ASK1 regulates influenza virus infection-induced apoptotic cell death

Apoptosis occurs in influenza virus (IV)-infected cells. There are a number of mechanisms for the regulation of apoptosis. However, the molecular mechanism of IV infection-induced apoptosis is still controversial. Apoptosis signal-regulating kinase1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the SEK1-c-Jun N-terminal kinase (JNK) and MKK3/MKK6-p38 MAPK signaling cascades. ASK1 has been implicated in cytokine- and stress-induced apoptosis. Here, we show the following: (1) IV infection activated ASK1 and concomitantly phosphorylated JNK and p38 MAPK in human bronchial epithelial cells; (2) the activation of JNK and p38 MAPK but not extracellular-regulated kinase (ERK) in embryonic fibroblasts (MEFs) derived from ASK1 knockout mice (ASK1(-/-) MEFs) was depressed compared to MEFs derived from wild type mice (ASK1(+/+) MEFs); and (3) ASK1(-/-) MEFs were defective in IV infection-induced caspase-3 activation and cell death. These results indicate that apoptosis in IV-infected BEC is mediated through ASK1-dependent cascades.     

Impaired autophagy due to constitutive mTOR activation sensitizes TSC2-null cells to cell death under stress

It has been well documented that cells deficient in either TSC1 or TSC2 are highly sensitive to various cell death stimuli. In this study, we utilized the TSC2 (-/-) mouse embryonic fibroblasts (MEFs) to study the involvement of autophagy in the enhanced susceptibility of TSC2-null cells to cell death. We first confirmed that both TSC1-null and TSC2-null MEFs are more sensitive to apoptosis in response to amino acid starvation (EBSS) and hypoxia. Second, we found that both the basal and inducible autophagy in TSC2 (-/-) MEFs is impaired, mainly due to constitutive activation of mTORC1. Third, suppression of autophagy by chloroquine and Atg7 knockdown sensitizes TSC2 (+/+) cells, but not TSC2 (-/-) cells, to EBSS-induced cell death. Conversely, the inhibition of mTORC1 by raptor knockdown and rapamycin activates autophagy and subsequently rescues TSC2 (-/-) cells. Finally, in starved cells, nutrient supplementations (insulin-like growth factor-1 (IGF-1) and leucine) enhanced cell death in TSC2 (-/-) cells, but reduced cell death in TSC2 (+/+) cells. Taken together, these data indicate that constitutive activation of mTORC1 in TSC2 (-/-) cells leads to suppression of autophagy and enhanced susceptibility to stress-mediated cell death. Our findings thus provide new insights into the complex relationships among mTOR, autophagy and cell death, and support the possible autophagy-targeted intervention strategies for the treatment of TSC-related pathologies.     

An adenosine triphosphatase of the sucrose nonfermenting 2 family, androgen receptor-interacting protein 4, is essential for mouse embryonic development and cell proliferation

An adenosine triphosphatase of the sucrose nonfermenting 2 protein family, androgen receptor-interacting protein 4 (ARIP4), modulates androgen receptor activity. To elucidate receptor-dependent and -independent functions of ARIP4, we have analyzed Arip4 gene-targeted mice. Heterozygous Arip4 mutants were normal. Arip4 is expressed mainly in the neural tube and limb buds during early embryonic development. Arip4-/- embryos were abnormal already at embryonic d 9.5 (E9.5) and died by E11.5. At E9.5 and E10.5, almost all major tissues of Arip4-null embryos were proportionally smaller than those of wild-type embryos, and the neural tube was shrunk in some Arip4-/- embryos. Dramatically reduced cell proliferation and increased apoptosis were observed in E9.5 and E10.5 Arip4-null embryos. Mouse embryonic fibroblasts (MEFs) isolated from Arip4-/- embryos ceased to grow after two to three passages and exhibited increased apoptosis and decreased DNA synthesis compared with wild-type MEFs. Comparison of gene expression profiles of Arip4-/- and wild-type MEFs at E9.5 revealed that putative ARIP4 target genes are involved in cell growth and proliferation, apoptosis, cell death, DNA replication and repair, and development. Collectively, ARIP4 plays an essential role in mouse embryonic development and cell proliferation, and it appears to coordinate multiple essential biological processes, possibly through a complex chromatin remodeling system.     

The inhibitor of growth protein 5 (ING5) depends on INCA1 as a co-factor for its antiproliferative effects

The proteins of the Inhibitor of Growth (ING) family are involved in multiple cellular functions such as cell cycle regulation, apoptosis, and chromatin remodeling. For ING5, its actual role in growth suppression and the necessary partners are not known. In a yeast-two-hybrid approach with human bone marrow derived cDNA, we identified ING5 as well as several other proteins as interaction partners of Inhibitor of cyclin A1 (INCA1) that we previously characterized as a novel interaction partner of cyclin A1/CDK2. ING5 expression in leukemic AML blasts was severely reduced compared to normal bone marrow. In line, ING5 inhibited bone marrow colony formation upon retroviral transduction. However, Inca1(-/-) bone marrow colony formation was not suppressed by ING5. In murine embryonic fibroblast (MEF) cells from Inca1(+/+) and Inca1(-/-) mice, overexpression of ING5 suppressed cell proliferation only in the presence of INCA1, while ING5 had no effect in Inca1(-/-) MEFs. ING5 overexpression induced a delay in S-phase progression, which required INCA1. Finally, ING5 overexpression enhanced Fas-induced apoptosis in Inca1(+/+) MEFs, while Inca1(-/-) MEFs were protected from Fas antibody-induced apoptosis. Taken together, these results indicate that ING5 is a growth suppressor with suppressed expression in AML whose functions depend on its interaction with INCA1.     

Embryonic fibroblasts from Gpx4+/- mice: a novel model for studying the role of membrane peroxidation in biological processes

A previous study using mice null for Gpx4 indicates that PHGPx plays a critical role in antioxidant defense and is essential for the survival of the mouse. In the present study, we further analyzed the stress response of MEFs (murine embryonic fibroblasts) derived from mice heterozygous for the Gpx4 gene (Gpx4(+/-) mice). MEFs from Gpx4(+/-) mice have a 50% reduction in PHGPx expression without any changes in the activities of other major antioxidant defense enzymes. Compared to MEFs from Gpx4(+/+) mice, MEFs from Gpx4(+/-) mice were more sensitive to exposure to the oxidizing agent t-butyl hydroperoxide (t-BuOOH), and t-BuOOH exposure induced increased apoptosis in MEFs from Gpx4(+/-) mice. When cultured at low cell density, MEFs from Gpx4(+/-) mice also showed retarded growth under normal culture conditions (20% oxygen) that was reversed by culturing under low oxygen (2% oxygen). In addition, oxidative damage was increased in the MEFs from the Gpx4(+/-) mice, as indicated by increased levels of F(2)-isoprostanes and 8-oxo-2-deoxyguanosine in these cells. Our data demonstrate that MEFs from Gpx4(+/-) mice are more sensitive to oxidative stress because of reduced expression of PHGPx.     

CIB1 is essential for mouse spermatogenesis

CIB1 is a 22-kDa calcium binding, regulatory protein with approximately 50% homology to calmodulin and calcineurin B. CIB1 is widely expressed and binds to a number of effectors, such as integrin alphaIIb, PAK1, and polo-like kinases, in different tissues. However, the in vivo functions of CIB1 are not well understood. To elucidate the function of CIB1 in whole animals, we used homologous recombination in embryonic stem cells to generate Cib1(-/-) mice. Although Cib1(-/-) mice grow normally, the males are sterile due to disruption of the haploid phase of spermatogenesis. This is associated with reduced testis size and numbers of germ cells in seminiferous tubules, increased germ cell apoptosis, and the loss of elongated spermatids and sperm. Cib1(-/-) testes also show increased mRNA and protein expression of the cell cycle regulator Cdc2/Cdk1. In addition, mouse embryonic fibroblasts (MEFs) derived from Cib1(-/-) mice exhibit a much slower growth rate compared to Cib1(+/+) MEFs, suggesting that CIB1 regulates the cell cycle, differentiation of spermatogenic germ cells, and/or differentiation of supporting Sertoli cells.     

Pivotal role of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 in apoptosis and autophagy

Programmed cell death (PCD) is involved in a variety of biologic events. Based on the morphologic appearance of the cells, there are two types of PCD as follows: apoptotic (type I) and autophagic (type II). However, the molecular machinery that determines the type of PCD is poorly defined. The purpose of this study was to show whether the presence of the cyclin-dependent kinase (CDK) inhibitor p21(WAF1/CIP1), a modulator of apoptosis, determines which type of PCD the cell undergoes. Treatment with C(2)-ceramide was associated with both the cleavage of caspase-3 and poly(ADP-ribose) polymerase and the degradation of autophagy-related Beclin 1 and Atg5 proteins, without a change in the cyclin-CDK activity, which culminated in apoptosis in p21(+/+) mouse embryonic fibroblasts (MEFs). On the other hand, C(2)-ceramide did not cleave caspase-3 or poly(ADP-ribose) polymerase and kept Beclin 1 and Atg5 proteins stable in p21(-/-) MEFs, events that this time culminated in autophagy. When expression of the p21 protein was inhibited by small interfering RNA or when the overexpression of Beclin 1 or Atg5 was induced, autophagy rather than apoptosis was initiated in the p21(+/+) MEFs treated with C(2)-ceramide. In contrast, the exogenous expression of p21 or the silencing of Beclin 1 and Atg5 with small interfering RNA increased the number of apoptotic cells and decreased the number of autophagic cells among C(2)-ceramide-treated p21(-/-) MEFs. gamma-Irradiation, which endogenously generates ceramide, induced a similar tendency in these MEFs. These results suggest that p21 plays an essential role in determining the type of cell death, positively for apoptosis and negatively for autophagy.     

p53 Retards cell-growth and suppresses etoposide-induced apoptosis in Pin1-deficient mouse embryonic fibroblasts

We studied the effects of Pin1, a regulatory molecule of the oncosuppressor p53, on both cell cycle arrest and apoptosis by treating primary mouse embryonic fibroblasts (MEFs) with etoposide. Etoposide induced G1 arrest in both wild-type and Pin1 null (pin1(-/-)) MEFs, and G2/M arrest and apoptotic cell death in MEFs lacking either p53 only (p53(-/-)) or both Pin1 and p53 (pin1(-/-)p53(-/-)). Both pin1(-/-) and pin1(-/-)p53(-/-) MEFs were enhanced the release of cytochrome c from the mitochondria, which might induce apoptosis. In response to etoposide treatment, apoptotic cell death was displayed in pin1(-/-)p53(-/-) MEFs but not in pin1(-/-) MEFs. These results suggest that p53 retards growth and suppresses etoposide-induced apoptosis in pin1(-/-) MEFs.     

Resistance to vesicular stomatitis virus infection requires a functional cross talk between the eukaryotic translation initiation factor 2alpha kinases PERK and PKR

Phosphorylation of the alpha (alpha) subunit of the eukaryotic translation initiation factor 2 (eIF2) leads to the inhibition of protein synthesis in response to diverse stress conditions, including viral infection. The eIF2alpha kinase PKR has been shown to play an essential role against vesicular stomatitis virus (VSV) infection. We demonstrate here that another eIF2alpha kinase, the endoplasmic reticulum-resident protein kinase PERK, contributes to cellular resistance to VSV infection. We demonstrate that mouse embryonic fibroblasts (MEFs) from PERK(-/-) mice are more susceptible to VSV-mediated apoptosis than PERK(+/+) MEFs. The higher replication capacity of VSV in PERK(-/-) MEFs results from their inability to attenuate viral protein synthesis due to an impaired eIF2alpha phosphorylation. We also show that VSV-infected PERK(-/-) MEFs are unable to fully activate PKR, suggesting a cross talk between the two eIF2alpha kinases in virus-infected cells. These findings further implicate PERK in virus infection, and provide evidence that the antiviral and antiapoptotic roles of PERK are mediated, at least in part, via the activation of PKR.     

In mouse embryonic fibroblasts, neither caspase-8 nor cellular FLICE-inhibitory protein (FLIP) is necessary for TNF to activate NF-κB, but caspase-8 is required for TNF to cause cell death, and induction of FLIP by NF-κB is required to prevent it

Binding of TNF to TNF receptor-1 can give a pro-survival signal through activation of p65/RelA NF-κB, but also signals cell death. To determine the roles of FLICE-inhibitory protein (FLIP) and caspase-8 in TNF-induced activation of NF-κB and apoptosis, we used mouse embryonic fibroblasts derived from FLIP and caspase-8 gene-deleted mice, and treated them with TNF and a smac-mimetic compound that causes degradation of cellular inhibitor of apoptosis proteins (cIAPs). In cells treated with smac mimetic, TNF and Fas Ligand caused wild-type and FLIP(-/-) MEFs to die, whereas caspase-8(-/-) MEFs survived, indicating that caspase-8 is necessary for death of MEFs triggered by these ligands when IAPs are degraded. By contrast, neither caspase-8 nor FLIP was required for TNF to activate p65/RelA NF-κB, because IκB was degraded, p65 translocated to the nucleus, and an NF-κB reporter gene activated normally in caspase-8(-/-) or FLIP(-/-) MEFs. Reconstitution of FLIP(-/-) MEFs with the FLIP isoforms FLIP-L, FLIP-R, or FLIP-p43 protected these cells from dying when treated with TNF or FasL, whether or not cIAPs were depleted. These results show that in MEFs, caspase-8 is necessary for TNF- and FasL-induced death, and FLIP is needed to prevent it, but neither caspase-8 nor FLIP is required for TNF to activate NF-κB.     

Deletion of the dual specific phosphatase-4 (DUSP-4) gene reveals an essential non-redundant role for MAP kinase phosphatase-2 (MKP-2) in proliferation and cell survival

Mitogen-activated protein kinase phosphatase-2 (MKP-2) is a type 1 nuclear dual specific phosphatase (DUSP) implicated in a number of cancers. We examined the role of MKP-2 in the regulation of MAP kinase phosphorylation, cell proliferation, and survival responses in mouse embryonic fibroblasts (MEFs) derived from a novel MKP-2 (DUSP-4) deletion mouse. We show that serum and PDGF induced ERK-dependent MKP-2 expression in wild type MEFs but not in MKP-2(-/-) MEFs. PDGF stimulation of sustained ERK phosphorylation was enhanced in MKP-2(-/-) MEFs, whereas anisomycin-induced JNK was only marginally increased. However, marked effects upon cell growth parameters were observed. Cellular proliferation rates were significantly reduced in MKP-2(-/-) MEFs and associated with a significant increase in cell doubling time. Infection with adenoviral MKP-2 reversed the decrease in proliferation. Cell cycle analysis revealed a block in G(2)/M phase transition associated with cyclin B accumulation and enhanced cdc2 phosphorylation. MEFs from MKP-2(-/-) mice also showed enhanced apoptosis when stimulated with anisomycin correlated with increased caspase-3 cleavage and γH2AX phosphorylation. Increased apoptosis was reversed by adenoviral MKP-2 infection and correlated with selective inhibition of JNK signaling. Collectively, these data demonstrate for the first time a critical non-redundant role for MKP-2 in regulating cell cycle progression and apoptosis.     

The tumor suppressor neurofibromin confers sensitivity to apoptosis by Ras-dependent and Ras-independent pathways

Neurofibromatosis type 1 (NF1) is characterized by a high incidence of benign and malignant tumors attributed to loss of function of Nf1, which encodes neurofibromin, a tumor suppressor with Ras-GAP activity. Neurofibromin deficiency typically causes chronic activation of Ras, considered the major contributor to manifestation of NF1. Resistance to radio- and chemotherapy are typical of NF1-associated tumors, but the underlying mechanism is unknown. Here, we investigated interrelationships between neurofibromin expression, Ras activity, and sensitivity to apoptosis. Neurofibromin-deficient mouse embryonic fibroblasts (MEFs) and human NF1 tumor cells were more resistant than neurofibromin-expressing cells to apoptosis. Moreover, Nf1(-/-), Nf1(+/-), and Nf1(+/+) MEFs exhibited gene-dosage-related resistance to apoptosis. Resistance of the Nf1-deficient cells was mediated by two survival pathways: a Ras-dependent pathway, and a Ras-independent pathway promoted by the lack of an NF1-GRD-independent proapoptotic action of neurofibromin. Therefore, besides its Ras-dependent growth inhibition, neurofibromin can exert tumor suppression via a proapoptotic effect.