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An Alternative Form of Paraptosis-Like Cell Death, Triggered by TAJ/TROY and Enhanced by PDCD5 Overexpression

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An Alternative Form of Paraptosis-Like Cell Death, Triggered by TAJ/TROY and Enhanced by PDCD5 Overexpression http://www.paper.edu.cn Research Article 1525 An alternative form of paraptosis-like cell death, triggered by TAJ/TROY and enhanced by PDCD5 overexpression Ying Wang1, Xianting Li1, Lu Wang2, Peiguo Ding1, Yingmei Zhang2, Wenling Han2 and Dalong Ma1,2,* 1Laboratory of Medical Immunology, School of Basic Medical Science, Peking University, Xueyuan Road 38, Beijing 100083, China 2Center for Human Disease Genomics, Health Science Center, Peking University, Beijing 100083, China *Author for correspondence (e-mail: [email protected]) Accepted 18 November 2003 Journal of Cell Science 117, 1525-1532 Published by The Company of Biologists 2004 doi:10.1242/jcs.00994 Summary Accumulating reports demonstrate that apoptosis does of caspase activation. In addition, TAJ/TROY suppressed not explain all the forms of programmed cell death clonogenic growth of HEK293 and HeLa cells. (PCD), particularly in individual development and Interestingly, overexpression of Programmed cell death 5 neurodegenerative disease. Recently, a novel type of PCD, (PDCD5), an apoptosis-promoting protein, enhanced designated ‘paraptosis’, was described. Here, we show that TAJ/TROY-induced paraptotic cell death. Moreover, overexpression of TAJ/TROY, a member of the tumor cellular endogenous PDCD5 protein was significantly necrosis factor receptor superfamily, induces non- upregulated in response to TAJ/TROY overexpression. apoptotic cell death with paraptosis-like morphology in These results provide novel evidence that TAJ/TROY 293T cells. Transmission electron microscopy studies reveal activates a death pathway distinct from apoptosis and that extensive cytoplasmic vacuolation and mitochondrial PDCD5 is an important regulator in both apoptotic and swelling in some dying cells and no condensation or non-apoptotic PCD. fragmentation of the nuclei. Characteristically, cell death triggered by TAJ/TROY was accompanied by phosphatidylserine externalization, loss of the Key words: Paraptosis, Non-apoptotic, PDCD5, TFAR19, mitochondrial transmembrane potential and independent TAJ/TROY Introduction neuropeptide substance P and its receptor, neurokinin-1 Apoptosis is a common, conserved, endogenous cell suicide receptor, mediated a non-apoptotic form of PCD resembling program required for proper embryonic development and paraptosis in some cases (Castro-Obregon et al., 2002). maintenance of homeostasis in adult tissues (Vaux and Activated microglia triggered neuronal cell death with Korsmeyer, 1999; Jacobson et al., 1997). Apoptosis is ultrastructural characteristics of marked vacuolation, slightly characterized by a biochemical cascade and morphological condensed chromatin and severely disintegrated perikarya, changes, including activation of caspases, translocation of following blockage of the caspase cascade (Tanabe et al., phosphatidylserine from the inner to the outer layer of the 1999). Apaf-1-deficient embryonic stem (ES) cells died through a non-apoptotic mechanism associated with loss of plasma membrane, blebbing of cytoplasmic membranes, mitochondrial membrane potential after treatment with diverse chromatin condensation and fragmentation into apoptotic cell death stimuli (Haraguchi et al., 2000). Overexpression of bodies (Kerr et al., 1972; Thornberry and Lazebnik, 1998; extramitochondrial Omi/HtrA2 induced atypical PCD, which Hengartner, 2000). Recently, a novel non-apoptotic was neither accompanied by an increase in caspase activity nor programmed cell death (PCD) process designated paraptosis inhibited by caspase inhibitors (Suzuki et al., 2001). In was described by Sperandio et al. (Sperandio et al., 2000). The addition, ceramide induced non-apoptotic PCD with necrotic- features of paraptosis differ from those of apoptosis and like morphology in human glioma cells in the presence of involve cytoplasmic vacuolation, mitochondrial swelling and pan-caspase inhibitors or during overexpression of Bcl-xL absence of caspase activation or typical nuclear changes, (Mochizuki et al., 2002). These examples support the theory including pyknosis and DNA fragmentation (Sperandio et al., that cells have other intrinsic programs for death that 2000; Wyllie and Golstein, 2001). There is increasing evidence are distinct from apoptosis. Despite the elucidation of that this alternative, non-apoptotic PCD exists in parallel with morphological characteristics, the precise biochemical apoptosis. For instance, T9 glioma cells expressing membrane mechanism for non-apoptotic PCD and the basic components macrophage colony-stimulating factor were killed by of the death machinery are currently unknown. polymorphonuclear leukocytes and macrophages with Tumor necrosis factor receptor (TNFR) superfamily proteins vacuolization of the mitochondria and endoplasmic reticulum play important regulatory roles in cellular responses ranging (ER) resembling paraptosis (Chen et al., 2002). Similarly, the from cell proliferation and differentiation to cell survival or 转载 中国科技论文在线 http://www.paper.edu.cn 1526 Journal of Cell Science 117 (8) apoptotic PCD (Baker and Reddy, 1998; Gravestein and Borst, TCGAGTCACAGGGAACCCAGTCG-3′ using a PCR reaction with 1998). However, little is known about whether members of 1 cycle of 94°C for 5 minutes and 35 cycles of 94°C for 1 minute, TNFR are involved in non-apoptotic or paraptotic PCD. 58°C for 1 minute and 72°C for 1.5 minutes, followed by an extension TAJ/TROY (HGNC-approved symbol TNFRSF19) is a novel at 72°C for 7 minutes from a fetal heart cDNA library (Clontech, member of the TNFR family that is highly expressed during USA). The PCR product were subcloned into pcDNA3.1-Myc-His (B) embryonic development and lacks a death domain in the vector (Invitrogen, USA). The cDNA encoding human Bax was generated by PCR and subcloned into pcDNA3.1-Myc-His (B). The cytoplasmic tail of its protein product (Eby et al., 2000; Hu et al., entire open reading frame of human PDCD5 was subcloned from 1999; Kojima et al., 2000). Similar to other members of the pCDI-PDCD5 (Liu et al., 1999) and inserted into pcDNA3.1-Myc-His TNFR family, TAJ/TROY activates JNK (Eby et al., 2000) and (B) with a C-terminal Myc tag using an EcoRI site. All plasmids were NF-κB (Eby et al., 2000; Kojima et al., 2000) pathways. Co- confirmed by DNA sequencing. HeLa, HEK293 and 293T cells were immunoprecipitation assay revealed that TAJ/TROY binds transiently transfected using the calcium phosphate precipitation TRAF1, TRAF2, TRAF3 and TRAF5 (Eby et al., 2000). method according to standard protocols (Xia et al., 2002). TAJ/TROY is capable of inducing apoptosis independent of DNA fragmentation and caspase activation (Eby et al., 2000), and Clonogenic assay might transduce signals associated with epidermal development through TRAF6 (Naito et al., 2002; Sinha et al., 2002). Briefly, the constructs were transfected into HEK293 and HeLa cells using the calcium phosphate precipitation method. After incubation PDCD5 (programmed cell death 5), formerly designated for 48 hours at 37°C, transfected cells were trypsinized and seeded TFAR19 (TF-1-cell apoptosis-related gene 19), is an apoptosis- into 100-mm dishes (1000 cells per dish). Selection medium with promoting gene cloned in our laboratory (Liu et al., 1999). G418 (600–800 µg ml–1) was added. 3 weeks later, colonies were An mRNA dot blot disclosed that PDCD5 is expressed fixed and stained with crystal violet, and clones containing more than ubiquitously in adult tissues and, at lower levels, in fetal tissues 50 cells were counted. Each group was assayed in triplicate dishes, (Liu et al., 1999). PDCD5 mRNA and protein levels are and each experiment was repeated twice. upregulated in response to various apoptotic stimuli (Li et al., 2000; Liu et al., 1999). Overexpression of this gene induces Electron microscopy increased sensitivity of specific tumor cells to apoptotic stimulation (Liu et al., 1999). These results suggest that For transmission electron microscopy, cells were initially fixed in 0.1 M sodium phosphate buffer containing 2.5% glutaraldehyde, pH 7.4. PDCD5 is a positive regulator in classic apoptotic PCD. Next, cells were fixed in 0.1 M sodium phosphate buffer containing However, whether PDCD5 is involved in non-apoptotic or 1% OsO4, pH 7.2. Cells were embedded into Ultracut (Leica, paraptotic PCD has never been investigated. Germany) and sliced into 60 nm sections. Ultrathin sections were In this report, we provide novel evidence that TAJ/TROY stained with uranyl acetate and lead citrate, and examined with a JEM- induces non-apoptotic cell death with paraptosis-like 1230 transmission electron microscope (JEOL, Japan). morphology in HEK293, HeLa and 293T cells. Significantly, TAJ/TROY-induced paraptotic cell death was enhanced efficiently upon overexpression of PDCD5, which plays a pro- Detection of phosphatidylserine externalization To detect phosphatidylserine (PS) externalization, transfected 293T apoptotic role in classical apoptosis. In addition, significant × 5 translational upregulation of PDCD5 was observed in cells (5 10 ) were harvested by trypsinization and washed twice with PBS. Washed cells were resuspended in 200 µl binding buffer (PBS TAJ/TROY-transfected cells. These results highlight a signal containing 1 mM calcium chloride). FITC-conjugated annexin V transduction system to study alternative PCD and suggest that (0.5 µg ml–1 final concentration) and propidium iodide (PI; 1 µg ml–1 PDCD5 is an important regulator implicated in both apoptotic final concentration) were added according to the manufacturer’s and paraptotic PCD. instructions
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