Increased expression of CD95-ligand and other apoptotic signaling factors by fumonisin B1, a hepatotoxic mycotoxin, in livers of mice lacking tumor necrosis factor alpha

Fumonisin B1 (FB1), a mycotoxin, is a potent inhibitor of ceramide synthase, and produces organ-, species-, and even gender-specific toxic responses in animals. The hepatotoxic response of FB1 in mice involves accumulation of free sphingoid bases and induction of inflammatory cytokines including tumor necrosis factor alpha (TNFalpha). The FB1-induced hepatotoxic responses were reduced in mice lacking TNFalpha receptor (TNFR) 1 or TNFR2. However, the hepatotoxicity was exacerbated in mice lacking TNFalpha. We therefore investigated the modulation of various other apoptotic signaling factors in TNFalpha-knockout (TKO) mice compared to wild-type (WT) strain after repeated daily subcutaneous injections of 2.25 mg/kg FB1 treatment for 5 days. Expression of various signaling genes in liver was evaluated by ribonuclease protection assay. Expression of CD95-ligand (FasL) was more than doubled in TKO animals after FB1 whereas it was unaltered in the WT group. FB1 did not alter CD95 expression in either strain; however, expressions of TRAIL, and downstream signaling factors FADD, TRADD, and caspase 8 were higher in FB1-treated TKO mice than in the corresponding WT animals. The TKO strain had a higher constitutive expression of apoptotic factors except CD95L. In addition to the CD95 and TNFalpha systems, the expression of apoptotic molecules bcl-2, b-myc, c-myc, bax, max, mad and IL1alpha was induced by FB1 in TKO mice to a greater extent than in WT animals; many of these factors also had a higher constitutive expression in TKO animals than WT mice. Results indicated that FB1 can induce CD95 modulated signaling when TNFalpha is absent. Differential constitutive expression of apoptotic genes in TKO mice may explain their increased sensitivity to FB1. These results are important in characterizing the modulating effect of TNFalpha on apoptotic signaling and in explaining the unexpected sensitivity of mice lacking this cytokine in response to hepatotoxic xenobiotics.     

Role of Inositol Trisphosphate Receptors in Autophagy in DT40 Cells

Previous studies have shown that small interfering RNA knockdown and pharmacological inhibition of inositol 1,4,5-trisphosphate receptors (IP₃Rs) stimulate autophagy. We have investigated autophagy in chicken DT40 cell lines containing targeted deletions of all three IP₃R isoforms (triple knock-out (TKO) cells). Using gel shifts of microtubule-associated protein 1 light chain 3 as a marker of autophagy, we find that TKO cells have enhanced basal autophagic flux even under nutrient-replete conditions. Stable DT40 cell lines derived from TKO cells containing the functionally inactive D2550A IP₃R mutant did not suppress autophagy in the same manner as wild-type receptors. This suggests that the channel function of the receptor is important in its regulatory role in autophagy. There were no marked differences in the phosphorylation state of AMP-activated protein kinase, Akt, or mammalian target of rapamycin between wild-type and TKO cells. The amount of immunoprecipitated complexes of Bcl-2-Beclin-1 and Beclin-1-Vps34 were also not different between the two cell lines. The major difference noted was a substantially decreased mTORC1 kinase activity in TKO cells based on decreased phosphorylation of S6 kinase and 4E-BP1. The discharge of intracellular stores with thapsigargin stimulated mTORC1 activity (measured as S6 kinase phosphorylation) to a greater extent in wild-type than in TKO cells. We suggest that basal autophagic flux may be negatively regulated by IP₃R-dependent Ca²⁺ signals acting to maintain an elevated mTORC1 activity in wild-type cells and that Ca²⁺ regulation of this enzyme is defective in TKO cells. The protective effect of a higher autophagic flux in cells lacking IP₃Rs may play a role in the delayed apoptotic response observed in these cells.     

Serine/threonine protein kinases and apoptosis

Over the past decade, our understanding of apoptosis, or programmed cell death, has increased greatly, with the identification of some of the major components of the apoptotic programme and the processes regulating their activation. Although apoptosis is an intrinsic process present in all cells, it can be regulated by extrinsic factors, including hormones, growth factors, cell surface receptors, and cellular stress. The actions of both pro- and antiapoptotic factors are often affected by modulation of the phosphorylation status of key elements of the apoptotic process. This minireview will focus on the role of protein kinases in apoptosis. Apoptosis is a multistep process and protein kinases have been implicated both in the upstream induction phase of apoptosis and in the downstream execution stage, as the direct targets for caspases. Due to the space constraints of this review it is not possible to discuss all of the kinases involved in the apoptotic process and we have focused here on the role of the serine/threonine protein kinases. The kinases of this family that have been suggested to play a role in apoptosis are the mitogen-activated protein kinase (MAPK) family, specifically p42/44 ERK, p38 MAPK and c-Jun N-terminal kinase (JNK), cyclic AMP-dependent protein kinase (PKA), protein kinase B (PKB), or Akt and protein kinase C (PKC). We have also considered briefly the potential for the regulation of these kinases by tyrosine protein kinases, such as c-abl.     

Myrtucommulone‐A Induces both Extrinsic and Intrinsic Apoptotic Pathways in Cancer Cells

Myrtucommulone‐A is the active compound derived from Myrtus communis. The molecular targets of myrtucommulone‐A is widely unknown, which impedes its potential therapeutic use. In this study, we demonstrated the cytotoxicity of MC‐A and its potential to induce apoptosis in cancer cells. Myrtucommulone‐A was also found to be antiproliferative and strongly inhibited cancer cell migration. Eighty four apoptotic pathway genes were used to assess the effect of myrtucommulone‐A on cancer cells. Myrtucommulone‐A mediated an increase in apoptotic genes including Fas, FasL, Gadd45a, Tnf, Tnfsf12, Trp53, and caspase 4. The increase in myrtucommulone‐A dose (25 μM versus 6.25 μM) also upregulated the expression of genes, which are involved mainly in apoptosis, regulation of apoptosis, role of mitochondria in apoptotic signaling, cytokine activity, and tumor necrosis factor signaling. Our data indicate that myrtucommulone‐A could be utilized as a potential therapeutic compound with its molecular targets in apoptotic pathways.     

Identification of a network involved in thapsigargin-induced apoptosis using a library of small interfering RNA expression vectors

We describe here the construction of a library of small interfering RNA expression vectors targeted to a few hundred apoptosis-related genes and the application of this library to an investigation of thapsigargin (TG)-induced apoptosis. Thapsigargin triggers endoplasmic reticulum stress, with subsequent apoptosis, but the molecular mechanisms underlying this process are incompletely understood. Using our library, we identified three anti-apoptotic genes, namely, NOXA, E2F1, and MAPK1, in addition to already characterized genes in the apoptotic pathway. In contrast to proposals by others, our data revealed (i) that TG-induced apoptosis is associated with Apaf1 in a caspase-3- and caspase-9-independent manner; (ii) that the E2F1-PUMA pathway might be involved; and (iii) that the ERK pathway, via MAP3K8 (mitogen-activated protein kinase kinase 8), is required for the induction by TG of apoptosis. Our study demonstrates clearly that unexpected and novel genes can be identified effectively by our method, and it provides evidence for the efficacy and utility of the comprehensive analysis of signaling networks and pathways using a library of small interfering RNA expression vectors.     

A p38 inhibitor allows to dissociate differentiation and apoptotic processes triggered upon LIF withdrawal in mouse embryonic stem cells

Mouse embryonic stem cells remain pluripotent when maintained in the presence of leukemia inhibitory factor (LIF). Upon LIF withdrawal, most cells differentiate into various lineages, while some die by apoptosis within 3 days. We have analyzed the activation pattern of the mitogen-activated protein kinase (MAPK) families and characterized the expression profile of selected genes modulated during differentiation or apoptosis. We show that p38 MAPKs are activated first, during the apoptotic crisis, while extracellular-regulated kinases and c-Jun N-terminal kinases are induced after the apoptotic crisis in differentiated cells. However, by using both p38 kinase inhibitors (PD169316 and SB203580) and a p38alpha(-/-) cell line, we demonstrate that p38alpha activation is rather a consequence than a cause of apoptosis. We thus reveal novel properties of PD169316, which induces cell survival without impairing cell differentiation, and identify PD169316-sensitive targets like the fibroblast growth factor-5, Brachyury and bcl-2 genes. Finally, we demonstrate that overexpression of the PD169316 - regulated bcl-2 gene prevents LIF withdrawal - induced cell death.     

Nox1 causes ileocolitis in mice deficient in glutathione peroxidase-1 and -2

We previously reported that mice deficient in two Se-dependent glutathione peroxidases, GPx1 and GPx2, have spontaneous ileocolitis. Disease severity depends on mouse genetic background. Whereas C57BL/6J (B6) GPx1/2-double-knockout (DKO) mice have moderate ileitis and mild colitis, 129S1Svlm/J (1 2 9) DKO mice have severe ileocolitis. Because GPx’s are antioxidant enzymes, we hypothesized that elevated reactive oxygen species trigger inflammation in these DKO mice. To test whether NADPH oxidase 1 (Nox1) contributes to colitis, we generated B6 triple-KO (TKO) mice to study their phenotype. Because the Nox1 gene is X-linked, we analyzed the effects of Nox1 on male B6 TKO mice and female B6 DKO mice with the Nox1^+/− (het-TKO) genotype. We found that the male TKO and female het-TKO mice are virtually disease-free when monitored from 8 through 50 days of age. Male TKO and female het-TKO mice have nearly no signs of disease (e.g., lethargy and perianal alopecia) that are often exhibited in the DKO mice; further, the slower growth rate of DKO mice is almost completely eliminated in male TKO and female het-TKO mice. Male TKO and female het-TKO mice no longer have the shortened small intestine present in the DKO mice. Finally, the pathological characteristics of the DKO ileum, including the high level of crypt apoptosis (analyzed by apoptotic figures, TUNEL, and cleaved caspase-3 immunohistochemical staining), high numbers of Ki-67-positive crypt epithelium cells, and elevated levels of monocytes expressing myeloperoxidase, are all significantly decreased in male TKO mice. The attenuated ileal and colonic pathology is also evident in female het-DKO mice. Furthermore, the male DKO ileum has eightfold higher TNF cytokine levels than TKO ileum. Nox1 mRNA is highly elevated in both B6 and 129 DKO ileum compared to wild-type mouse ileum. Taking these results together, we propose that ileocolitis in the DKO mice is caused by Nox1, which is induced by TNF. The milder disease in female het-TKO intestine is probably due to random or imprinted X-chromosome inactivation, which produces mosaic Nox1 expression.     

c-Abl Tyrosine Kinase Mediates Neurotoxic Prion Peptide-Induced Neuronal Apoptosis via Regulating Mitochondrial Homeostasis

Prion diseases are neurodegenerative disorders characterized by the accumulation of a disease-associated prion protein and apoptotic neuronal death. Previous studies indicated that the ubiquitous expression of c-Abl tyrosine kinase transduces a variety of extrinsic and intrinsic cellular signals. In this study, we demonstrated that a synthetic neurotoxic prion fragment (PrP106-126) activated c-Abl tyrosine kinase, which in turn triggered the upregulation of MST1 and BIM, suggesting the activation of the c-Abl-BIM signaling pathway. The peptide fragment was found to result in cell death via mitochondrial dysfunction in neuron cultures. Knockdown of c-Abl using small interfering RNA protected neuronal cells from PrP106-126-induced mitochondrial dysfunction, production of reactive oxygen species, and apoptotic events inducing translocation of Bax to the mitochondria, cytochrome c release into the cytosol, and activation of caspase-9 and caspase-3. Blocking the c-Abl tyrosine kinase also prevented neuronal cells from PrP106-126-induced apoptotic morphological changes. This is the first study reporting that c-Abl tyrosine kinase as a novel upstream activator of MST1 and BIM plays an important role in prion-induced neuron apoptosis via mitochondrial dysfunction. Our findings suggest that c-Abl tyrosine kinase is a potential therapeutic target for prion disease.     

Ras protects Rb family null fibroblasts from cell death: a role for AP-1

The retinoblastoma protein (Rb) controls cell proliferation, differentiation, and senescence and provides an essential tumor suppressive function that cells must eliminate to attain unlimited proliferative potential. Elimination of the Rb pathway also results in apoptosis, however, thereby providing an efficient surveillance mechanism to sense the loss of Rb. To become tumorigenic cells must thus overcome not only Rb function but also the apoptotic response caused by the loss of Rb function. We show that oncogenic Ras (RasV12) potently blocks cell death in Rb family member knockout mouse embryo fibroblasts (TKO cells). Activation of phosphatidylinositol 3-kinase and Raf by oncogenic Ras mediated this protection, implying that multiple Ras effector pathways are required, in concert, for this pro-survival signal. Although activation of Raf by selective Ras mutants protected TKO cells from cell death, pharmacologic inhibition of MEK had little effect on RasV12 protection, suggesting that a Raf-dependent, MEK-independent pathway was important for this effect. We show that this Raf-dependent protection occurred through activation of c-Jun and thus AP-1 activation. These observations could account for the dependence of Ras transformation on c-Jun activity and for the roles of AP-1 in oncogenesis. Our results support the concept of two oncogenic events cooperating to achieve a balance between immortalization and survival.     

Apoptosis and differentiation commitment: novel insights revealed by gene profiling studies in mouse embryonic stem cells

Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF). LIF starvation leads to apoptosis of some of the ES-derived differentiated cells, together with p38alpha mitogen-activated protein kinase (MAPK) activation. Apoptosis, but not morphological cell differentiation, is blocked by a p38 inhibitor, PD169316. To further understand the mechanism of action of this compound, we have identified its specific targets by microarray studies. We report on the global expression profiles of genes expressed at 3 days upon LIF withdrawal (d3) compared to pluripotent cells and of genes whose expression is modulated at d3 under anti-apoptotic conditions. We showed that at d3 without LIF cells express, earlier than anticipated, specialized cell markers and that when the apoptotic process was impaired, expression of differentiation markers was altered. In addition, functional tests revealed properties of anti-apoptotic proteins not to alter cell pluripotency and a novel role for metallothionein 1 gene, which prevents apoptosis of early differentiated cells.     

The selective inhibition of nuclear PKCζ restores the effectiveness of chemotherapeutic agents in chemoresistant cells

The atypical protein kinase C (PKC) isoform zeta (PKCζ) has been implicated in the intracellular transduction of mitogenic and apoptotic signals by acting on different signaling pathways. The key role of these processes in tumorigenesis suggests a possible involvement of PKCζ in this event. PKCζ is activated by cytotoxic treatments, inhibits apoptotic cell death and reduces the sensitivity of cancer cells to chemotherapeutic agents. Here, using pharmacological and DNA recombinant approaches, we show that oxidative stress triggers nuclear translocation of PKCζ and induces resistance to apoptotic agents. Accordingly, chemoresistant cells show accumulation of PKCζ within the nucleus, and a nuclear-targeted PKCζ transfected in tumor cells decreases sensitivity to apoptosis. We thus developed a novel recombinant protein capable of selectively inhibiting the nuclear fraction of PKCζ that restored the susceptibility to apoptosis in cells in which PKCζ was enriched in the nuclear fraction, including chemoresistant cells. These findings establish the importance of PKCζ as a possible target to increase the effectiveness of anticancer therapies and highlight potential sites of intervention.     

MK-STYX, a catalytically inactive phosphatase regulating mitochondrially dependent apoptosis

Evasion of apoptosis is a significant problem affecting an array of cancers. In order to identify novel regulators of apoptosis, we performed an RNA interference (RNAi) screen against all kinases and phosphatases in the human genome. We identified MK-STYX (STYXL1), a catalytically inactive phosphatase with homology to the mitogen-activated protein kinase (MAPK) phosphatases. Despite this homology, MK-STYX knockdown does not significantly regulate MAPK signaling in response to growth factors or apoptotic stimuli. Rather, RNAi-mediated knockdown of MK-STYX inhibits cells from undergoing apoptosis induced by cellular stressors activating mitochondrion-dependent apoptosis. This MK-STYX phenotype mimics the loss of Bax and Bak, two potent guardians of mitochondrial apoptotic potential. Similar to loss of both Bax and Bak, cells without MK-STYX expression are unable to release cytochrome c. Proapoptotic members of the BCL-2 family (Bax, Bid, and Bim) are unable to trigger cytochrome c release in MK-STYX-depleted cells, placing the apoptotic deficiency at the level of mitochondrial outer membrane permeabilization (MOMP). MK-STYX was found to localize to the mitochondria but is neither released from the mitochondria upon apoptotic stress nor proximal to the machinery currently known to control MOMP, indicating that MK-STYX regulates MOMP using a distinct mechanism.     

Wing-to-Leg homeosis by spineless causes apoptosis regulated by Fish-lips, a novel leucine-rich repeat transmembrane protein

Growth, patterning, and apoptosis are mutually interactive during development. For example, cells that select an abnormal fate in a developing field are frequently removed by apoptosis. An important issue in this process that needs to be resolved is the mechanism used by cells to discern their correct fate from an abnormal fate. In order to examine this issue, we developed an animal model that expresses the dioxin receptor homolog Spineless (Ss) ectopically in the Drosophila wing. The presence of mosaic clones ectopically expressing ss results in a local transformation of organ identity, homeosis, from wing into a leg or antenna. The cells with misspecified fates subsequently activate c-Jun N-terminal kinase to undergo apoptosis in an autonomous or nonautonomous manner depending on their position within the wing, suggesting that a cell-cell interaction is, at least in some cases, involved in the detection of misspecified cells. Similar position dependence is commonly observed when various homeotic genes controlling the body segments are ectopically expressed. The autonomous and nonautonomous apoptosis caused by ss is regulated by a novel leucine-rich repeat family transmembrane protein, Fish-lips (Fili) that interacts with surrounding normal cells. These data support a mechanism in which the lack of some membrane proteins helps to recognize the presence of different cell types and direct these cells to an apoptotic fate in order to exclude them from the normal developing field.