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• W1966822894 abstract "Tribbles, an atypical protein kinase superfamily member, coordinates cell proliferation, migration, and morphogenesis during the development of Drosophila and Xenopus embryos. Although Tribbles are highly conserved throughout evolution, the physiological functions of mammalian Tribbles family remain largely unclear. Here we report that human TRB2 is a pro-apoptotic molecule that induces apoptosis of cells mainly of the hematopoietic origin. TRB2 mRNA is selectively induced by removal of granulocyte macrophage colony-stimulating factor (GM-CSF) or interleukin-2 from human erythroleukemia-derived TF-1 cell line or activated primary CD4+ T cells, respectively. It is, however, not induced by many other treatments that trigger apoptosis of these two cell types. Overexpression of TRB2 activates many apoptotic events observed in GM-CSF-deprived TF-1 cells, including loss of mitochondrial membrane potential, Mcl-1 cleavage/degradation, and activation of Bax and a number of caspases. Specific knockdown of TRB2 significantly suppresses GM-CSF deprivation-induced apoptosis and all apoptotic events mentioned above. Finally, we demonstrate that TRB2-induced cleavage and degradation of Mcl-1 are mediated via a caspase-dependent but proteasome-independent mechanism, and overexpression of Mcl-1 or its upstream activator Akt can markedly overcome the apoptogenic effect of TRB2. Altogether, these results suggest that the TRB2-Mcl-1 axis plays an important role in survival factor withdrawal-induced apoptosis of TF-1 cells. Tribbles, an atypical protein kinase superfamily member, coordinates cell proliferation, migration, and morphogenesis during the development of Drosophila and Xenopus embryos. Although Tribbles are highly conserved throughout evolution, the physiological functions of mammalian Tribbles family remain largely unclear. Here we report that human TRB2 is a pro-apoptotic molecule that induces apoptosis of cells mainly of the hematopoietic origin. TRB2 mRNA is selectively induced by removal of granulocyte macrophage colony-stimulating factor (GM-CSF) or interleukin-2 from human erythroleukemia-derived TF-1 cell line or activated primary CD4+ T cells, respectively. It is, however, not induced by many other treatments that trigger apoptosis of these two cell types. Overexpression of TRB2 activates many apoptotic events observed in GM-CSF-deprived TF-1 cells, including loss of mitochondrial membrane potential, Mcl-1 cleavage/degradation, and activation of Bax and a number of caspases. Specific knockdown of TRB2 significantly suppresses GM-CSF deprivation-induced apoptosis and all apoptotic events mentioned above. Finally, we demonstrate that TRB2-induced cleavage and degradation of Mcl-1 are mediated via a caspase-dependent but proteasome-independent mechanism, and overexpression of Mcl-1 or its upstream activator Akt can markedly overcome the apoptogenic effect of TRB2. Altogether, these results suggest that the TRB2-Mcl-1 axis plays an important role in survival factor withdrawal-induced apoptosis of TF-1 cells. Tribbles has been shown to coordinate cell proliferation, migration, and morphogenesis during the development of Drosophila and Xenopus embryos (1Grosshans J. Wieschaus E. Cell. 2000; 101: 523-531Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 2Rorth P. Szabo K. Texido G. Mol. Cell. 2000; 6: 23-30Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 3Mata J. Curado S. Ephrussi A. Rorth P. Cell. 2000; 101: 511-522Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 4Seher T.C. Leptin M. Curr. Biol. 2000; 10: 623-629Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 5Saka Y. Smith J.C. Dev. Biol. 2004; 273: 210-225Crossref PubMed Scopus (22) Google Scholar). The protein sequence of Tribbles suggests that it is a member of the protein kinase superfamily, but its sequence diverges from the conventional kinase consensus in subdomains I and II, which are essential for ATP binding (6Kostich M. English J. Madison V. Gheyas F. Wang L. Qiu P. Greene J. Laz T.M. Genome Biol. 2002; 3RESEARCH0043Crossref PubMed Google Scholar). The mammalian orthologs of Tribbles, TRB1, TRB2, and TRB3, all appear to contain the consensus serine/threonine kinase catalytic core, but lack a conserved ATP-binding pocket. Accordingly, no kinase activity has been demonstrated for these proteins by in vitro kinase assay (1Grosshans J. Wieschaus E. Cell. 2000; 101: 523-531Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 7Du K. Herzig S. Kulkarni R.N. Montminy M. Science. 2003; 300: 1574-1577Crossref PubMed Scopus (714) Google Scholar). Recent studies on mammalian Tribbles proteins have revealed that they may play some important roles in metabolism and growth regulation. In 293 cells, TRB3 is induced by NF-κB and functions to negatively regulate NF-κB-dependent transcription (8Wu M. Xu L.G. Zhai Z. Shu H.B. J. Biol. Chem. 2003; 278: 27072-27079Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Under fasting conditions, TRB3 is induced through the PCG-1/peroxisome proliferator-activated receptor-α pathway and inhibits Akt/PKB 2The abbreviations used are: PKB, protein kinase B; GM-CSF, granulocyte macrophage colony-stimulating factor; IL, interleukin; BM, bone marrow; siRNA, short interfering RNA; EGFP, enhanced green fluorescent protein; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; HA, hemagglutinin; TNF, tumor necrosis factor; DSS, disuccinimidyl suberate; TUNEL, deoxynucleotidyltransferase-mediated dUTP nick-end labeling; IRES, internal ribosome-entering site; MMP, mitochondrial membrane potential; An-V, annexin V; μF, microfarads. activation in liver (7Du K. Herzig S. Kulkarni R.N. Montminy M. Science. 2003; 300: 1574-1577Crossref PubMed Scopus (714) Google Scholar, 9Koo S.H. Satoh H. Herzig S. Lee C.H. Hedrick S. Kulkarni R. Evans R.M. Olefsky J. Montminy M. Nat. Med. 2004; 10: 530-534Crossref PubMed Scopus (477) Google Scholar). TRB1 and TRB3 were shown to either inhibit or activate the activities of mitogen-activated protein kinase kinase via protein-protein interaction (10Kiss-Toth E. Bagstaff S.M. Sung H.Y. Jozsa V. Dempsey C. Caunt J.C. Oxley K.M. Wyllie D.H. Polgar T. Harte M. O'Neill L.A. Qwarnstrom E.E. Dower S.K. J. Biol. Chem. 2004; 279: 42703-42708Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). During endoplasmic reticulum stress-induced apoptosis, human TRB3 expression was up-regulated, and knockdown expression of TRB3 by RNA interference rescued cell viability (11Ohoka N. Yoshii S. Hattori T. Onozaki K. Hayashi H. EMBO J. 2005; 24: 1243-1255Crossref PubMed Scopus (752) Google Scholar). Conversely, whereas TRB3 mRNA was induced by nutrient starvation, overexpression of TRB3 prevented nutrient starvation-induced cell death (12Schwarzer R. Dames S. Tondera D. Klippel A. Kaufmann J. Cell. Signal. 2006; 18: 899-909Crossref PubMed Scopus (81) Google Scholar). trb2 is recently reported to be an oncogene, which induces acute myelocytic leukemia in the chimeric mice bearing TRB2-transduced bone marrow, a result likely because of conversion of C/EBPα p42 to C/EBPα p30 in TRB2-transduced cells (13Keeshan K. He Y. Wouters B.J. Shestova O. Xu L. Sai H. Rodriguez C.G. Maillard I. Tobias J.W. Valk P. Carroll M. Aster J.C. Delwel R. Pear W.S. Cancer Cell. 2006; 10: 401-411Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). In the absence of a specific cytokine, cytokine-dependent hematopoietic cells actively undergo apoptosis, which can be blocked by treatment with actinomycin D or cycloheximide, implying a requirement for de novo RNA/protein synthesis in this apoptotic pathway (14Ishida Y. Agata Y. Shibahara K. Honjo T. EMBO J. 1992; 11: 3887-3895Crossref PubMed Scopus (2203) Google Scholar, 15Han J.H. Gileadi C. Rajapaksa R. Kosek J. Greenberg P.L. Exp. Hematol. 1995; 23: 265-272PubMed Google Scholar). Several genes have been reported to be induced in mRNA levels following cytokine deprivation and may play some important roles in the cell-death pathway. For example, hrk, the BH3-only Bcl-2 family gene, is up-regulated during interleukin 3 (IL-3) deprivation of hematopoietic progenitors and is involved in the regulation of apoptosis (16Sanz C. Benito A. Inohara N. Ekhterae D. Nunez G. Fernandez-Luna J.L. Blood. 2000; 95: 2742-2747Crossref PubMed Google Scholar). 24p3, which encodes a secreted lipocalin, is induced by IL-3 deprivation in several IL-3-dependent cell lines and primary bone marrow cells and is able to induce apoptosis of a variety of leukocytes through an autocrine mechanism (17Devireddy L.R. Teodoro J.G. Richard F.A. Green M.R. Science. 2001; 293: 829-834Crossref PubMed Scopus (330) Google Scholar). RC3, a calcium/calmodulin-binding protein, is induced by IL-2 deprivation in several IL-2-dependent cell lines and activated T cells, which is part of the apoptosis pathway (18Devireddy L.R. Green M.R. Mol. Cell. Biol. 2003; 23: 4532-4541Crossref PubMed Scopus (35) Google Scholar). We also launched a gene expedition study by transcription profiling of a human granulocyte macrophage colony-stimulating factor (GM-CSF)-dependent cell line TF-1 cultured in the absence of GM-CSF. Here we report the identification of TRB2 as an important modulator of apoptosis in TF-1 cells. Cell Lines and Culture Conditions—TF-1 is a cytokine-dependent hematopoietic cell line whose normal growth in vitro requires culture medium supplemented with human GM-CSF or IL-3. TF1-bcl-2 is a TF-1 derivative ectopically overexpressing Bcl-2 (19Huang H.M. Li J.C. Hsieh Y.C. Yang-Yen H.F. Yen J.J. Blood. 1999; 93: 2569-2577Crossref PubMed Google Scholar). Ba/F3, 32D, and FDCP-1 are all murine IL3-dependent cell lines. Cytokine-independent suspension cell lines (Jurkat, WEHI-3, U937, and K562) were maintained in RPMI 1640 medium containing 10% fetal bovine serum and 55 μm β-mercaptoethanol. All cytokine-dependent suspension cell lines were cultured in the same medium but supplemented with specific survival cytokines. Adherent cell lines (HeLa, 293, H1299, and HepG2) and MEL were all maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Chemicals—Dimethyl sulfoxide (Me2SO) and digitonin were purchased from Sigma. Z-VAD-fmk was purchased from Bachem. TNF-α, actinomycin D, camptothecin, cycloheximide, dexamethasone, etoposide, and staurosporine were all purchased from BioVision. Disuccinimidyl suberate (DSS) and MG132 were purchased from Pierce and Calbiochem, respectively. Cytokine Deprivation and Re-stimulation—For cytokine depletion experiments, factor-dependent cells were grown to a saturation density, washed three times in medium without cytokine, and seeded in cytokine-free medium with 0.5% fetal bovine serum for the indicated length of time. For cytokine re-stimulation experiments, GM-CSF was added to the starved TF-1 cells to a concentration of 20 ng/ml. Isolation and Activation of Mouse Primary CD4+ T Cells—Splenic CD4+ T cells were isolated by incubating splenocytes with anti-CD4 (L3T4) microbeads followed by purification with an LS-positive selection column and a MACS separator (Miltenyi Biotec, Auburn, CA) according to the manufacturer's recommendations. Purified CD4+ cells were activated in plates coated with anti-CD3 (2C11, 5 μg/ml) and anti-CD28 antibody (37.51, 2.5 μg/ml) for 3 days and then transferred to plates without antibody coating for further cultivation in the presence or absence of IL-2. Expression Vectors—pTRB2-IRES-EGFP was generated by subcloning mouse trb2 cDNA into the BamHI site of the pIRES2-EGFP vector (Clontech). For construction of pEGFP-TRB2 (T2E), the coding region of human TRB2 cDNA without a stop codon was ligated into the BamHI sites of the pEGFP-N3 vector (Clontech). To construct expression plasmids encoding the TRB2-EGFP mutants, standard PCR-assisted mutagenesis-coupled cloning methods were carried out using primer 5′-GCAAGGTGTTTGATATCAGC-3′ in combination with the following primers (underlined nucleotides differ from the wild-type sequence): TRB2-K177A, 5′-CTACTTTTTCCGCAGCGCGAGGTCCCGCAGCA-3′, and TRB2-K177R, 5′-CTACTTTTTCCGCAGCCTGAGGTCCCGCAGCA-3′. These PCR products and a third primer 5′-AGCTGGGTTCAATGTCATGG-3′ were used in a second PCR using pEGFP-TRB2 as template. The resultant PCR products were restricted with EcoRV and BstXI before they were used to replace the corresponding fragment of the pEGFP-TRB2. All mutated nucleotides were confirmed by sequencing. Expression plasmids encoding HA-tagged mouse mcl-1 (20Liu H. Peng H.W. Cheng Y.S. Yuan H.S. Yang-Yen H.F. Mol. Cell. Biol. 2005; 25: 3117-3126Crossref PubMed Scopus (198) Google Scholar) and HA-tagged myr-Akt were as described previously (21Wang J.M. Chao J.R. Chen W. Kuo M.L. Yen J.J. Yang-Yen H.F. Mol. Cell. Biol. 1999; 19: 6195-6206Crossref PubMed Google Scholar). Gene Transfer—Gene transfers into hematopoietic cells were carried out by electroporation using a Bio-Rad Genepulser set at 180 V and 975 μF (for TF-1 and TF-1-bcl-2 cells) or at 200 V and 975 μF (for other hematopoietic cells) or as otherwise indicated. Transfections of adhesion cells were performed by using Lipofectamine (Invitrogen) according to the manufacturer's protocol. Northern and Western Blot Analyses—For Northern blot analysis, total RNA was prepared using the TRIzol reagent kit (Invitrogen) and analyzed by the standard protocol using cDNA probes specific to TRB1, TRB2, TRB3, and G3PDH. For Western blot analysis, cells to be analyzed were lysed in a buffer containing 25 mm Tris (pH 7.6), 150 mm NaCl, 2.5 mm MgCl2, 0.5 mm EDTA, 0.5% Nonidet P-40, 5 mm β-glycerophosphate, 1 mm dithiothreitol, 5% glycerol, and protease inhibitors (7Du K. Herzig S. Kulkarni R.N. Montminy M. Science. 2003; 300: 1574-1577Crossref PubMed Scopus (714) Google Scholar), and 100 μg of cell lysates, unless otherwise indicated, were analyzed by Western blotting using antibodies as indicated in each figure. Antibodies—Polyclonal antibody specifically recognizing TRB2 was generated by immunizing rabbits with bacterially produced recombinant TRB2 protein by a standard protocol, and was affinity-purified using specific antigen cross-linked to CNBr-activated Sepharose (GE Healthcare). The specificity of this antibody was reconfirmed by Western blot analysis with or without TRB2 antigen competition (data not shown). This antibody can recognize both human and murine TRB2. Other antibodies used in this study include those specific to Bcl-2, Bcl-xL, Bax, GFP, human Mcl-1, cytochrome c, p38 (all from Santa Cruz Biotechnology), activated Bax (Clontech), caspase-3, caspase-8, caspase-9, cleaved poly(ADP-ribose) polymerase (all from Cell Signaling Technologies, Boston), hemagglutinin (HA) tag (Roche Diagnostics), and actin (Sigma). Antibody specific to mouse mcl-1 (20Liu H. Peng H.W. Cheng Y.S. Yuan H.S. Yang-Yen H.F. Mol. Cell. Biol. 2005; 25: 3117-3126Crossref PubMed Scopus (198) Google Scholar) or mitochondrial import receptor Tom70 was as described previously (22Chou C.H. Lee R.S. Yang-Yen H.F. Mol. Biol. Cell. 2006; 17: 3952-3963Crossref PubMed Scopus (38) Google Scholar). Antibody specific to p53 was a gift from Sheau-Yann Shieh, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan. Annexin V and TUNEL Staining and Caspase Activity Assays—To analyze cells that have undergone apoptosis, annexin V staining and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assays were carried out using the annexin V-cy3 apoptosis detection kit (BioVision, CA) and the in situ cell death detection kit (TMR red, Roche Diagnostics), respectively. In some cases, cells were stained with 4′,6-diamidino-2-phenylindole to visualize nuclei and examined by confocal microscopy. The activated caspase-3, caspase-8, and caspase-9 were evaluated with the CaspGLOW™ Red Active caspase-3, caspase-8, and caspase-9 staining kits (BioVision, CA) by flow cytometry. Flow cytometric analysis was performed with a BD FACSCalibur system. Cytochrome c Release and Detection of Oligomerized Bax—Staining of cytochrome c was carried out essentially as described by Willis et al. (23Willis S.N. Chen L. Dewson G. Wei A. Naik E. Fletcher J.I. Adams J.M. Huang D.C. Genes Dev. 2005; 19: 1294-1305Crossref PubMed Scopus (1053) Google Scholar). In some cases, cells to be analyzed were fractionated into cytosolic and mitochondrial fractions (24Everett H. Barry M. Lee S.F. Sun X. Graham K. Stone J. Bleackley R.C. McFadden G. J. Exp. Med. 2000; 191: 1487-1498Crossref PubMed Scopus (117) Google Scholar), and cytochrome c partitioned in these two fractions was analyzed by Western blotting using anti-cytochrome c antibody. To detect oligomerized Bax, cells to be analyzed were cross-linked with disuccinimidyl suberate essentially as described by Sundararajan and White (25Sundararajan R. White E. J. Virol. 2001; 75: 7506-7516Crossref PubMed Scopus (80) Google Scholar). Following cross-linking, cells were lysed and cell lysates analyzed by Western blotting using Bax-specific antibody. RNA Interference—Double-stranded RNA duplex corresponding to human TRB2 (5′-gctggaaagcctggaagac-3′) and nontargeting (nontargeting number 1, D-001210-01-20) siRNA were purchased from Dharmacon (Chicago). Cells to be analyzed (5 × 106) were mixed with 1 nmol of siRNA in 0.4 ml of Optimal MEM and subjected to electroporation at 250 V, 400 μF with Bio-Rad GenePulser apparatus. TRB2 Is an Immediate Early Gene Induced by GM-CSF Deprivation—In the TF-1 cell line, deprivation of human GM-CSF induces profound apoptosis within 24 h. This apoptotic process can be suppressed significantly by the protein synthesis inhibitor cycloheximide, suggesting that de novo gene expression is required to mediate this death program. To identify genes whose expression is up-regulated after cytokine deprivation, we studied the expression profiles of 8000 genes using a cDNA microarray (Taiwan Genome Sciences, Inc., Taipei) with mRNA isolated from TF-1 cells cultured in medium with or without GM-CSF. Among several candidates, we confirmed by Northern blot analysis that the TRB2 gene was highly inducible upon GM-CSF deprivation. After removal of GM-CSF, the mRNA levels of TRB2 were up-regulated by 3 h and reached a peak at 24 h (Fig. 1A, lanes 2–5). However, when the cytokine-deprived cells were re-stimulated with GM-CSF, the TRB2 mRNA returned to a base-line level at 3 h (Fig. 1B, lanes 3–5). Furthermore, the induction of TRB2 mRNA by GM-CSF deprivation did not require new protein synthesis, as cycloheximide could not suppress this induction (Fig. 1C, compare lanes 6 and 9). In fact, cycloheximide alone slightly induced TRB2 expression (Fig. 1C, lane 3). This result suggests that TRB2 is an immediate early gene activated by GM-CSF deprivation. Consistent with the increased expression of TRB2 mRNA, the increased TRB2 protein level following GM-CSF deprivation of TF-1 was also observed (Fig. 1D, lanes 2 and 3). Induction of TRB2 expression was also observed in TF-1 cells that had been previously maintained in another survival cytokine IL-3 or in TF-1 derivatives stably overexpressing IL5R α chain (19Huang H.M. Li J.C. Hsieh Y.C. Yang-Yen H.F. Yen J.J. Blood. 1999; 93: 2569-2577Crossref PubMed Google Scholar) maintained in IL-5 (data not shown). We next examined whether TRB2 could also be induced in other cytokine-dependent cells upon removal of their dependent cytokines. To our surprise, most cell lines examined, including murine GM-CSF-dependent C2GM cells, IL-2-dependent HT-2 cell line, and IL-3-dependent Ba/F3, 32D, and FDCP-1 cells do not manifest this property (data not shown). However, the trb2 mRNA and the TRB2 protein were clearly induced in the activated primary murine (Fig. 1, E and F, respectively) or human (data not shown) CD4+ T cells upon IL-2 deprivation. TRB2 Induction Is Highly Selective—To explore whether TRB2 is a general apoptosis-responsive gene, TF-1 cells were treated with various apoptotic stimuli, including actinomycin D, camptothecin, cycloheximide, dexamethasone, etoposide, staurosporine, and UV irradiation (Fig. 2A). These apoptotic agents, except cycloheximide and dexamethasone, could induce a substantial degree of apoptosis, which was comparable with that induced by GM-CSF deprivation (-GM, Fig. 2A). Intriguingly, the results of Northern blot analysis demonstrated that TRB2 expression was induced profoundly by GM-CSF starvation, and induced slightly by cycloheximide and dexamethasone, but not by other apoptotic treatments (Fig. 2A, lanes 5–19). Because another human tribbles ortholog, TRB3 (also named NIPK or SKIP3), could be induced in PC6-3 cells by nerve growth factor deprivation (26Mayumi-Matsuda K. Kojima S. Suzuki H. Sakata T. Biochem. Biophys. Res. Commun. 1999; 258: 260-264Crossref PubMed Scopus (61) Google Scholar), we investigated whether other members of tribbles are inducible in our experiments. Northern blot analysis showed that in the absence of GM-CSF TRB1 mRNA was only slightly induced (Fig. 2A, top panel), whereas the mRNA of TRB3 was depressed (Fig. 2A, 3rd panel, compare lanes 1 and 2, and 3 and 4). Likewise, the RNA level of TRB1 could be induced slightly by camptothecin (Fig. 2A, 3rd panel, lanes 7 and 8), cycloheximide (lanes 9 and 10), and etoposide (lanes 13 and 14). On the other hand, TRB3 was clearly induced by dexamethasone (Fig. 2A, 3rd panel, lanes 11 and 12) but not by other treatments. In contrast, camptothecin and staurosporine could reduce the expression of TRB3, like the effect of GM-CSF deprivation (Fig. 2A, lanes 3, 4, 7, 8, 15, and 16). To extend our observation in TF-1 cells, activated mouse primary CD4+ T cells were subjected to the same treatments and analyzed. As shown in Fig. 2B, murine trb2 was induced strongly by IL-2 starvation (lanes 3 and 4), induced slightly by cycloheximide and etoposide, but not induced by other treatments (lanes 5–16), suggesting that a similar induction mechanism may apply to both human and mouse TRB2 genes. We further tested whether TRB2 can be induced by stimuli that activate the extrinsic death pathway. To address this issue, TF-1 cells were treated with cycloheximide plus tumor necrosis factor-α (TNF-α, Fig. 2C) to activate a TNF receptor 1-dependent death signal, or the activated murine CD4+ T cells were treated with IL-2 and CD3 antibody (Fig. 2D) to elicit a CD95-dependent activation-induced cell death. As shown in Fig. 2, C and D, in neither case did we observe any significant induction of TRB2 (lanes 3, 4, 7, and 8 of Fig. 2C and lanes 4 and 6–8 of Fig. 2D). Altogether, these results suggest that the induction of mammalian tribbles orthologs is highly selective. Ectopic Expression of TRB2 Induces Apoptosis of Many Cell Lines of Hematopoietic Origin—The correlation between induction of TRB2 and apoptosis after cytokine deprivation in TF-1 and activated primary CD4+ T cells prompted us to investigate whether ectopic expression of TRB2 is sufficient to induce apoptosis. To address this issue, a bicistronic expression plasmid pTRB2-IRES-EGFP containing an internal ribosome-entering site (IRES) sequence, which can simultaneously produce the authentic TRB2 and the EGFP (Clontech) (Fig. 3A, panel a), was constructed and introduced into rapidly growing Ba/F3 cells. As shown in Fig. 3B, the expression of TRB2 was readily detectable with anti-TRB2 antibody in Western blot analysis (lane 2). In IL-3-free culture medium overexpression of EGFP alone (Vec) induced apoptosis of 31.6% (±3.4) (n = 5) of transfected cells (i.e. GFP(+) population), whereas overexpression of TRB2 (T2) increased apoptosis to 65.6% (±4.9) (n = 5). In these experiments, we observed consistently that TRB2 expression significantly enhanced IL-3 withdrawal-induced apoptosis of Ba/F3 cells. However, no significant apoptosis-inducing effect was observed for TRB2 when cells were cultured in medium containing IL-3 (data not shown). We next examined whether a similar apoptosis-enhancing effect could be observed for the TRB2-EGFP fusion protein (T2E, Fig. 3D, lane 2), which is encoded by the plasmid pTRB2-EGFP (Fig. 3A, panel b). In the IL-3-containing medium, transfection of Ba/F3 cells with 30 μg of pEGFP-N3 vector usually resulted in 20–30% GFP(+) cells (12 or 20 h post-transfection), whereas under the same conditions, transfection with the pTRB2-EGFP plasmid resulted in only 5–15% GFP(+) cells (see one example shown in supplemental Fig. S1). Of note, although transfection of cells with lower amounts of EGFP or T2E expression vectors (5–15 μg) resulted in lower percentages of GFP(+) cells, the same trend was always observed, i.e. significantly less GFP(+) cells were observed in cells transfected with T2E than with EGFP expression vector (supplemental Fig. S1). Furthermore, we noticed that for cells transfected with the control vector, a majority of GFP(+) cells were TUNEL(-) under confocal microscopy. However, the TUNEL(+)/GFP(+) population in pTRB2-EGFP-transfected cells was significantly increased, even when cells were cultured in the presence of IL-3 (Fig. 3E). Flow cytometric analysis revealed that in one representative experiment, expression of T2E in Ba/F3 cells resulted in 61% of GFP(+) cells to be TUNEL-positive, whereas overexpression of EGFP proteins resulted in only 3% cell death (Fig. 3F, top panels, T2E versus E). Similar percentages of cell death were observed when the apoptotic cells were quantified by the annexin V staining assay (Fig. 3F, bottom panels). Furthermore, T2E-induced cell death was also consistently observed in experiments performed with TF-1 cells (Fig. 3G). Next, we examined whether the apoptotic effect of T2E was mediated through caspase-dependent pathways. As shown in Fig. 3G, T2E-induced apoptosis of either Ba/F3 or TF-1 cells cultured in cytokine-containing medium could be markedly suppressed when cells were treated with the pan-caspase inhibitor Z-VAD-fmk (Fig. 3G, white versus gray columns). These data suggest that overexpression of the TRB2-EGFP fusion protein could cause cell death with typical apoptotic characteristics. Because TRB2 is a kinase-like protein, we wondered whether any potential kinase activity of TRB2 would be required for its pro-apoptotic function. To address this issue, the lysine residue at the putative catalytic center of subdomain VIB of TRB2, which has the motif RDLKL, was mutated into either arginine (K177R) or alanine (K177A) to create a kinase-dead mutant, and the apoptogenic activity of these two kinase-dead mutants was then compared with that of the wild-type protein. As shown in Fig. 3, H and I, under the same experimental conditions when wild-type and both mutants (all expressed as EGFP fusion proteins, i.e. T2E, K177A-E and K177R-E) were transiently expressed at a similar level in Ba/F cells (Fig. 3H), they all resulted in a similar degree of apoptosis (Fig. 3I), suggesting that the pro-apoptotic function of TRB2 does not require its putative kinase activity. The ability of the TRB2 fusion proteins (T2E) to induce cell death was further investigated in a few other murine and human cell lines. As shown in Fig. 4, T2E induced apoptosis in all cytokine-dependent hematopoietic cell lines tested, including 32D, FDCP-1, and HT-2 (lanes 1–3), although these cell lines did not express TRB2 upon cytokine deprivation (data not shown). Furthermore, the primary murine CD4+ T cells were very sensitive to T2E (Fig. 4, lane 4). On the other hand, some cytokine-independent leukemic cell lines, e.g. WEHI3 and Jurkat (Fig. 4, lanes 5 and 6), were very sensitive to T2E-induced apoptosis, whereas other cell lines, including K562 and U937 (lanes 8 and 9), were highly resistant. Interestingly, most nonhematopoietic cell lines tested, including HeLa, 293, H1299, and HepG2, were highly resistant to T2E-induced apoptosis (Fig. 4, lanes 10–13). These data suggest that the apoptotic activity of T2E is largely restricted to hematopoietic cells. TRB2-EGFP Triggers Apoptosis via Mitochondria Dys-function—A variety of external and internal signals converge on mitochondria to trigger or inhibit apoptosis (27Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). To understand the mechanism of the apoptotic effect of TRB2, we investigated the ability of T2E to damage mitochondria. The mitochondrial dye MitoTracker Red was used to stain the viable cells, because apoptotic cells lost membrane potential and lost the staining of MitoTracker Red. As shown in one representative result (Fig. 5A), expression of T2E in Ba/F3 cells increased dramatically the proportion of cells that lost the membrane potential, from 10 to 78% (see ΔΨmL). Loss of mitochondrial membrane potential (MMP, ΔΨm) correlated strongly with activation of caspases, including caspase-3, caspase-8, and caspase-9 (Fig. 5A), and the percentage of cells that have undergone apoptosis as revealed by the annexin V (An-V) staining method. T2E-induced activation of caspase-3, caspase-8, and caspase-9, as evident from the generation of specific cleavage products, was also demonstrated in TF-1 cells by Western blotting with antibody specifically recognizing each caspase (Fig. 5B). Of note, under our experimental conditions, although the cleaved form of caspase-3 was only slightly detectable, the production of cleaved poly(ADP-ribose) polymerase, a typical caspase 3 substrate, was quite prominent (Fig. 5B, 2nd to the bottom panel), suggesting that caspase-3 was also highly activated by T2E in TF-1 cells. We next examined whether T2E could induce activation of Bax, a proapoptotic molecule that, in response to apoptotic stimuli, will change conformation and oligomerize, which leads to mitochondria dysfunction (28Griffiths G.J. Dubrez L. Morgan C.P. Jones N.A. Whitehouse J. Corfe B.M. Dive C. Hickman J.A. J. Cell Biol. 1999; 144: 903-914Crossref PubMed Scopus (394) Google Schola" @default.
• W1966822894 created "2016-06-24" @default.
• W1966822894 creator A5001758552 @default.
• W1966822894 creator A5055314744 @default.
• W1966822894 creator A5055561346 @default.
• W1966822894 creator A5068463170 @default.
• W1966822894 creator A5069505210 @default.
• W1966822894 creator A5074666172 @default.
• W1966822894 date "2007-07-01" @default.
• W1966822894 modified "2023-09-28" @default.
• W1966822894 title "Survival Factor Withdrawal-induced Apoptosis of TF-1 Cells Involves a TRB2-Mcl-1 Axis-dependent Pathway" @default.
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