The Drosophila Inhibitor of Apoptosis (IAP) DIAP2 Is Dispensable for Cell Survival, Required for the Innate Immune Response to Gram-negative Bacterial Infection, and Can Be Negatively Regulated by the Reaper/Hid/Grim Family of IAP-binding Apoptosis Inducers

Many inhibitor of apoptosis (IAP) family proteins inhibit apoptosis. IAPs contain N-terminal baculovirus IAP repeat domains and a C-terminal RING ubiquitin ligase domain. Drosophila IAP DIAP1 is essential for the survival of many cells, protecting them from apoptosis by inhibiting active caspases. Apoptosis initiates when proteins such as Reaper, Hid, and Grim bind a surface groove in DIAP1 baculovirus IAP repeat domains via an N-terminal IAP-binding motif. This evolutionarily conserved interaction disrupts DIAP1-caspase interactions, unleashing apoptosis-inducing caspase activity. A second Drosophila IAP, DIAP2, also binds Rpr and Hid and inhibits apoptosis in multiple contexts when overexpressed. However, due to a lack of mutants, little is known about the normal functions of DIAP2. We report the generation of diap2 null mutants. These flies are viable and show no defects in developmental or stress-induced apoptosis. Instead, DIAP2 is required for the innate immune response to Gram-negative bacterial infection. DIAP2 promotes cytoplasmic cleavage and nuclear translocation of the NF-{kappa}B homolog Relish, and this requires the DIAP2 RING domain. Increasing the genetic dose of diap2 results in an increased immune response, whereas expression of Rpr or Hid results in down-regulation of DIAP2 protein levels. Together these observations suggest that DIAP2 can regulate immune signaling in a dose-dependent manner, and this can be regulated by IBM-containing proteins. Therefore, diap2 may identify a point of convergence between apoptosis and immune signaling pathways

Fetal-derived trophoblast use the apoptotic cytokine tumor necrosis factor-alpha-related apoptosis-inducing ligand to induce smooth muscle cell death.

Remodeling of the uterine spiral arteries during pregnancy transforms them from high to low resistance vessels that lack vasoconstrictive properties. This process is essential to meet the demand for increased blood flow imposed by the growing fetus. Loss of endothelial and smooth muscle cells (SMC) is evident in remodeled arteries but the mechanisms underlying this transformation remain unknown. This study investigated the hypothesis that fetal trophoblast invading from the placenta instigate remodeling by triggering cell death in vascular SMC. Specifically, a role for trophoblast-derived death inducing cytokine tumor necrosis factor-α–related apoptosis-inducing ligand (TRAIL) was investigated. Expression of the activating TRAIL receptors R1 and R2 was detected by flow cytometry on human aortic SMC and by immunohistochemistry on spiral artery SMC. Recombinant human TRAIL induced human aortic SMC apoptosis, which was inhibited by antibodies against TRAIL-R1 or -R2. Perfusion of denuded spiral artery segments with recombinant human TRAIL also induced SMC apoptosis. Trophoblasts isolated from first trimester placenta expressed membrane-associated TRAIL and induced apoptosis of human aortic SMC; apoptosis was significantly inhibited by a recombinant human TRAIL-R1:Fc construct. Trophoblast within the first trimester placental bed also expressed TRAIL. These data show that: 1) TRAIL causes SMC death; 2) trophoblast produce the apoptotic cytokine TRAIL; and 3) trophoblast induce SMC apoptosis via a TRAIL-dependent mechanism. We conclude that TRAIL produced by trophoblast causes apoptosis of SMC and thus may contribute to SMC loss during spiral artery remodeling in pregnancy

Constitutively Active Galpha q and Galpha 13 Trigger Apoptosis through Different Pathways

We investigated the effect of expression of constitutively active Galpha mutants on cell survival. Transfection of constitutively active Galphaq and Galpha13 in two different cell lines caused condensation of genomic DNA and nuclear fragmentation. Endonuclease cleavage of genomic DNA was followed by labeling the DNA fragments and subsequent flow cytometric analysis. The observed cellular phenotype was identical to the phenotype displayed by cells undergoing apoptosis. To distinguish between the apoptosis-inducing ability of the two Galpha-subunits, the signaling pathways involved in this cellular function were investigated. Whereas Galpha q induced apoptosis via a protein kinaseC-dependent pathway, Galpha13 caused programmed cell death through a pathway involving the activation of the small G-protein Rho. Both of the pathways leading to apoptosis were blocked by overexpression of bcl-2. In contrast to other apoptosis-inducing systems, expression of constitutively active Galphaq and Galpha13 triggered apoptosis in high serum as well as in defined medium

A cardinal role for cathepsin D in co-ordinating the host-mediated apoptosis of macrophages and killing of pneumococci

The bactericidal function of macrophages against pneumococci is enhanced by their apoptotic demise, which is controlled by the anti-apoptotic protein Mcl-1. Here, we show that lysosomal membrane permeabilization (LMP) and cytosolic translocation of activated cathepsin D occur prior to activation of a mitochondrial pathway of macrophage apoptosis. Pharmacological inhibition or knockout of cathepsin D during pneumococcal infection blocked macrophage apoptosis. As a result of cathepsin D activation, Mcl-1 interacted with its ubiquitin ligase Mule and expression declined. Inhibition of cathepsin D had no effect on early bacterial killing but inhibited the late phase of apoptosis-associated killing of pneumococci in vitro. Mice bearing a cathepsin D-/- hematopoietic system demonstrated reduced macrophage apoptosis in vivo, with decreased clearance of pneumococci and enhanced recruitment of neutrophils to control pulmonary infection. These findings establish an unexpected role for a cathepsin D-mediated lysosomal pathway of apoptosis in pulmonary host defense and underscore the importance of apoptosis-associated microbial killing to macrophage function

Effect of Insulin-Like Growth Factor-1 (IGF-1) on the expression of NFκB and cFLIP in Bovine Granulosa Cells

Infertility, often attributed to follicular atresia, is a growing problem in the agricultural industry. Programmed cell death, also known as apoptosis, is a contributing factor of follicular atresia. It occurs in both the granulosa cells and the oocyte that comprise ovarian follicles. Here, mechanisms influencing the process of apoptosis, via the death receptor Fas, were explored using bovine granulosa cells (bGCs) because Fas-induced apoptosis is a plausible mechanism of follicular atresia. Cell culture techniques, optimized for bGCs, were developed and used throughout the current study. In brief, cultures of bGCs were exposed to Fas ligand (100ng/mL) for 24 hrs. This induced cell death, as measured by MTS assay (p=0.024, n=3 experiments). Subsequent experiments in which doses of insulin-like growth factor-1 (IGF-1) were co-administered indicated that 100ng/ml IGF-1 provides the greatest protection against Fas-induced apoptosis (p= 0.001, n=3 experiments). Currently, we are testing the hypothesis that IGF-1 protects bGCs from Fas-induced apoptosis by stimulating the expression of cellular FLICE-like inhibitory protein (cFLIP) and the activation of nuclear factor-κB (NFκB), two molecules thought to protect granulosa cells from apoptosis. IGF-1-stimulated expression of cFLIP and NFκB will be assessed by immunoblots and in-cell western assays. This project was supported by the Hamel Center for Undergraduate Research (SD) and USDA grant no. 2013-67016-21071 (DHT)

Scotin, a novel p53-inducible proapoptotic protein located in the ER and the nuclear membrane

p53 is a transcription factor that induces growth arrest or apoptosis in response to cellular stress. To identify new p53-inducible proapoptotic genes, we compared, by differential display, the expression of genes in spleen or thymus of normal and p53 nullizygote mice after γ-irradiation of whole animals. We report the identification and characterization of human and mouse Scotin homologues, a novel gene directly transactivated by p53. The Scotin protein is localized to the ER and the nuclear membrane. Scotin can induce apoptosis in a caspase-dependent manner. Inhibition of endogenous Scotin expression increases resistance to p53-dependent apoptosis induced by DNA damage, suggesting that Scotin plays a role in p53-dependent apoptosis. The discovery of Scotin brings to light a role of the ER in p53-dependent apoptosis

Kaposi's sarcoma-associated herpesvirus oncoprotein K13 protects against B cell receptor induced growth arrest and apoptosis through NF-κB activation

Kaposi's sarcoma-associated herpesvirus (KSHV) has been linked to the development of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease (MCD). We have characterized the role of KSHV-encoded viral FLICE inhibitory protein K13 in the modulation of anti-IgM induced growth arrest and apoptosis in B cells. We demonstrate that K13 protects WEHI 231, an immature B cell line, against anti-IgM induced growth arrest and apoptosis. The protective effect of K13 was associated with the activation of the NF-κB pathway and was deficient in its mutant, K13-58AAA, and a structural homolog, vFLIP E8, which lack NF-κB activity. K13 upregulated the expression of NF-κB subunit RelB and blocked the anti-IgM induced decline in c-Myc and rise in p27(Kip1) that have been associated with growth arrest and apoptosis. K13 also upregulated the expression of Mcl-1, an anti-apoptotic member of the Bcl2 family. Finally, K13 protected the mature B cell line Ramos against anti-IgM induced apoptosis through NF-κB activation. Inhibition of anti-IgM induced apoptosis by K13 may contribute to the development of KSHV-associated lymphoproliferative disorders

Effect of Vitamin E on Oocytes Apoptosis in nicotine-treated Mice

Objective(s): Cigarette and nicotine enhances embryogenesis, fertility, pregnancy loss and ultrastructure alterations of oocyte. This study was performed to determine the effect of daily supplementation of vitamin E on oocytes apoptosis in nicotine-treated mice. Materials and Methods: In this experimental study, 24 NMARI adult female mice were randomly allocated into four experimental groups. For 30 days, animals in control group (C) were received saline through subcutaneous injection, group I received vitamin E (60 mg/kg/day orally), group II received nicotine (5 mg/kg/day, subcutaneous) and animals of group III received nicotine with vitamin E (60 mg/kg/day orally). After 30 days, the animals were superovulated with PSMG (10 Units) and HCG (10 Units). Next day animals were sacrificed and oocytes were flushed. Collected oocytes were examined through TUNEL assay for the determination of apoptosis through the use of fluorescent microscope. Results: The number of retrieved oocytes was 139, 148, 97 and 127 in control, experimental group I, II and III, respectively. Nicotine treatment increased apoptosis in oocytes up to 13.4% whereas oocytes apoptosis was 3.6% in controls. Supplementation with vitamin E in nicotine-treated mice reduced the oocytes apoptosis to 5.5%. Conclusion: This study showed that nicotine exposure (5 mg/kg/day for 30 days) can increase apoptosis in oocytes, and supplementation with vitamin E (60 mg/kg/day orally) can reduce the oocytes apoptosis in nicotine-treated mice

Bioactive Compounds From Torbangun [Plectranthus Amboinicus (Lour.) Spreng] Chloroform Fraction Induce Apoptosis in Breast Cancer (Mcf-7 Cells) in Vitro

Torbangun (Plectranthus amboinicus (Lour.) Spreng) is a medicinal plant that has been traditionally used in tropical countries to cure various illnesses. The objective of this study was to identify the active compounds in the chloroform fraction which have effect on the apoptosis-related genes expression of breast cancer MCF-7 cells. Apoptosis was observed morphologically using Hoechst nuclear staining. Expression of the genes was analyzed using Real-Time PCR. Chemical compounds of the plant fractions were determined using LC-MS. Result of cell morphology observation clearly indicated apoptosis after the treatment of the plant fraction. Increased expression of anti-apoptotic gene Bcl-2 could not prevent the cells from apoptosis. Expressions of p53 and p21 genes were increased significantly. The expressions of caspase 9, caspase 7 and caspase 1 were increased at concentration-dependent manner. Most of the compounds in the chloroform fraction are identified as diterpenoids which may contribute to the apoptosis inducing activity of the fraction

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Apoptosis is one type of programmed cell death, important during tissue development and to maintain the tissue homeostasis. Apoptosis comprises a complex network of internal signaling pathways, and an important part of this signaling network is the action of voltage‐gated ion channels. The aim of this thesis was to explore the role of ion channels and the role of intracellular metal ions during apoptosis in Xenopus laevis oocytes. The reasons for using these oocytes are that they are large, robust, easy to handle, and easy to study electrophysiologically. Apoptosis was induced either chemically by incubation of the oocytes in staurosporine (STS) or mechanically by centrifugation of the oocytes. Ion currents were measured by a two‐electrode voltage clamp technique, intracellular ion concentrations were measured either directly by in‐house developed K+‐selective microelectrodes or indirectly by the electrophysiological technique, and apoptosis was measured by caspase‐3 activation. Paper I describes that the intracellular K+ concentration was reduced by about 30 % during STS‐induced apoptosis. However, this reduction was prevented by excessive expression of exogenous ion channels. Despite the magnitude of the intracellular K+ concentration, either normal or reduced level, the oocytes displayed normal signs of apoptosis, suggesting that the intracellular K+ reduction was not required for the apoptotic process. Because the intracellular K+ concentration was not critical for apoptosis we searched for other ion fluxes by exploring the electrophysiological properties of X. laevis oocytes. Paper II, describes a non‐inactivating Na+ current activated at positive membrane voltages that was upregulated by a factor of five during STS‐induced apoptosis. By preventing influx of Na+, the apoptotic signaling network involving capsase‐3 was prevented. To molecularly identify this voltage‐gated Na channel, the X. tropicalis genome and conserved regions of the human SCNA genes were used as a map. Paper III, shows that the voltage‐gated Na channel corresponds to the SCN2A gene ortholog and that supression of this SCN2A ortholog using miRNA prevented cell death. In conclusion, this thesis work demonstrated that a voltage‐gated Na channel is critical for the apoptotic process in X. laevis oocytes by increasing the intracellular Na+ concentration