A death receptor-associated anti-apoptotic protein, BRE, inhibits mitochondrial apoptotic pathway

BRE, brain and reproductive organ-expressed protein, was found previously to bind the intracellular juxtamembrane domain of a ubiquitous death receptor, tumor necrosis factor receptor 1 (TNF-R1), and to down-regulate TNF-alpha-induced activation of NF-kappaB. Here we show that BRE also binds to another death receptor, Fas, and upon overexpression conferred resistance to apoptosis induced by TNF-alpha, anti-Fas agonist antibody, cycloheximide, and a variety of stress-related stimuli. However, down-regulation of the endogenous BRE by small interfering RNA increased apoptosis to TNF-alpha, but nottoetoposide, indicating that the physiological antiapoptotic role of this protein is specific to death receptor-mediated apoptosis. We further demonstrate that BRE mediates antiapoptosis by inhibiting the mitochondrial apoptotic machinery but without translocation to the mitochondria or nucleus or down-regulation of the cellular level of truncated Bid. Dissociation of BRE rapidly from TNF-R1, but not from Fas, upon receptor ligation suggests that this protein interacts with the death inducing signaling complex during apoptotic induction. Increased association of BREwith phosphorylated, sumoylated, and ubiquitinated proteins after death receptor stimulation was also detected. We conclude that in contrast to the truncated Bid that integrates mitochondrial apoptosis to death receptor-triggered apoptotic cascade, BRE inhibits the integration. We propose that BRE inhibits, by ubiquitination-like activity, components in or proximal to the death-inducing signaling complexes that are necessary for activation of the mitochondria.     

Dual role of BRUCE as an antiapoptotic IAP and a chimeric E2/E3 ubiquitin ligase

Apoptotic cell death and survival is controlled by pro- and antiapoptotic proteins. Because these proteins act on each other, cell fate is dictated by the relative activity of pro- versus antiapoptotic proteins. Here we report that BRUCE, a conserved 528 kDa peripheral membrane protein of the trans-Golgi network, protects cells against apoptosis and functions as an inhibitor of apoptosis (IAP). By using wild-type and mutant forms we show that BRUCE inhibits caspase activity and apoptosis depending on its BIR domain. Upon apoptosis induction, BRUCE is antagonized by three mechanisms: first, through binding to Smac; second, by the protease HtrA2; and third, by caspase-mediated cleavage. In addition to its IAP activity BRUCE has the distinctive property of functioning as a chimeric E2/E3 ubiquitin ligase with Smac being a substrate. Our work suggests that, owing to its two activities and its localization, BRUCE may function as a specialized regulator of cell death pathways.     

衣原体调控宿主细胞凋亡的机制研究进展

衣原体是一类专性细胞内寄生的原核微生物,常引起性传播疾病、失明和肺炎等疾病。衣原体可能已经进化出调控宿主细胞的若干机制,通过改变宿主细胞蛋白位置,干扰宿主细胞凋亡信号通路等来抑制细胞凋亡,维持其自身的存活,核转录因子NF-κB也涉及到衣原体感染细胞凋亡抑制;衣原体可以经半胱氨酸蛋白酶依赖途径等机制诱导细胞凋亡,进而感染邻近细胞。     

Structure of the Chlamydia protein CADD reveals a redox enzyme that modulates host cell apoptosis

The Chlamydia protein CADD (Chlamydia protein associating with death domains) has been implicated in the modulation of host cell apoptosis via binding to the death domains of tumor necrosis factor family receptors. Transfection of CADD into mammalian cells induces apoptosis. Here we present the CADD crystal structure, which reveals a dimer of seven-helix bundles. Each bundle contains a di-iron center adjacent to an internal cavity, forming an active site similar to that of methane mono-oxygenase hydrolase. We further show that CADD mutants lacking critical metal-coordinating residues are substantially less effective in inducing apoptosis but retain their ability to bind to death domains. We conclude that CADD is a novel redox protein toxin unique to Chlamydia species and propose that both its redox activity and death domain binding ability are required for its biological activity.     

Cyclosporin A, an Immunosuppressive Drug, Induces Programmed Cell Death in Rat C6 Glioma Cells by a Mechanism that Involves the AP-1 Transcription Factor

Abstract: Cyclosporin A (CsA) is a clinically important immunosuppressive drug widely used to prevent graft rejection following organ or bone marrow transplantation. Although there are reports of serious neurologic alterations associated with the use of the drug, the precise mechanism of its action on the CNS still remains unknown. We studied the effects of CsA on the growth of C6 glioma cells. We found that CsA inhibits the growth of C6 glioma cells in a dose-dependent manner and induces morphological changes such as shrinkage of the cell body and loss of extensions followed by cell death. The analysis of DNA from CsA-treated cells revealed a ladder-like pattern of fragmented DNA. Acridine orange staining showed the occurrence of apoptotic changes in nuclear morphology. Apoptotic morphological alterations were prevented by the treatment with cycloheximide. Altogether, our findings suggest that the CsA-induced cell death of C6 glioma cells bears all the features characteristic of programmed cell death. We also observed a significant increase in the DNA-binding activity of AP-1 during CsA-induced apoptosis. The AP-1 induction preceded the appearance of apoptotic, morphological changes and was accounted for by an increase in the expression of c-Jun protein. The occurrence of increased levels of AP-1 complex and c-Jun protein during CsA-induced programmed cell death suggests its involvement in the induction of apoptosis.     

Modulation of P2Z/P2X(7) receptor activity in macrophages infected with Chlamydia psittaci

Given the role that extracellular ATP (ATP(o))-mediated apoptosis may play in inflammatory responses and in controlling mycobacterial growth in macrophages, we investigated whether ATP(o) has any effect on the viability of chlamydiae in macrophages and, conversely, whether the infection has any effect on susceptibility to ATP(o)-induced killing via P2Z/P2X(7) purinergic receptors. Apoptosis of J774 macrophages could be selectively triggered by ATP(o), because other purine/pyrimidine nucleotides were ineffective, and it was inhibited by oxidized ATP, which irreversibly inhibits P2Z/P2X(7) purinergic receptors. Incubation with ATP(o) but not other extracellular nucleotides inhibits the growth of intracellular chlamydiae, consistent with previous observations on ATP(o) effects on growth of intracellular mycobacteria. However, chlamydial infection for 1 day also inhibits ATP(o)-mediated apoptosis, which may be a mechanism to partially protect infected cells against the immune response. Infection by Chlamydia appears to protect cells by decreasing the ability of ATP(o) to permeabilize macrophages to small molecules and by abrogating a sustained Ca(2+) influx previously associated with ATP(o)-induced apoptosis.     

N-acetylcysteine increases the biosynthesis of recombinant EPO in apoptotic Chinese hamster ovary cells

Sodium butyrate (NaBu) is known to enhance the rate of biosynthesis of recombinant proteins in Chinese hamster ovary cells (CHO). Here we demonstrate that supplementation with NaBu during rapid growth brings about abrupt death of the cells. The death of the cells is due to apoptosis, as assessed by intranucleosomal DNA fragmentation. The promotion of apoptotic death of the cells could be partially blocked by treatment with the well-known antioxidant, N-acetylcysteine (NAC). Strikingly, the NAC treatment enhanced the production of recombinant EPO two-fold compared with that of the culture without NAC supplementation. These results showed that NaBu treatment supplemented with NAC not only inhibits apoptosis, but also exerts a synergistic effect on the biosynthesis of recombinant EPO.     

N-acetylcysteine increases the biosynthesis of recombinant EPO in apoptotic Chinese hamster ovary cells

Sodium butyrate (NaBu) is known to enhance the rate of biosynthesis of recombinant proteins in Chinese hamster ovary cells (CHO). Here we demonstrate that supplementation with NaBu during rapid growth brings about abrupt death of the cells. The death of the cells is due to apoptosis, as assessed by intranucleosomal DNA fragmentation. The promotion of apoptotic death of the cells could be partially blocked by treatment with the well-known antioxidant, N-acetylcysteine (NAC). Strikingly, the NAC treatment enhanced the production of recombinant EPO two-fold compared with that of the culture without NAC supplementation. These results showed that NaBu treatment supplemented with NAC not only inhibits apoptosis, but also exerts a synergistic effect on the biosynthesis of recombinant EPO.     

Apoptosis in mammalian eye development: lens morphogenesis, vascular regression and immune privilege

Formation of the mammalian eye requires a complex series of tissue interactions that result in an organ of exquisite sensory capability. The early steps in eye development involve extensive cell death associated with morphogenesis. Later, suppression of programmed cell death is essential for tissue differentiation and in the adult, the immune privileged status of the eye is maintained in part through factors that induce inflammatory cell apoptosis. Experimental evidence suggests that suppression of apoptosis in cells of the lens lineage by fibroblast growth factors is one component of their action during lens morphogenesis. Fibroblast growth factors are also required for normal lens fiber-cell differentiation. This includes a degenerative step for organelles that is presumably an adaptation for the clearance of light scattering elements from the optic axis. The process of organelle degeneration may be related to apoptosis in a few of its features. Actively-induced apoptosis becomes important for eye development as the temporary ocular vasculatures regress. This too, is presumably an adaptation for the disposal of cells that would disturb the passage of light to the retina. Ocular macrophages appear to be essential for the induction of apoptosis in the endothelial cells comprising the ocular vasculatures. In the adult, inflammatory cells entering the eye are exposed to the pro-apoptotic agents transforming growth factor-beta2 and Fas ligand. The expression of these molecules in the eye, and their action in killing inflammatory cells, has evolved as a means of preventing inflammation and subsequent loss of vision. Thus, the eye offers a unique and versatile system for studying the role of programmed cell death in lens development, vascular regression and immune privilege.     

Insulin-like growth factor-I and Bcl-X(L) inhibit c-jun N-terminal kinase activation and rescue Schwann cells from apoptosis

We previously reported that Schwann cells undergo apoptosis after serum withdrawal. Insulin-like growth factor-I, via phosphatidylinositol-3 kinase, inhibits caspase activation and rescues Schwann cells from serum withdrawal-induced apoptosis. In this study, we examined the role of c-jun N-terminal protein kinase (JNK) in Schwann cell apoptosis induced by serum withdrawal. Activation of both JNK1 and JNK2 was detected 1 h after serum withdrawal with the maximal level detected at 2 h. A dominant negative JNK mutant, JNK (APF), blocked JNK activation induced by serum withdrawal and Schwann cell apoptosis, suggesting JNK activation participates in Schwann cell apoptosis. Serum withdrawal-induced JNK activity was caspase dependent and inhibited by a caspase 3 inhibitor, Ac-DEVD-CHO. Because insulin-like growth factor-I and Bcl-X(L) are both Schwann cell survival factors, we tested their effects on JNK activation during apoptosis. Insulin-like growth factor-I treatment decreased both JNK1 and JNK2 activity induced by serum withdrawal. LY294002, a phosphatidylinositol-3 kinase inhibitor, blocked insulin-like growth factor-I inhibition on JNK activation, suggesting that phosphatidylinositol-3 kinase mediates the effects of insulin-like growth factor-I. Overexpression of Bcl-X(L) also resulted in less Schwann cell death and inhibition of JNK activation after serum withdrawal. Collectively, these results suggest JNK activation is involved in Schwann cell apoptosis induced by serum withdrawal. Insulin-like growth factor-I and Bcl family proteins rescue Schwann cells, at least in part, by inhibition of JNK activity.     

CHM-1, a New Vascular Targeting Agent, Induces Apoptosis of Human Umbilical Vein Endothelial Cells via p53-mediated Death Receptor 5 Up-regulation

CHM-1 (2′-fluoro-6,7-methylenedioxy-2-phenyl-4-quinolone) has been identified as a potent antitumor agent in human hepatocellular carcinoma; however, its role in tumor angiogenesis is unclear. This study investigated the effects of CHM-1 and the mechanisms by which it exerts its antiangiogenic and vascular disrupting properties. Using a xenograft model antitumor assay, we found that CHM-1 significantly inhibits tumor growth and microvessel formation. Flow cytometry, immunofluorescence microscopy, and cell death enzyme-linked immunosorbent assay kit revealed that CHM-1 inhibits growth of human umbilical vein endothelial cells (HUVEC) by induction of apoptotic cell death in a concentration-dependent manner. CHM-1 also suppresses HUVEC migration and capillary-like tube formation. We were able to correlate CHM-1-induced apoptosis in HUVEC with the cleavage of procaspase-3, -7, and -8, as well as with the cleavage of poly(ADP-ribose) polymerase by Western blotting assay. Such sensitization was achieved through up-regulation of death receptor 5 (DR5) but not DR4 or Fas. CHM-1 was also capable of increasing the expression level of p53, and most importantly, the induction of DR5 by CHM-1 was abolished by p53 small interfering RNA. Taken together, the results of this study indicate that CHM-1 exhibits vascular targeting activity associated with the induction of DR5-mediated endothelial cell apoptosis through p53 up-regulation, which suggests its potential as an antivascular and antitumor therapeutic agent.     

Vaccinia-induced epidermal growth factor receptor-MEK signalling and the anti-apoptotic protein F1L synergize to suppress cell death during infection

F1L is a functional Bcl-2 homologue that inhibits apoptosis at the mitochondria during vaccinia infection. However, the extent and timing of cell death during ΔF1L virus infection suggest that additional viral effectors cooperate with F1L to limit apoptosis. Here we report that vaccinia growth factor (VGF), a secreted virulence factor, promotes cell survival independently of its role in virus multiplication. Analysis of single and double knockout viruses reveals that VGF acts synergistically with F1L to protect against cell death during infection. Cell survival in the absence of F1L is dependent on VGF activation of the epidermal growth factor receptor. Furthermore, signalling through MEK kinases is necessary and sufficient for VGF-dependent survival. We conclude that VGF stimulates an epidermal growth factor receptor-MEK-dependent pro-survival pathway that synergizes with F1L to counteract an infection-induced apoptotic pathway that predominantly involves the BH3-only protein Bad.     

Protease Activity of Procaspase-8 Is Essential for Cell Survival by Inhibiting Both Apoptotic and Nonapoptotic Cell Death Dependent on Receptor-interacting Protein Kinase 1 (RIP1) and RIP3

Caspase-8 has an important role as an initiator caspase during death receptor-mediated apoptosis. Moreover, it has been reported to contribute to the regulation of cell fate in various types of cells including T-cells. In this report, we show that caspase-8 has an essential role in cell survival in mouse T-lymphoma-derived L5178Y cells. The knockdown of caspase-8 expression decreased the growth rate and increased cell death, both of which were induced by the absence of protease activity of procaspase-8. The cell death was associated with reactive oxygen species (ROS) accumulation, caspase activation, and autophagosome formation. The cell death was inhibited completely by treatment with ROS scavengers, but only partly by treatment with caspase inhibitors, expression of Bcl-xL, and knockdown of caspase-3 or Atg-7 which completely inhibits apoptosis or autophagosome formation, respectively, indicating that apoptosis and autophagy-associated cell death are induced simultaneously by the knockdown of caspase-8 expression. Further analysis indicated that RIP1 and RIP3 regulate this multiple cell death, because the cell death as well as ROS production was completely inhibited by not only treatment with the RIP1 inhibitor necrostatin-1, but also by knockdown of RIP3. Thus, in the absence of protease activity of procaspase-8, RIP1 and RIP3 simultaneously induce not only nonapoptotic cell death conceivably including autophagic cell death and necroptosis but also apoptosis through ROS production in mouse T-lymphoma cells.     

Down-regulation of IL-2 receptor α (CD25) characterizes human γδ-T cells rendered resistant to apoptosis after CD2 engagement in the presence of IL-12

We recently identified a CD2-mediated, IL-12-dependent signaling pathway that inhibits apoptosis in mitogen-stimulated human gammadelta-T cells. Here we show that gammadelta-T cells which acquire resistance to mitogen-induced apoptosis upregulate IL-12 receptor beta 1 subunit (IL-12Rbeta1); in contrast, gammadelta-T cells which remain sensitive to mitogen-induced apoptosis fail to express IL-12Rbeta1. Next we show that gammadelta-T cells which are rendered resistant to mitogen-induced apoptosis attenuate their expression of the IL-2 receptor alpha chain (IL-2Ralpha/CD25), this in part accounting for their acquired resistance to IL-2-induced death. In contrast, apoptosis-sensitive gammadelta-T cells are shown to persist in their expression of IL-2Ralpha/CD25, thus remaining sensitive to IL-2-induced death. Moreover, we show that apoptosis-resistant, but not apoptosis-sensitive, gammadelta-T cells display an enhanced responsiveness to IL-15, a finding in keeping with the known function of IL-15 as a growth and survival factor. Finally, we present evidence to suggest that this differential responsiveness to IL-15 occurs in part by the increased expression of the IL-15Ralpha chain on apoptosis-resistant gammadelta-T cells, compared to apoptosis-sensitive gammadelta-T cells. The biological and clinical implications of these findings are discussed.     

Wnt/beta-catenin signaling inhibits death receptor-mediated apoptosis and promotes invasive growth of HNSCC

The Wnt/beta-catenin signaling pathway plays a critical role in cell proliferation and oncogenesis. It has been found to be chronically activated in a variety of human cancers, including head and neck squamous cell carcinoma (HNSCC). Previously, we have found that the activation of the Wnt/beta-catenin signaling pathway inhibits mitochondria-mediated apoptosis. In this study, we extended our studies to determine whether the Wnt/beta-catenin signaling pathway inhibited death receptor-mediated apoptosis in HNSCC cells. We found that Wnt/beta-catenin inhibited not only tumor necrosis factor (TNF)/c-Myc-mediated apoptosis, but also cell detachment-mediated apoptosis (anoikis) which is dependent on the death receptor signaling pathway. Interestingly, we also observed that the Wnt/beta-catenin signaling pathway induced HNSCC cell scattering and promoted cell invasion in the Matrigel, both of which are hallmarks for the invasive growth of HNSCC. Consistently, the over-expression of beta-catenin promoted HNSCC tumor growth in nude mice. Taken together, our results suggest that the Wnt/beta-catenin signaling pathway plays dual functions in HNSCC development: promoting both cell survival and invasive growth of HNSCC cells.     

Apoptosis resistance in Chlamydia-infected cells: a fate worse than death?

Chlamydia has long been studied as an intracellular pathogen causing widespread diseases. In the last three decades, the field of apoptosis has rapidly emerged, and as a consequence, research on infectious diseases in general and on Chlamydia –host interaction in particular shifted to apoptosis modulation. Ten years ago, the first paper describing the drastic inhibition of apoptosis in Chlamydia -infected cells was published. In a reversal of roles, here was a pathogen that was strongly protecting cells in an organism against destruction by the organism's immune system. Since then, numerous studies have described apoptosis inhibition by Chlamydia and the mechanisms involved, but still there is a lack of general consensus on the subject. With a section of studies even reporting the induction of cell death by Chlamydia and not its inhibition, the field became even more diverse and complicated. In this review, an attempt is made to discuss the recent findings on apoptosis modulation by chlamydial species.     

Lack of effective anti-apoptotic activities restricts growth of Parachlamydiaceae in insect cells

The fundamental role of programmed cell death in host defense is highlighted by the multitude of anti-apoptotic strategies evolved by various microbes, including the well-known obligate intracellular bacterial pathogens Chlamydia trachomatis and Chlamydia (Chlamydophila) pneumoniae. As inhibition of apoptosis is assumed to be essential for a successful infection of humans by these chlamydiae, we analyzed the anti-apoptotic capacity of close relatives that occur as symbionts of amoebae and might represent emerging pathogens. While Simkania negevensis was able to efficiently replicate within insect cells, which served as model for metazoan-derived host cells, the Parachlamydiaceae (Parachlamydia acanthamoebae and Protochlamydia amoebophila) displayed limited intracellular growth, yet these bacteria induced typical features of apoptotic cell death, including formation of apoptotic bodies, nuclear condensation, internucleosomal DNA fragmentation, and effector caspase activity. Induction of apoptosis was dependent on bacterial activity, but not bacterial de novo protein synthesis, and was detectable already at very early stages of infection. Experimental inhibition of host cell death greatly enhanced parachlamydial replication, suggesting that lack of potent anti-apoptotic activities in Parachlamydiaceae may represent an important factor compromising their ability to successfully infect non-protozoan hosts. These findings highlight the importance of the evolution of anti-apoptotic traits for the success of chlamydiae as pathogens of humans and animals.