p73 Induces Apoptosis via PUMA Transactivation and Bax Mitochondrial Translocation
2004; Elsevier BV; Volume: 279; Issue: 9 Linguagem: Inglês
10.1074/jbc.m307469200
ISSN1083-351X
AutoresGerry Melino, Francesca Bernassola, Marco Ranalli, Karen S. Yee, Wei Zong, Marco Corazzari, Richard A. Knight, Douglas R. Green, Craig B. Thompson, Karen H. Vousden,
Tópico(s)Cell death mechanisms and regulation
Resumop73, an important developmental gene, shares a high sequence homology with p53 and induces both G1 cell cycle arrest and apoptosis. However, the molecular mechanisms through which p73 induces apoptosis are unclear. We found that p73-induced apoptosis is mediated by PUMA (p53 up-regulated modulator of apoptosis) induction, which, in turn, causes Bax mitochondrial translocation and cytochrome c release. Overexpression of p73 isoforms promotes cell death and bax promoter transactivation in a time-dependent manner. However, the kinetics of apoptosis do not correlate with the increase of Bax protein levels. Instead, p73-induced mitochondrial translocation of Bax is kinetically compatible with the induction of cell death. p73 is localized in the nucleus and remains nuclear during the induction of cell death, indicating that the effect of p73 on Bax translocation is indirect. The ability of p73 to directly transactivate PUMA and the direct effect of PUMA on Bax conformation and mitochondrial relocalization suggest a molecular link between p73 and the mitochondrial apoptotic pathway. Our data therefore indicate that PUMA-mediated Bax mitochondrial translocation, rather than its direct transactivation, correlates with cell death. Finally, human ΔNp73, an isoform lacking the amino-terminal transactivation domain, inhibits TAp73-induced as well as p53-induced apoptosis. The ΔNp73 isoforms seem therefore to act as dominant negatives, repressing the PUMA/Bax system and, thus, finely tuning p73-induced apoptosis. Our findings demonstrate that p73 elicits apoptosis via the mitochondrial pathway using PUMA and Bax as mediators. p73, an important developmental gene, shares a high sequence homology with p53 and induces both G1 cell cycle arrest and apoptosis. However, the molecular mechanisms through which p73 induces apoptosis are unclear. We found that p73-induced apoptosis is mediated by PUMA (p53 up-regulated modulator of apoptosis) induction, which, in turn, causes Bax mitochondrial translocation and cytochrome c release. Overexpression of p73 isoforms promotes cell death and bax promoter transactivation in a time-dependent manner. However, the kinetics of apoptosis do not correlate with the increase of Bax protein levels. Instead, p73-induced mitochondrial translocation of Bax is kinetically compatible with the induction of cell death. p73 is localized in the nucleus and remains nuclear during the induction of cell death, indicating that the effect of p73 on Bax translocation is indirect. The ability of p73 to directly transactivate PUMA and the direct effect of PUMA on Bax conformation and mitochondrial relocalization suggest a molecular link between p73 and the mitochondrial apoptotic pathway. Our data therefore indicate that PUMA-mediated Bax mitochondrial translocation, rather than its direct transactivation, correlates with cell death. Finally, human ΔNp73, an isoform lacking the amino-terminal transactivation domain, inhibits TAp73-induced as well as p53-induced apoptosis. The ΔNp73 isoforms seem therefore to act as dominant negatives, repressing the PUMA/Bax system and, thus, finely tuning p73-induced apoptosis. Our findings demonstrate that p73 elicits apoptosis via the mitochondrial pathway using PUMA and Bax as mediators. p73 is a member of the p53 family (1Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrar P. McKeon F Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1530) Google Scholar). The two proteins show a high degree of sequence homology, particularly in the central sequence-specific DNA binding domain, the amino-terminal activation domain, and the carboxyl-terminal oligomerization domain (1Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrar P. McKeon F Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1530) Google Scholar). Even though p73 shows an evident developmental role, the strong structural similarity suggests that, at least in part, the function of p73 may closely resemble that of p53 (2Yang A. Kaghad M. Caput D. McKeon F. Trends Genet. 2002; 18: 90-95Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 3Kaelin Jr., W.G. Oncogene. 1999; 18: 7701-7705Crossref PubMed Scopus (158) Google Scholar). Indeed, like p53, p73 induces G1 cell growth arrest (1Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrar P. McKeon F Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1530) Google Scholar, 4Jost C.A. Marin M.C. Kaelin Jr., W.G. Nature. 1997; 389: 191-194Crossref PubMed Scopus (895) Google Scholar), activates the transcription of some endogenous p53 target genes such as p21Waf1/Cip1, RGC (ribosomal gene cluster), mdm2, bax, cyclin G, GADD45 (growth arrest- and DNA damage-inducible 45), IGF-BP3 (insulin-like growth factor-binding protein 3), and 14-3-3σ (4Jost C.A. Marin M.C. Kaelin Jr., W.G. Nature. 1997; 389: 191-194Crossref PubMed Scopus (895) Google Scholar, 5Zhu J. Jiang J. Zhou W. Chen X. Cancer Res. 1998; 58: 5061-5065PubMed Google Scholar, 6Di Como C.J. Gaiddon C. Prives C. Mol. Cell. Biol. 1999; 19: 1438-1449Crossref PubMed Scopus (379) Google Scholar, 7Zeng X. Chen L. Jost C.A. Maya R. Keller D. Wang X. Kaelin Jr., W.G. Oren M. Chen J. Lu H. Mol. Cell. Biol. 1999; 19: 3257-3266Crossref PubMed Scopus (301) Google Scholar, 8Dobbelstein M. Wienzek S. Konig C. Roth J. Oncogene. 1999; 18: 2101-2106Crossref PubMed Scopus (144) Google Scholar), and induces apoptosis irrespective of p53 status (1Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrar P. McKeon F Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1530) Google Scholar, 4Jost C.A. Marin M.C. Kaelin Jr., W.G. Nature. 1997; 389: 191-194Crossref PubMed Scopus (895) Google Scholar). The structural integrity of the p73 DNA binding domain is required for these activities, suggesting that p73 recognizes the p53-responsive DNA elements. The levels of p73 are not changed by exposure to DNA-damaging agents such as actinomycin D or UV irradiation, which increase p53 levels (1Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrar P. McKeon F Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1530) Google Scholar) Moreover, steady-state levels of p73 are not reduced by complex formation with Mdm2 (7Zeng X. Chen L. Jost C.A. Maya R. Keller D. Wang X. Kaelin Jr., W.G. Oren M. Chen J. Lu H. Mol. Cell. Biol. 1999; 19: 3257-3266Crossref PubMed Scopus (301) Google Scholar, 8Dobbelstein M. Wienzek S. Konig C. Roth J. Oncogene. 1999; 18: 2101-2106Crossref PubMed Scopus (144) Google Scholar), which targets p53 for ubiquitin-mediated proteolysis (9Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3629) Google Scholar, 10Kubbutat M.H.G. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2798) Google Scholar, 11Honda R. Tanaka H. Yasuda H. FEBS Lett. 1997; 420: 25-27Crossref PubMed Scopus (1576) Google Scholar). Recent studies have shown that p73 can be stabilized and tyrosine phosphorylated by c-Abl following DNA damage, leading to an enhanced p73-mediated apoptotic response (12Agami R. Blandino G. Oren M. Shaul Y. Nature. 1999; 399: 809-813Crossref PubMed Scopus (502) Google Scholar, 13Gong J.C. Costanzo A. Yang H.Q. Melino G. Kaelin Jr., W.G. Levrero M. Wang J.Y. Nature. 1999; 399: 806-809Crossref PubMed Scopus (826) Google Scholar, 14Yuan Z.M. Shioya H. Ishiko T. Sun X. Gu J. Huang Y.Y. Lu H. Kharbanda S. Weichselbaum R. Kufe D. Nature. 1999; 399: 814-817Crossref PubMed Scopus (538) Google Scholar). Therefore there are both similarities and differences in the mechanisms of p53- and p73-mediated apoptosis. bax, a pro-apoptotic Bcl-2 family member, is a p53 and a p73 target gene (15Oltvai Z.N. Milliman C.L. Korsmeyer S.J. Cell. 1993; 74: 609-619Abstract Full Text PDF PubMed Scopus (5823) Google Scholar, 16Yin X.M. Oltvai Z.N. Veis-Novack D.J. Linette G.P. Korsmeyer S.J. Cold Spring Harbor Symp. Quant. Biol. 1994; 59: 387-393Crossref PubMed Scopus (72) Google Scholar). In unstressed cells, the Bax protein exists as an inactive monomer in the cytosol and is induced to homo-oligomerize and translocate to mitochondria upon death stimuli, thus leading to cytochrome c release and caspase activation (17Hsu Y.T. Wolter K.G. Youle R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3668-3672Crossref PubMed Scopus (1021) Google Scholar, 18Wolter K.G. Hsu Y. Smith C.L. Nechushtan A. Xi X. Youle R.J. J. Cell Biol. 1997; 139: 1281-1292Crossref PubMed Scopus (1562) Google Scholar, 19Rosse T. Olivier R. Monney L. Rager M. Conus S. Fellay I. Jansen B. Borner C. Nature. 1998; 391: 496-499Crossref PubMed Scopus (787) Google Scholar, 20Juergensmeier J.M. Xie Z. Deveraux Q. Ellerby L. Bredesen D. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4997-5002Crossref PubMed Scopus (1367) Google Scholar). Cytochrome c release results from the induction of the mitochondrial permeability transition, an event associated with disruption of the mitochondrial inner transmembrane potential ΔΨm (21Pastorino J.G. Chen S.T. Tafani M. Snyder J.W. Farber J.L. J. Biol. Chem. 1998; 273: 7770-7775Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar) that has been implicated in a variety of apoptotic phenomena (22Zamzami N. Marchetti P. Castedo M. Zamin C. Vayssiere J.L. Petit P.X. Kroemer G. J. Exp. Med. 1995; 181: 1661-1672Crossref PubMed Scopus (1089) Google Scholar, 23Zarotti M. Szabo I. Biochim. Biophys. Acta. 1995; 1241: 139-176Crossref PubMed Scopus (2176) Google Scholar). Unfortunately however, very little is known about the molecular events through which p73 induces apoptosis. Here we report a study on p73-induced apoptosis in Saos-2 cells demonstrating that the γ isoform is the most effective both in the induction of apoptosis and in bax transactivation. However, at least in this model, bax transactivation does not seem to be crucial for the induction of death. We show that, in cells undergoing p73-dependent apoptosis, p73 displays a nuclear localization pattern, whereas Bax translocates from the cytosol to mitochondria, thus causing cytochrome c release. The BH3-only protein PUMA (p53 up-regulated modulator of apoptosis) is transcriptionally induced during p73-mediated cell death and favors a conformational change and relocalization of Bax to the mitochondria. Moreover, ΔNp73, 1The abbreviations used are: ΔNp73, p73 isoform lacking most of the N-terminal transactivation domain; Δ84p73β, p73β isoform with 84 amino-terminal residues deleted; dox, doxycycline; GFP, green fluorescent protein; HA, hemagglutinin A; Luc, luciferase; MEF, mouse embryo fibroblast; PI, propidium iodide; Tap73, transactivating p73 isoform; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling. 1The abbreviations used are: ΔNp73, p73 isoform lacking most of the N-terminal transactivation domain; Δ84p73β, p73β isoform with 84 amino-terminal residues deleted; dox, doxycycline; GFP, green fluorescent protein; HA, hemagglutinin A; Luc, luciferase; MEF, mouse embryo fibroblast; PI, propidium iodide; Tap73, transactivating p73 isoform; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling. a recently cloned p73 isoform (24Grob T.J. Novak U. Maisse C. Barcaroli D. Luthi A.U. Pirnia F. Hugli B. Graber H.U. De Laurenzi V. Fey M.F. Melino G. Tobler A. Cell Death Differ. 2001; 12: 1213-1223Crossref Scopus (306) Google Scholar) that lacks the transactivation domain and acts as a p73 dominant negative, inhibits p73-induced apoptosis. Here we provide evidence for a transcription-independent effect of p73 on Bax able to activate the mitochondrial pathway during cell death. Cell Culture—Saos-2 and HeLa cells stably expressing Bax-GFP or the cytochrome c-GFP fusion protein were cultured in a 1:1 mixture of Ham's F-12/Dulbecco's minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO2 in air. Saos-2 cells with doxycycline (dox)-inducible expression of p73 isoforms (25Nakano K. Bàlint E. Ashcroft M. Vousden K.H. Oncogene. 2000; 19: 4283-4289Crossref PubMed Scopus (199) Google Scholar) or PUMA (prepared as in Ref. 25Nakano K. Bàlint E. Ashcroft M. Vousden K.H. Oncogene. 2000; 19: 4283-4289Crossref PubMed Scopus (199) Google Scholar) were cultured in the same medium supplemented with 10% heat-inactivated, tetracycline-free fetal bovine serum (Tet system-approved fetal bovine serum, Clontech). Both p73 and PUMA inducible cell lines were HA-tagged in order to monitor the steady-state levels of protein induction by Western and laser densitometry. Mouse embryo fibroblasts (MEFs) for null p53, Bax, Bak, Bax/Bak, and U2OS cells were grown in similar conditions. To induce protein expression, the tetracycline-inducible cell lines were treated with the tetracycline analog dox at 2.5 μg/ml as indicated. Plasmids—Human p73 isoforms (including Δ84p73β) and p53 cDNAs in pCDNA3 (18Wolter K.G. Hsu Y. Smith C.L. Nechushtan A. Xi X. Youle R.J. J. Cell Biol. 1997; 139: 1281-1292Crossref PubMed Scopus (1562) Google Scholar, 26De Laurenzi V. Costanzo A. Barcaroli D. Terrinoni A. Falco M. Annicchiarico-Petruzzelli M. Levrero M. Melino G. J. Exp. Med. 1998; 188: 1763-1768Crossref PubMed Scopus (360) Google Scholar), ΔNp73 (24Grob T.J. Novak U. Maisse C. Barcaroli D. Luthi A.U. Pirnia F. Hugli B. Graber H.U. De Laurenzi V. Fey M.F. Melino G. Tobler A. Cell Death Differ. 2001; 12: 1213-1223Crossref Scopus (306) Google Scholar),and PUMA cDNA (27Nakano K. Vousden K.H. Mol. Cell. 2001; 7: 683-694Abstract Full Text Full Text PDF PubMed Scopus (1839) Google Scholar, 28Yu J. Zhang L. Hwang P.M. Kinzler K.W. Vogelstein B. Mol. Cell. 2001; 7: 673-682Abstract Full Text Full Text PDF PubMed Scopus (1075) Google Scholar) have been described previously. Western Blot Analysis—Saos-2 subclones with dox-inducible expression of p73 isoforms were treated with dox (2.5 μg/ml) for 48 h and lysed in buffer A (50 mm Tris, pH 8, 150 mm NaCl, 0.5% Nonidet P-40, 0.5 mg/ml leupeptin, 1 mg/ml aprotinin, and 0.5 mm phenylmethylsulfonyl fluoride) for 1 h on ice. The lysate was cleared by centrifugation, and 20-μg aliquots of cell extract, as determined by the Bradford method, were resolved by electrophoresis in a 12% SDS-polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and probed with anti-HA antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) to quantitate the steady-state levels of protein expression. To analyze the expression of Bax in the mitochondrial fraction, cells were resuspended in 3 ml of ice-cold buffer B (250 mm sucrose, 20 mm HEPES, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 17 μg/ml phenylmethylsulfonyl fluoride, 8 μg/ml aprotinin, and 2 μg/ml leupeptin, pH 7.4. Cells were passed through an ice-cold cylinder cell homogenizer (H&Y Enterprise, Redwood City, CA). Intact cells and nuclei were pelleted for 10 min at 750 × g. The supernatant was spun at 10,000 × g for 20 min. The pellet was lysed in buffer A as reported above and represents the mitochondrial fraction. 20 μg of this lysate was resolved by electrophoresis in a 12% SDS-polyacrylamide gel, transferred to a PVDF membrane, and probed with anti-Bax, an anti-procaspase antibody (Pharmingen), or an anti-Bax conformational epitope (antibody 6A7, donated by C. Thompson). Immunocomplexes were detected by a chemiluminescence-based system (Amersham Biosciences) according to the manufacturer's instructions. Densitometric analysis was carried out using Kodak one-dimensional 2.0 software. Determination of Apoptosis—To estimate DNA fragmentation, Saos-2 cells with dox-inducible expression of p73 isoforms were treated with dox (2.5 μg/ml) for 48 h, collected at 800 × g for 10 min, and fixed with a 1:1 phosphate-buffered saline and methanol-acetone (4:1 (v/v)) solution at -20 °C. Hypodiploid sub-G1 events were evaluated by flow cytometry after propidium iodide (PI) staining, as described previously (13Gong J.C. Costanzo A. Yang H.Q. Melino G. Kaelin Jr., W.G. Levrero M. Wang J.Y. Nature. 1999; 399: 806-809Crossref PubMed Scopus (826) Google Scholar, 24Grob T.J. Novak U. Maisse C. Barcaroli D. Luthi A.U. Pirnia F. Hugli B. Graber H.U. De Laurenzi V. Fey M.F. Melino G. Tobler A. Cell Death Differ. 2001; 12: 1213-1223Crossref Scopus (306) Google Scholar, 26De Laurenzi V. Costanzo A. Barcaroli D. Terrinoni A. Falco M. Annicchiarico-Petruzzelli M. Levrero M. Melino G. J. Exp. Med. 1998; 188: 1763-1768Crossref PubMed Scopus (360) Google Scholar). For each point 20,000 events were collected, excluding doublets and aggregates, by electronic gate. Bax Staining—Saos-2 cells with inducible expression of p73γ and p53 were stimulated to produce the protein for the indicated times using dox (2.5 μg/ml) and then fixed in 4% paraformaldehyde in phosphate-buffered saline (pH 7.4) for 15 min. Cells were then permeabilized for 2 min in 0.1% Triton X-100 in phosphate-buffered saline and incubated for 1 h with anti-Bax antibody followed by 30 min of incubation with anti-rabbit ALEXA 488 antibody (Molecular Probes). 10,000 events were collected by monitoring 530-nm fluorescence on a FACS-Calibur flow cytometer (BD Biosciences), as indicated above. Subcellular Localization—Cells were grown overnight on a glass coverslip and, after the indicated treatments, fixed in 4% paraformaldehyde in phosphate-buffered saline (pH 7.4) for 15 min. Saos-2 p73γ-inducible cells were induced with dox (2.5 μg/ml) for 24 h and then treated, when indicated, with 25 μm cisplatin for 24 h. Immunofluorescence was carried out using an anti-HA or anti-Bax antibody combined with TUNEL reaction according to the manufacturer's instructions (in situ cell death detection kit, TMR red, Roche Applied Science). HeLa cells stably expressing Bax-GFP were cotransfected by using LipofectAMINE 2000 (Invitrogen) with p73γ and pDS-Red1-Mito (3:1). Confocal images were acquired by using a PCM-2000 confocal microscope (Nikon) and excited with a 488-nm argon-ion laser line or a 542-nm helium-neon laser detecting with the appropriate filter set (515/30 green filter and 595/70 red filter). Images were then processed by using PCM-2000 software (Nikon). Luciferase Assay—Saos-2 cells with dox-inducible p73 isoforms or p53 were cotransfected with a reporter plasmid containing luciferase cDNA under the control of the bax promoter (Bax-Pr/Luc) and a Renilla plasmid as internal standard (40:1). p73 or p53 expression was then induced by treatment with dox (2.5 μg/ml). Saos-2 cells were cotransfected with expression plasmids encoding p73α or p53 with or without ΔNp73α, ΔNp73β, or ΔNp73γ, together with Bax-Pr/Luc (bax-Pr/Luc: p73/p53:ΔNp73:Renilla = 1:1:8:0.25). N1E-115 cells were cotransfected with an expression plasmid encoding p73α, p73β, p73γ, p73δ, or p53, with or without Δ84p73β together with Bax-Pr/Luc. Luciferase assays were carried out 48 h after transfection with the dual-luciferase assay system (Promega) according to the manufacturer's instructions. Luminescence was detected by using an EG&G Berthold Lumat LB 9507 luminometer, averaging the signal for 10s. p73 Expression Induces Apoptosis in Saos-2 Cells—To study the mechanisms through which p73 induces apoptosis, we took advantage of three Saos-2-derived cell lines stably transfected with the HA-tagged, dox-inducible TAp73 isoforms α, β, or γ (25Nakano K. Bàlint E. Ashcroft M. Vousden K.H. Oncogene. 2000; 19: 4283-4289Crossref PubMed Scopus (199) Google Scholar). Saos-2 cells are p53-null and do not express p73 at either mRNA and protein levels, therefore forming a suitable model for evaluation of the induction of apoptosis by p73. We measured p73 expression in Saos-2 cells incubated with the tetracycline analog dox (2.5 μg/ml). Cells were collected at different time points and assayed for protein expression using the anti-HA antibody. p73 protein levels were undetectable in untreated cells, whereas the expression of all the three p73 isoforms was induced in a time-dependent manner following treatment with dox (Fig 1A). Densitometric analysis of p73 induction revealed that β and γ isoforms were the most highly induced upon dox treatment (Fig. 1B). To test whether caspases play a role during p73-induced cell death, we measured the proenzyme expression levels of caspase 6, 7, and 8 upon dox stimulation of Saos-2 p73-inducible cell lines (Fig. 1, C-E). We found a significant reduction in procaspase 6 steadystate levels (less significant for the other procaspases) 48 h after treatment of Saos-2 p73β and p73γ. (Fig. 1, D and E). Consistent with the p73β- and γ-triggered processing of procaspase 6 and, to a lesser extent, procaspase 8, both isoforms induced apoptosis (Fig. 1F) after 48 h of dox stimulation. Therefore, expression of p73, in particular the isoforms β and γ, induces cell death and caspase activation in Saos-2 cells. p73-induced bax Expression Does Not Correlate with Apoptotic Kinetics—To investigate the possible involvement of Bax in p73-induced apoptosis, we performed a transactivating assay using a reporter plasmid (bax-Pr/Luc) containing the full-length bax-promoter placed upstream of a luciferase cDNA. We transfected Saos-2 cells expressing the dox-inducible p73α, β, and γ isoforms or p53 as a positive control, and the luciferase assay was performed after 24 h. As shown in Fig. 2A, all three p73 isoforms transactivated the bax promoter, although to a lesser degree than p53, with the p73γ isoform being the most potent transcriptional activator. This is in keeping with previous literature. Saos-2 cells inducible for p73γ and p53 expression were also analyzed for Bax protein levels following stimulation with dox by flow cytometric analysis. Interestingly, Bax up-regulation was induced in a time-dependent manner, reaching a maximum at 96 h (Fig. 2, B and C), thus indicating that the kinetics of Bax expression did not correlate with the induction of apoptosis (Fig. 1F). Although there are structural and functional similarities between p73 and p53, p73 is a less efficient transcriptional activator of the bax promoter (Fig. 2A) and a significantly slower inducer of Bax protein expression (Fig 2, B and C) than p53. Hence, at least in our cellular model, the direct transactivation of the bax promoter by p73 does not seem to be the crucial apoptotic effector mechanism driven by p73. p73 Indirectly Induces Bax Mitochondrial Relocalization— Bax translocation from cytosol to mitochondria is a critical step in p53-mediated apoptosis (29Deng Y. Wu X. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12050-12055Crossref PubMed Scopus (175) Google Scholar). Bax contributes to apoptosis by interacting with Bcl-2/Bcl-xL and thus controlling mitochondrial protein export. Using confocal and subcellular fractionation studies, we investigated whether Bax re-localization could mediate p73-induced apoptosis in cells stably expressing a Bax-GFP fusion protein (Bax-GFP). Bax-GFP HeLa cells were cotransfected with p73γ along with a plasmid encoding a red protein that localizes to the mitochondria (pDsRed1-Mito). Confocal microscopy revealed that Bax-GFP was diffusely distributed throughout the cytosol in the absence of p73 overexpression (Fig. 3A, green fluorescence). However, Bax-GFP moved to a completely punctate distribution, co-localizing with mitochondria 48 h after p73 up-regulation (Fig. 3B). To further confirm Bax relocalization into mitochondria in apoptotic cells, we performed a Western blot analysis on the purified mitochondrial fraction from the Saos-2 p73γ subclone 48 h after dox stimulation. As shown in Fig. 3C, induction of p73 revealed a significant increase in Bax protein levels in the mitochondrial fraction as compared with untreated cells. Identical results were obtained using the Bax-GFP HeLa cells (data not shown). Evidence for transcription-independent, p53-mediated apoptosis has been accumulating (30Sansome C. Zaika A. Marchenko N.D. Moll U.M. FEBS Lett. 2001; 488: 110-115Crossref PubMed Scopus (155) Google Scholar, 31Mihara M. Erster S. Zaika A. Petrenko O. Chittenden T. Pancoska P. Moll U.M. Mol. Cell. 2003; 11: 577-590Abstract Full Text Full Text PDF PubMed Scopus (1444) Google Scholar). Because during DNA damage- and hypoxia-stimulated apoptosis a fraction of p53 translocates to mitochondria and directly induces cytochrome c release, we sought to examine whether p73 undergoes similar distribution changes. At the same time, the experiment would elucidate if Bax translocation is directly induced by the p73 protein per se. Interestingly, p73 (Fig. 3, green fluorescence) was clearly restricted to the nuclear compartment and did not re-localize during apoptosis triggered either by p73γ induction (Fig. 3, D and E) or by cisplatin treatment of Saos-2 cells overexpressing p73γ (Fig. 3F). Transient transfection of Saos-2 cells with an expression plasmid encoding HA-p73γ gave identical results to those obtained with Saos-2 stably transfected with p73γ (data not shown). Thus, unlike p53, p73 does not translocate to mitochondria in response to death signals. Moreover, Bax translocation to the mitochondria is not caused by a direct p73-Bax interaction. Finally, we monitored cellular distribution of the endogenous Bax protein in the p73γ-inducible clones 48 h after dox stimulation. Fig. 3G shows that Bax (green fluorescence) was spread throughout the cytosol in control cells. On the contrary, Bax displayed a punctate distribution pattern in p73γ-overexpressing cells similar to that observed in Fig. 3B (Fig. 3H). Comparable results were obtained in Saos-2 cells transfected with an expression plasmid encoding HA-p73γ (data not shown). These data indicate that p73 and Bax do not co-localize, even at later stages of p73-induced cell death. To more directly investigate the interaction between Bax and p73, we overexpressed both proteins in Saos-2 cells. As shown in Fig. 3I, we did not observe any co-localization of p73 (green fluorescence) in mitochondria (red fluorescence). These results indicate that p73 induces the mitochondrial translocation of Bax through indirect mechanisms. PUMA Is Induced by p73 and Causes a Conformational Change and Mitochondrial Translocation of Bax—To identify the mediators through which p73 indirectly induces Bax mitochondrial translocation, we examined the modulation of several BH3-only proteins (data not shown) that bind to Bcl2/Xl via their BH3 domain, thereby inactivating their protective functions (27Nakano K. Vousden K.H. Mol. Cell. 2001; 7: 683-694Abstract Full Text Full Text PDF PubMed Scopus (1839) Google Scholar, 32Cheng E.H. Wei M.C. Weiler S. Flavell R.A. Mak T.W. Lindsten T. Korsmeyer S.J. Mol. Cell. 2001; 8: 705-711Abstract Full Text Full Text PDF PubMed Scopus (1406) Google Scholar). The recently identified PUMA gene encodes two BH3 domain-containing proteins, PUMAα and PUMAβ, that are induced following p53 activation and play a role in mediating p53-induced cell death through the cytochrome c/Apaf-1-dependent pathway (27Nakano K. Vousden K.H. Mol. Cell. 2001; 7: 683-694Abstract Full Text Full Text PDF PubMed Scopus (1839) Google Scholar). We therefore investigated the possibility that p73 could directly transactivate PUMA. As shown in Fig. 4A, PUMA was transcriptionally induced by p73 within 12 h. The specific roles of the individual PUMA isoforms are not clear at the moment. We then examined whether PUMA affects Bax translocation to mitochondria during p73-induced apoptosis. As shown in Fig. 4, B-D, overexpression of both PUMA isoforms in HeLa cells stably transfected with Bax-GFP led to Bax movement from the cytosol to mitochondria 24 h after transfection. In addition, we found that PUMA caused the release of cytochrome c from mitochondria as visualized in HeLa cells stably transfected with cytochrome c-GFP (Fig. 4, E-G). Therefore, PUMA seems to mediate the effect of p73 on Bax, causing mitochondrial release of cytochrome c to activate the final steps of death in the apoptosome. To investigate how PUMA physiologically regulates Bax cellular distribution during apoptosis, we took advantage of the 6A7 anti-Bax antibody, which recognizes the activated membrane-bound form of Bax (33Hsu Y-T. Youle R.J. J. Biol. Chem. 1998; 273: 10777-10783Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). As shown in Fig. 5A, overexpression of PUMA in U2OS cells led to translocation of the endogenous Bax protein to mitochondria. Interestingly, the active membrane-bound conformation of Bax co-localized with PUMA (Fig. 5A). Identical results were obtained in a Tet-On, PUMA-inducible H1299-derived cell line. Induction of PUMA by dox stimulation resulted in Bax conformational modification and co-localization with PUMA on the mitochondria (Fig. 5B). The data suggest that PUMA causes a conformational modification of Bax, which then results in its accumulation on the mitochondria. PUMA-induced Apoptosis Requires Functional Bax/Bak—To formally prove that PUMA-induced cytochrome c release and cell death indeed relies on Bax during p73-mediated apoptosis, we first cotransfected bax-/- MEFs with p73γ and cytochrome c-GFP. We found that p73-induced cytochrome c release was delayed, but not abrogated, in the absence of bax (Fig. 6A, and data not shown). When cells were exposed to DNA damage (25 μm cisplatin), apoptosis was indeed slower in bax-/-, even though the same plateau was reached (Fig. 6B). In this experiment, both in bax+/+ and bax-/- cells, p73 steady-state protein levels were induced within 12 h (Fig. 6C). This suggests that, indeed, p73 is physiolog
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