: PMC3579889  PMID: 23451280
25.  Inflammasome-Dependent Release of the Alarmin HMGB1 in Endotoxemia 
Endotoxin administration recapitulates many of the host responses to sepsis. Inhibitors of the cysteine protease caspase 1 have long been sought as a therapeutic because mice lacking caspase 1 are resistant to LPS-induced endotoxic shock. According to current thinking, caspase 1-mediated shock requires the proinflammatory caspase 1 substrates IL-1β and IL-18. We show, however, that mice lacking both IL-1β and IL-18 are normally susceptible to LPS-induced splenocyte apoptosis and endotoxic shock. This finding indicates the existence of another caspase 1-dependent mediator of endotoxemia. Reduced serum high mobility group box 1 (HMGB1) levels in caspase 1-deficient mice correlated with their resistance to LPS. A critical role for HMGB1 in endotoxemia was confirmed when mice deficient for IL-1β and IL-18 were protected from a lethal dose of LPS by pretreatment with HMGB1-neutralizing Abs. We found that HMGB1 secretion from LPS-primed macrophages required the inflammasome components apoptotic speck protein containing a caspase activation and recruitment domain (ASC), caspase 1 and Nalp3, whereas HMGB1 secretion from macrophages infected in vitro with Salmonella typhimurium was dependent on caspase 1 and Ipaf. Thus, HMGB1 secretion, which is critical for endotoxemia, occurs downstream of inflammasome assembly and caspase 1 activation.
PMCID: PMC3428148  PMID: 20802146

Results 1-25 (1193453)