Research Article 1525 An alternative form of paraptosis-like cell , 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 does of activation. In addition, TAJ/TROY suppressed not explain all the forms of programmed clonogenic growth of HEK293 and HeLa cells. (PCD), particularly in individual development and Interestingly, overexpression of 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 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 , 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 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 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 (Biosea, China). After incubation for 20 minutes at room temperature, 400 µl binding buffer was added, and samples were immediately analysed on a FACSCalibur flow cytometer (Becton Materials and Methods Dickinson, USA) with excitation using a 488 nm argon ion laser. PI Cell lines and reagents was added to samples to distinguish necrotic and late apoptotic events HEK293, HeLa and 293T (a kind gift from T. Matsuda, Japan) cells (annexin V–, PI+; annexin V+, PI+) from early apoptotic events were maintained in Dulbecco’s modified Eagle medium (Life (annexin V+, PI–). Technologies, USA) supplemented with penicillin (100 units ml–1), streptomycin (100 µg ml–1), L-glutamine (2 mM) and 10% fetal bovine serum (Hyclone, USA). All experiments were performed on Measurement of mitochondrial membrane potential logarithmically growing cells. z-VAD-fmk and Ac-DEVD-AMC were Rhodamine 123 was used to evaluate changes in mitochondrial 5 obtained from Pharmingen, and Rhodamine 123 was purchased from membrane potential (∆Ψm). Cell suspensions (5×10 ) were incubated Sigma. Monoclonal antibodies against caspase-3 (catalog number for 15 minutes at 37°C in 1 ml PBS containing 1 µM rhodamine 123 610322), XIAP (catalog number 610763) and PARP (catalog number and subsequently analysed with a FACSCalibur flow cytometer. P76420) were obtained from Transduction Laboratories (Becton Results were expressed as the proportion of cells exhibiting low Dickinson, USA). Monoclonal antibody against β-actin was obtained mitochondrial membrane potential estimated by the reduced from Sigma. Rainbow protein marker was obtained from Amersham rhodamine 123 uptake. Pharmacia. Anti-PDCD5 (clone 3A3) monoclonal antibody and labeled FITC-3A3 were prepared in our laboratory (Chen et al., 2001). DEVDase activity analysis 293T cells (3×105) were transfected with 2 µg empty vector and Constructs and transient transfection TAJ/TROY or Bax expression vectors. Cells were subsequently Human TAJ/TROY cDNA was amplified with 10 pmol of following washed twice with pre-cold PBS and harvested in lysis buffer (10 mM primer 5′-AAGATGGCTTTAAAAGTGCTACTAG-3′ and 5′-AGTC- Tris HCl, pH 7.5, 10 mM Na2HPO4/NaH2PO4, 130 mM NaCl, PDCD5 enhances TAJ/TROY induced paraptosis 1527

1% Triton-X100, 1 mM PMSF). Cell lysates were clarified by centrifugation at 18,000 g for 20 minutes at 4°C. Cell lysates containing 15 µg protein were incubated at 37°C in buffer containing 25 mM HEPES, pH 7.5, 1 mM EDTA, 100 mM NaCl, 0.1% CHAPS and 10 mM dithiothreitol with the fluorogenic substrate Ac-DEVD- AMC. To measure this signal, we used a FLUOStar fluorometer (BMG Labtechnologies, Germany) with an excitation filter of 380 nm and emission filter of 460 nm. Results were calculated as a proportion of the control over 90 minutes (T90 to T0). Samples were prepared in triplicate.

Protein extraction and immunoblotting Cells were pelleted by centrifugation, lysed in lysis buffer (20 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF) and incubated for 30 minutes on ice. Lysates were centrifuged at 18,000 g for 10 minutes at 4°C and the supernatant was measured using the BCA protein assay reagent (Pierce, USA). Equal amounts of protein (50 µg) were separated by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Hybond ECL; Amersham Pharmacia, UK). Membranes were blocked in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) containing 5% non-fat milk for 1 hour and incubated overnight at 4°C with the appropriate primary antibody. Blots were washed (three times for 10 minutes each) with TBS-T and incubated for 1 hour with the appropriate horseradish-peroxidase- conjugated secondary antibody (Promega, USA) prepared in TBS- T/5% non-fat milk. Blots were washed (three times for 10 minutes each) before detection of immunocomplexes using the enhanced chemiluminescence system (Amersham Pharmacia, UK).

Immunofluorescence analysis Transfected cells were washed in PBS, fixed in freshly prepared 2% paraformaldehyde (containing 0.1% Triton X-100) for 30 minutes at room temperature, pre-incubated in 2% fetal bovine serum for 1 hour and incubated with FITC-3A3 for 1 hour. Following antibody staining, cells were analysed using a FACSCalibur flow cytometer.

Results TAJ/TROY as a paraptotic cell death inducer in cultured cells Fig. 1. Overexpression of TAJ/TROY induces non-apoptotic cell TAJ/TROY-transfected cells underwent marked rounding and death. (A) Colony formation after TAJ/TROY transfection. eventually detached from the culture dish. There were no HEK293 and HeLa cells were transfected with an empty vector or signs of classic apoptotic changes, such as cell shrinkage or expression vectors encoding Bax or TAJ/TROY. Overexpression of membrane blebbing, as observed in Bax-transfected cells. TAJ/TROY significantly decreased clonogenic survival of the Ultrastructural analyses revealed that TAJ/TROY-induced cell cells. The figure shows a representative experiment with the means death was distinct from apoptosis. Transmission electron and standard deviations from triplicate plates. (B) Electron micrographs of TAJ/TROY-induced cell death. HEK293, HeLa and microscopy of HEK293, HeLa and 293T cells at 40 hours 293T cells were transfected with a vector control or plasmids after transfection with TAJ/TROY confirmed that these cells encoding Bax or TAJ/TROY along with a GFP-encoding vector. did not undergo apoptosis, unlike Bax-transfected cells, Whereas Bax-transfected 293T cells (b) displayed classic which displayed features typical of apoptosis, including characteristics of apoptosis, including chromatin condensation and chromatin condensation with margination at the nuclear apoptosis body, TAJ/TROY-transfected HEK293 (e), HeLa (g) and periphery, nuclear fragmentation and apoptotic body 293T (c) cells showed extensive cytoplasmic vacuolization and formation (Fig. 1B). Compared with untreated cells, swelling of mitochondria or ER, along with absence of nuclear significant swelling of mitochondria and ER were observed fragmentation, membrane blebbing or apoptotic-body formation. in TAJ/TROY-transfected cells and the nuclei were intact. The following micrographs of empty-vector-transfected HEK293 Moreover, chromatin condensation and nuclear fragmentation (d), HeLa (f) and 293T (a) cells provided normal cell controls. Higher-magnification images of the vacuolization suggested that were absent and some cells displayed extensive cytosolic some might be formed from the remnants of the vacuolization. Clonogenic survival assays were performed mitochondrial (h) or ER (i). The transfection efficiency of the to determine the death-inducing effect of TAJ/TROY on indicated plasmids ranged from 60% to 70% as determined by the HEK293 and HeLa cells over an extended period of growth. expression of co-transfected GFP. Scale bars, 2 µM (a-g), 0.2 µM As shown in Fig. 1A, a significant decrease in the number of (h,i). 1528 Journal of Cell Science 117 (8) survival colonies was observed in the TAJ/TROY transfected 3-like activity in Bax-transfected cells indicated priming of the cells compared with control cells. Taken together, we propose caspase cascade for activation. The pan-caspase inhibitor, z- that TAJ/TROY-transfected cells died through a paraptotic VAD-fmk, effectively inhibited this caspase-3-like activity. To mechanism. confirm the activation of caspase-3, western blots were performed. We detected decreased procaspase-3 in lysates of Bax-overexpressing cells but not in those of TAJ/TROY- Cell death triggered by TAJ/TROY is accompanied by overexpressing or mock cells (Fig. 3B). Because proteolytic exposure of PS cleavage of specific substrates by activated caspases is A biochemical hallmark of apoptotic cell death is the responsible for cellular dysfunction and structural destruction translocation of PS from the cytoplasmic surface of the cell apoptosis (Thornberry and Lazebnik, 1998), we analysed the membrane to the external cell surface (Fadok et al., 1998). cleavage of PARP and XIAP as representative substrates in Exposure of PS at the surface of apoptotic cells is easily mock, TAJ/TROY- or Bax-transfected cells. Both substrates determined by flow cytometry using fluorescence-labeled were cleaved in Bax-transfected cells and that PARP cleavage annexin V, which specifically binds PS (Smith et al., 2002). To leaded to approximate 80 kDa fragments that is a feature of characterize TAJ/TROY-induced cell death further, we detected classic apoptosis (Fig. 3B). No significant caspase-specific PS surface exposure by FITC/annexin-V staining of cells 40 processing of PARP and XIAP was observed in TAJ/TROY- hours after transfection. Plasma membrane integrity was transfected cells, strongly suggesting that the caspase cascade simultaneously assessed by PI dye exclusion using two-color is not activated during TAJ/TROY-induced cell death. fluorescence-activated cell sorting (FACS) analysis. In TAJ/TROY-transfected cells, the proportion of annexin-V- positive/PI-negative cells was evidently increased (Fig. 2). Mitochondrial changes in TAJ/TROY-transfected cells Simultaneously, the integrity of the membrane in these cells An increasing amount of evidence shows that mitochondria was maintained, based on the finding that the PI-positive play central roles in the regulation of both apoptotic and non- population was similar to that observed in mock-transfected apoptotic cell death (Green and Reed, 1998; Sperandio et al., cells. 2000; Obrero et al., 2002). Among the sequence of events taking place in mitochondria during the course of cell death, loss of the ∆Ψm appear to be the major event closely associated TAJ/TROY-induced cell death is caspase-independent with cell death (Susin et al., 1996). Accordingly, at 30 hours Activation of effector caspases, such as caspases 3 and 7, is after transfection, we examined the involvement of responsible for the proteolytic cleavage of a diverse range of mitochondria in TAJ/TROY-induced non-apoptotic cell death structural and regulatory proteins in apoptosis (Salvesen and by monitoring ∆Ψm. During Bax-induced apoptosis, we Dixit, 1997). To determine whether PS externalization is detected loss of ∆Ψm (Fig. 4A). Interestingly, ∆Ψm loss was related to caspase-3-mediated proteolysis, we analysed the additionally observed in TAJ/TROY-induced cell death (Fig. activation of caspase-3 in TAJ/TROY- and Bax-transfected 4A), suggesting that mitochondria are involved in the cells approximately 40 hours after transfection. DEVD regulation of non-apoptotic cell death. Ultrastructural analysis cleavage assay is a quantitative method used to detect caspase- at 40 hours after transfection revealed significant mitochondrial 3-like activity (Rehm et al., 2002). As shown in Fig. 3A, swelling in TAJ/TROY-transfected cells and ER swelling also DEVD cleavage activity in lysates of TAJ/TROY- was observed (Fig. 4B). These results suggested that some overexpressing cells was at background levels, whereas activity vacuoles might be derived from the mitochondria or ER. in lysates from Bax-overexpressing cells was nearly ten times higher than that in mock cell lysates. The significant caspase- Overexpression of PDCD5 enhances TAJ/TROY- induced cell death PDCD5 is a pro-apoptotic regulator. We examined whether the protein has synergistic effects on TAJ/TROY-mediated paraptotic cell death. 36 hours after transfection, cells were photographed under a light microscope. Co-transfection of TAJ/TROY and PDCD5 resulted in a more severe cytotoxic effect on 293T cells than TAJ transfection alone (data not shown). Annexin- V staining revealed more positive cells in the co- transfection group than the TAJ/TROY transfection group. However, there were no differences in the Fig. 2. Cell death induced by TAJ/TROY is accompanied by PS exposure in number of annexin-V-positive cells between mock- the membrane. 40 hours after transfection, cells were analysed by flow transfected and PDCD5-transfected cells (Fig. 5A). cytometry with FITC-labeled annexin V and propidium iodide. (bottom left) Therefore, PDCD5 clearly acts synergistically with Viable cells (PI–, annexin-V/FITC–). (bottom right) Early apoptotic or paraptotic cells (PI–, annexin-V/FITC+). (top right) Non-viable, late TAJ/TROY in 293T cell killing, functioning as a positive apoptotic/necrotic cells (PI+, annexin-V/FITC+). (top left) Necrotic cells. regulator rather than an initiator. To determine whether Numbers in the quadrants indicate the proportions of cells in the enhanced cytotoxicity occurs through caspase-3- corresponding areas. Data from one experiment are presented. Experiments mediated proteolysis, we analysed the activation of were performed in triplicate, with similar results. caspase-3 in TAJ/TROY-transfected or TAJ/TROY- and PDCD5 enhances TAJ/TROY induced paraptosis 1529

Fig. 3. TAJ-induced non-apoptotic cell death is independent of caspase activation. (A) DEVDase activity assay. 293T cells were transfected with the empty vector or indicated constructs and, 40 hours after transfection, experiments were performed as described in Materials and Methods. All data are presented as mean±standard deviation (s.d.). (B) Cellular proteins were extracted from 293T cells that were transfected with mock, Bax or TAJ/TROY plasmid. Protein levels were detected by western blots with various antibodies as described in Materials and Methods. Fig. 4. Mitochondrial changes in TAJ/TROY-transfected cells. (A) Transfected cells were subjected to mitochondrial transmembrane potential assessment 30 hours after transfection using a potentiometric dye, rhodamine 123. Loss of ∆Ψm was visualized as PDCD5-co-transfected cells using the DEVD cleavage assay. a reduction in the fluorescence signal in the FL1 channel. The proportions of cells with reduced rhodamine 123 staining are shown. As shown in Fig. 5B, DEVD cleavage activity in all treated Data are presented from one experiment. Experiments were cells was similar to that observed in mock-transfected cells. performed in triplicate with similar results. (B) Ultrastructural Our results suggest that caspase activity is not involved in the mitochondrial characteristics of TAJ/TROY-induced cell death. death enhancement of PDCD5. HEK293, HeLa and 293T cells were transfected with a vector control or plasmids encoding Bax or TAJ/TROY along with a GFP-encoding vector. Extensive mitochondria swelling was seen in TAJ/TROY- Upregulation of PDCD5 protein in TAJ/TROY-transfected transfected HEK293 (b,c), HeLa (e) and 293T (g-i) cells. The cells following micrographs of empty-vector-transfected HEK293 (a), HeLa (d) and 293T (f) cells provide normal cell controls. Scale bars, We have demonstrated previously (Liu et al., 1999) that µ µ µ withdrawal of growth factor induced an increase in PDCD5 1 M (a,b,d,e), 0.5 M (c,f-h), 0.2 M (i). protein in apoptotic TF-1 cells. To determine whether PDCD5 protein expression is enhanced in response to TAJ/TROY overexpression, we used an FITC-labeled anti-PDCD5 monoclonal antibody to assess protein levels in treated 293T Discussion cells. Using a FACScalibur flow cytometer, we showed that Although it is established that caspase-regulated apoptosis is endogenous PDCD5 in TAJ/TROY-overexpressing cells was an important form of PCD, recent studies indicate that, in many significantly upregulated at 30 hours after transfection instances, PCD is caspase independent and non-apoptotic compared with mock-transfected and PDCD5-overexpressing (Crawford and Bowen, 2002; Davidson et al., 2002; Liu et al., cells (Fig. 6A). Transfection of a Bax-encoding plasmid also 2003; Guegan and Przedborski, 2003; Borner and Monney, induced a relatively weak increase in PDCD5 protein (Fig. 6A). 1999; Kitanaka and Kuchino, 1999). In this report, we 1530 Journal of Cell Science 117 (8)

Fig. 6. Upregulation of PDCD5 protein in TAJ/TROY-transfected cells. (A) 293T cells were transfected with empty plasmid vector or indicated plasmids. 30 hours after transfection, endogenous PDCD5 levels were measured with a FITC-labeled anti-PDCD5 monoclonal antibody by flow cytometry. Median values of fluorescence intensity represent the expression of cellular PDCD5. 293T cells transfected with PDCD5 expression plasmid were used as a positive control. Significant upregulation of endogenous PDCD5 was detected in TAJ/TROY-overexpressing cells. (B) Schematic overlap of fluorescence histograms. It helped to observe the increase of median Fig. 5. Overexpression of PDCD5 enhances TAJ-induced cell death. values of fluorescence intensity in TAJ/TROY-transfected cells. 293T cells were transfected with vector control and plasmids encoding TAJ/TROY, PDCD5 or both TAJ/TROY and PDCD5. Following transfection (36 hours), cells were processed for flow cytometry analysis (A) and DEVDase activity assay (B). PCD, non-apoptotic PCD or caspase-independent PCD that have appeared in recent literature are still ambiguous. We assume that paraptosis is one type of caspase-independent PCD demonstrate that TAJ/TROY induces a form of non-apoptotic among many. The precise classification, nomenclature and cell death characterized by paraptosis-like morphology. This description of cell should be re-assessed by scientists type of cell death involved extensive cytoplasmic vacuolation, involved in this field. mitochondria swelling, caspase activity deficiency, loss of In contrast to apoptotic PCD, in which the central mitochondrial membrane potential and surface exposure of PS mechanism and molecules involved in the transduction of death but displayed none of the classical nuclear characteristics of signals have been largely delineated, information regarding the apoptosis. Our results, together with those of Eby et al. (Eby regulation of caspase-independent paraptotic PCD is currently et al., 2000), who previously reported TAJ-induced apoptosis limited. Our studies demonstrate that paraptosis-like cell death independent of oligonucleosomal DNA fragmentation and is accompanied by mitochondria swelling and loss of the ∆Ψm, caspase activation, provide strong evidence that TAJ/TROY suggesting the involvement of mitochondrial products in this overexpression leads to activation of a cell death program that death pathway. Ultrastructural characteristics revealed that is totally distinct from apoptosis. This cell death might mitochondria lost membrane potential via a swelling way, represent an alternative form of paraptotic PCD, which has whereas, in classic apoptosis, the mitochondria maintains its been overlooked until now. However, terms such as atypical shape and volume. Further studies are required to determine PDCD5 enhances TAJ/TROY induced paraptosis 1531 whether known or unknown proteins localized at the involved in the paraptotic death enhancement by PDCD5 (Fig. mitochondrial intermembrane initiate an intrinsic paraptosis- 5B). Future studies are required to elucidate the mechanism of like PCD cascade or disruption of mitochondrial electron the promoting activity of PDCD5. transport and energy metabolism directly kills the cell. The relationship between apoptotic and paraptotic PCD An early loss of normal cell membrane asymmetry with remains to be clarified. Out of 7075 differentially expressed translocation of PS to the outer leaflet of the phospholipid genes, less than 2% are common to both cell death programs bilayer membrane is a general feature of apoptosis, regardless (Sperandio et al., 2000). We propose that differentially of the initiating stimulus (Martin et al., 1995). Sperandio et al. expressed genes are responsible for the distinct cell death compared apoptosis, paraptosis and necrosis with respect to pathways. So, PDCD5 might be one of the key molecules chromatin condensation, DNA fragmentation, mitochondrial connecting the two cell death programs. Programmed cell swelling, caspase-3 activity and the effect of apoptosis death in Dictyostelium discoideum (Cornillon et al., 1994; inhibitors (Sperandio et al., 2000). However, the issue of Levraud et al., 2003) and Schizosaccharomyces pombe whether cells undergoing paraptosis display PS translocation (Jurgensmeier et al., 1997) also feature cytoplasmic to the plasma membrane remains to be clarified. Using vacuolation, suggesting that paraptosis is an ancient form of annexin-V staining and FACS analysis, we observed PS PCD. Because PDCD5 shares significant sequence homology exposure in TAJ/TROY-transfected cells. Although data on the with the corresponding proteins of several species (ranging mechanisms leading to PS externalization in apoptotic cells from archaea to mammals), we predict that this protein and its are limited, it is proposed that depression of the orthologs are core regulators of PCD in phylogenetically old aminophospholipid translocase and calcium-mediated, non- metazoa, unicellular eukaryotes as well as vertebrates. specific flip-flop of phospholipids play a role (Bratton et al., In conclusion, our findings suggest that TAJ/TROY 1997). We speculate that the pathway(s) leading to PS overexpression induces paraptosis-like cell death and that externalization cross-link the cell death programs of apoptosis PDCD5 plays a promoting role in this process. Biochemical and paraptosis. Outer leaflet PS is the most extensively and genetic experiments that aim to clarify the molecular characterized signal in tissues for the non-inflammatory mechanisms of paraptotic PCD should shed light on engulfment of apoptotic cells (Fadok et al., 1992; Fadok et al., carcinogenesis, , development and 2001). It is possible that of paraptotic cells by evolutionary aspects of cell death programs. macrophages is additionally mediated by PS. Although annexin-V binding is a widely used method to evaluate We thank Y. S. Gao for the DNA sequencing, B. H. Hu, X. Chang, apoptosis, our results suggest that cells undergoing paraptosis X. R. Zhao and H. H. Hao for technical assistance with the electron also display surface exposure of PS. Thus, multiparameter microscope, and Elixigen (Shanghai) for assistance in the revision of analyses should be used to identify apoptotic or non-apoptotic the manuscript. This work was supported by a grant from the National Natural Science Foundation of China (39870427) and the National cells in the future. High Technology Research and Development Program of China Here, we demonstrate for the first time that PDCD5 (2002BA711A01). overexpression enhances caspase-independent, paraptosis-like cell death triggered by TAJ/TROY. We cloned the novel PDCD5, an increased expression gene observed during the References apoptosis of TF-1 cells induced by growth factor withdrawal Baker, S. J. and Reddy, E. P. (1998). Modulation of life and death by the (Liu et al., 1999). Further studies showed that transient or TNF receptor superfamily. Oncogene 17, 3261-3270. stable overexpression in cell lines or addition of recombinant Borner, C. and Monney, L. (1999). Apoptosis without caspases: an inefficient PDCD5 protein to culture medium facilitates apoptosis molecular guillotine? Cell Death Differ. 6, 497-507. Bratton, D. L., Fadok, V. A., Richter, D. A., Kailey, J. 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