The Molecular Mechanism of Noxa-induced Mitochondrial Dysfunction in p53-Mediated Cell Death

Abstract

Genotoxic stresses stabilize the p53 tumor suppressor protein which, in turn, transactivates target genes to cause apoptosis. Although Noxa, a “BH3-only” member of the Bcl-2 family, was shown to be a target of p53-mediated transactivation and to function as a mediator of p53-dependent apoptosis through mitochondrial dysfunction, the molecular mechanism by which Noxa causes mitochondrial dysfunction is largely unknown. Here we show that two domains (BH3 domain and mitochondrial targeting domain) in Noxa are essential for the release of cytochrome c from mitochondria. Noxainduced cytochrome c release is inhibited by permeability transition pore inhibitors such as CsA or MgCl2, and Noxa induces an ultra-structural change of mitochondria yielding “swollen” mitochondria that are unlike changes induced by tBid. This indicates that Noxa may activate the permeability transition-related pore to release cytochrome c from mitochondria into cytosol. Moreover, Bak-oligomerization, which is an essential event for tBid-induced cytochrome c release in the extrinsic death signaling pathway, is not associated with Noxa-induced cytochrome c release. This finding suggests that the pathway of Noxa-induced mitochondrial dysfunction is distinct from the one of tBid-induced mitochondrial dysfunction. Thus, we propose that there are at least two different pathways of mitochondrial dysfunction; one mediated through Noxa in response to genotoxic stresses and the other through tBid in response to death ligands. Genotoxic stresses stabilize the p53 tumor suppressor protein which, in turn, transactivates target genes to cause apoptosis. Although Noxa, a “BH3-only” member of the Bcl-2 family, was shown to be a target of p53-mediated transactivation and to function as a mediator of p53-dependent apoptosis through mitochondrial dysfunction, the molecular mechanism by which Noxa causes mitochondrial dysfunction is largely unknown. Here we show that two domains (BH3 domain and mitochondrial targeting domain) in Noxa are essential for the release of cytochrome c from mitochondria. Noxainduced cytochrome c release is inhibited by permeability transition pore inhibitors such as CsA or MgCl2, and Noxa induces an ultra-structural change of mitochondria yielding “swollen” mitochondria that are unlike changes induced by tBid. This indicates that Noxa may activate the permeability transition-related pore to release cytochrome c from mitochondria into cytosol. Moreover, Bak-oligomerization, which is an essential event for tBid-induced cytochrome c release in the extrinsic death signaling pathway, is not associated with Noxa-induced cytochrome c release. This finding suggests that the pathway of Noxa-induced mitochondrial dysfunction is distinct from the one of tBid-induced mitochondrial dysfunction. Thus, we propose that there are at least two different pathways of mitochondrial dysfunction; one mediated through Noxa in response to genotoxic stresses and the other through tBid in response to death ligands. The p53 mutation is found in more than half of all human cancer patients. Cancers with loss of p53 function are often resistant to chemotherapeutic agents mainly because of the absence of p53-dependent apoptosis (1Clarke A.R. Purdie C.A. Harrison D.J. Morris R.G. Bird C.C. Hooper M.L. Wyllie A.H. Nature. 1993; 362: 849-852Crossref PubMed Scopus (2285) Google Scholar, 2Lowe S.W. Schmitt E.M. Smith S.W. Osborne B.A. Jacks T. Nature. 1993; 362: 847-849Crossref PubMed Scopus (2753) Google Scholar, 3Lowe S.W. Bodis S. McClatchey A. Remington L. Ruley H.E. Fisher D.E. Housman D.E. Jacks T. Science. 1994; 266: 807-810Crossref PubMed Scopus (1537) Google Scholar). The p53-dependent apoptosis largely depends on the capability of p53 to function as a transcription factor, although recent reports show that the transcription-independent function of p53 plays a role in this process. There are many target genes (e.g. Apaf-1, DR5, p53AIP, PUMA, and Noxa) transactivated by p53 that are proposed to mediate p53-dependent apoptosis in various cancer cells (4El-Deiry W.S. Cell Death Differ. 2001; 8: 1066-1075Crossref PubMed Scopus (121) Google Scholar). A recent report shows a close correlation between p53 level and Noxa induction that could be a key determinant of chemically induced hepatocellular carcinoma in mice, suggesting that Noxa is a crucial mediator of p53-dependent apoptosis in the liver (5Eferl R. Ricci R. Kenner L. Zenz R. David J.P. Rath M. Wagner E.F. Cell. 2003; 112: 181-192Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). Noxa induced by p53 is up-regulated by x-ray irradiation and is heavily dependent on p53 and its homologue p73 (6Flores E.R. Tsai K.Y. Crowley D. Sengupta S. Yang A. McKeon F. Jacks T. Nature. 2002; 416: 560-564Crossref PubMed Scopus (717) Google Scholar). Noxa belongs to the “BH3-only” branch of the Bcl-2 protein family, although mouse Noxa has two BH3 domains, whereas other BH3-only proteins have a single BH3 domain. Like many Bcl-2 family proteins that translocate to mitochondria and modulate mitochondrial function, Noxa translocates to mitochondria and then leads to cytochrome c release and caspase-9 activation (7Oda E. Ohki R. Murasawa H. Nemoto J. Shibue T. Yamashita T. Tokino T. Taniguchi T. Tanaka N. Science. 2000; 288: 1053-1058Crossref PubMed Scopus (1678) Google Scholar). Most of the BH3-only proteins such as Bid and Bim appear to induce efflux of mitochondrial deathstimulating proteins through Bax or Bak homo-oligomerization leading to the permeabilization of the mitochondrial outer membrane (8Wei M.C. Zong W.X. Cheng E.H. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Crossref PubMed Scopus (3299) Google Scholar, 9Wei M.C. Lindsten T. Mootha V.K. Weiler S. Gross A. Ashiya M. Thompson C.B. Korsmeyer S.J. Genes Dev. 2000; 14: 2060-2071Crossref PubMed Google Scholar). However, the detailed molecular mechanism by which Noxa induces mitochondrial dysfunction remains to be solved. In these studies, we characterize Noxa-induced mitochondrial dysfunction in order to provide insight into the molecular mechanism by which p53 induces apoptosis. We show that Noxa causes mitochondrial dysfunction by a distinct pathway in mediating p53-dependent apoptosis, unlike mitochondrial dysfunction induced by tBid in response to FasL or tumor necrosis factor-α. Cell Culture and Mitochondrial Localization—HeLa cells were seeded onto an 8-well Lab-Tek chamber slide glass (Nunc) and were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen). The indicated GFP 1The abbreviations used are: GFPgreen fluorescent proteinZ-VAD-fmkbenzyloxycarbonyl-VAD-fluoromethyl ketoneMTDmitochondrial targeting domainPTpermeability transitionCsAcyclosporin A.-Noxa plasmids were transfected into HeLa cells using Effectene (Qiagen) according to the manufacturer's instructions and cultured for 2 additional days. In some experiments, Mitotracker Red CMXROS (Molecular Probes Inc.) was added to the medium at a final concentration of 200 nm, and cells were incubated for a further 10 min. In these experiments, the dye was visualized without a fixation step. After washing with PBS, fresh medium was added. Mitochondrial localization was analyzed with a laser scanning confocal microscope (Leica Microsystems TCS NT). green fluorescent protein benzyloxycarbonyl-VAD-fluoromethyl ketone mitochondrial targeting domain permeability transition cyclosporin A. Immunostaining of Cytochrome c—HeLa cells were rinsed with PBS three times and fixed with ice-cold 2% buffered paraformaldehyde (pH 7.4) for 10 min. After washing with PBS and blocking with a buffer containing 0.1% saponin and 0.05% BSA in PBS, pH 7.4, for 30 min, the cells were incubated with anti-cytochrome c antibody (dilution 1:250, BD Biosciences) for 1 h at 30 °C and then washed with the blocking buffer. Tetramethylrhodamine-labeled anti-mouse IgG antibody (dilution 1:250, BD Biosciences) was added to the cells, and they were incubated for 1 h more. After washing with PBS, cells were examined with the laser scanning confocal microscope. Apoptosis Assay—HeLa cells were cultured in 24-well plates and transfected with the indicated plasmid constructs. Each well was harvasted after 48 h of culture, and lysates were prepared using the luciferase assay system (Promega). Luciferase activities were determined by using a Micro-Lumat Plus LB96V microplate luminometer (Berthold Technologies) and were normalized against control β-galactosidase activities. Purification of Recombinant Human Noxa—Recombinant human Noxa protein was expressed in Escherichia coli BL21(DE3) cells transformed with pET30(+)a-human Noxa (1–54). It was induced by the addition of 0.8 mm isopropyl-1-thio-β-d-galactopyranoside for 3 hours. Purification was carried out according to the company's instructions (Novagen). Cytochrome c Release Assay and Assay of Endonuclease G Activity—Isolated mitochondria treated with recombinant Noxa protein were incubated at 30 °C for 1 h in buffer containing 250 mm sucrose, 10 mm HEPES-NaOH, pH 7.5, 2 mm KH2PO4, 5 mm sodium succinate, and 25 μm EGTA. After centrifugation at 10,000 × g for 10 min, supernatants were subjected to SDS-PAGE, and released cytochrome c was visualized by Western blot analysis. Analysis of Bak-oligomerization—Mitochondria were isolated from either mouse or rat liver and treated with either tBid or Noxa before incubation for 1 h at 30 °C. After centrifugation, mitochondrial pellets were resuspended in buffer B containing 250 mm sucrose, 10 mm HEPES-NaOH, pH 7.5, 2 mm KH2PO4, 5 mm sodium succinate, and 25 μm EGTA. A cross-linking agent of bismaleimide hexane (10 μm) or alternatively, Me2SO as a control, was then added. After further incubation for 30 min at room temperature with shaking, mitochondrial pellets were harvested by centrifugation and then analyzed by Western blot analyses using anti-Bak antibody. Alignment of Expressed Sequence Tag Clones of Noxa Homologues—Expressed sequence tag clones of cow, chicken, and pig Noxa were identified by a Blast search at the NCBI web site using human Noxa/APR as the reference. GenBank accession numbers of identified expressed sequence tag clones containing cow, chicken, and pig homologues of Noxa are BF075905, AJ450393, and AF319660, respectively. It has been reported that mouse Noxa has two BH3 domains and induces apoptosis, whereas human Noxa has only one BH3 domain. First, to determine whether human Noxa is able to induce cell death in HeLa cells, GFP-Noxa (1–54) was transfected into HeLa cells. As shown in Fig. 1A, GFP-Noxa (1–54) induced cell death, and Bcl-xL blocked Noxa-induced cell death. In addition, the pan-caspase inhibitor z-VAD-fmk inhibited Noxa-induced cell death, indicating that caspases play a crucial role in Noxa-induced cell death. These results indicate that one BH3 domain in human Noxa is sufficient to induce apoptosis. To determine whether cytochrome c release accompanies human Noxa-induced cell death, GFP-Noxa (1–54) was transfected into HeLa cells, and the cells were immunostained using anti-cytochrome c antibody. HeLa cells transfected with human Noxa showed diffuse cytochrome c distribution throughout the cytosol, but control cells (transfected with GFP alone) maintained the typical mitochondrial distribution of cytochrome c (Fig. 1B), indicating that cytochrome c release from mitochondria is an event induced during human Noxa-mediated cell death. To further confirm that human Noxa targets to mitochondria and causes mitochondrial dysfunction, we checked whether GFP-Noxa (1–54) was present in mitochondria after transfection. GFP-Noxa (1–54) showed a punctate distribution typical of mitochondria (Fig. 1C, left panel, green color), and mitochondrial localization of Noxa was confirmed by overlapping staining (Fig. 1C, right panel, yellow color) with Mitotracker (Fig. 1C, middle panel, red color). This finding is in agreement with a previous report showing the mitochondrial localization of mouse Noxa when using an adenovirus expressing mouse Noxa and suggests that human Noxa is functionally identical to mouse Noxa despite having a single BH3 domain (7Oda E. Ohki R. Murasawa H. Nemoto J. Shibue T. Yamashita T. Tokino T. Taniguchi T. Tanaka N. Science. 2000; 288: 1053-1058Crossref PubMed Scopus (1678) Google Scholar). Although the data indicated that human Noxa causes the release of cytochrome c from mitochondria in HeLa cells, it was not clear whether Noxa is able to directly trigger efflux of death-stimulating proteins from the inter-membrane space of mitochondria or whether another, non-mitochondrial, factor is also necessary. To address this question, we adopted the in vitro system of Noxa-induced mitochondrial dysfunction. In this system, recombinant human Noxa protein was able to induce cytochrome c release directly from isolated mitochondria. Noxa-induced cytochrome c release was inhibited by BclxLΔTM (Fig. 2, A and B). These results strongly suggest that the presence of human Noxa in mitochondria is enough to induce mitochondrial dysfunction. It has been shown that a death ligand-activated BH3-only protein, tBid, targets to the mitochondria and that the DEF helices of tBid play a crucial role in this localization. We hypothesized that human Noxa may have a mitochondrial targeting domain (MTD), similar to tBid. To determine the MTD of Noxa, various expression plasmids of Noxa fused to GFP were transfected into HeLa cells (Fig. 3A). In Fig. 1 we showed that Noxa (1–54) displays the punctate distribution that is typical of a mitochondrial staining pattern, and mitochondrial distribution of Noxa (1–54) was confirmed by overlapped staining with Mitotracker. This same punctate distribution disappeared in Noxa (1–30) and Noxa (1–40); however, Noxa (21–54) and Noxa (31–54) maintained a similar pattern, indicating that Noxa (31–54) contains the MTD. To define the MTD of Noxa, Noxa (31–54) was dissected as shown in Fig. 3B. Noxa (31–40) and Noxa (31–45) showed a dispersed cytoplasmic distribution, whereas Noxa (41–54) and Noxa (41–50) displayed the punctate pattern. These results demonstrate that human Noxa (41–50) contains the entire MTD. This MTD seems to be conserved between human, mouse, cow, pig, and chicken (Fig. 3C), suggesting that the MTD of Noxa is functionally significant for induction of cell death in p53-dependent apoptosis. To verify whether human Noxa needs the MTD to kill cells, plasmids containing various GFP-Noxa constructs and a reporter gene were co-transfected into HeLa cells. Noxa (1–54) and Noxa (21–54), which have intact BH3 and MTD domains, were able to induce cell death (Fig. 3D). We further confirmed that the cell killing induced by Noxa (1–54) and Noxa (21–54) was associated with an efflux of cytochrome c from mitochondria (Fig. 4). Interestingly, Noxa (11–40) and Noxa (21–40), which have the BH3 domain but not the MTD domain, failed to induce cell death (Fig. 3D). In addition, Noxa (31–54) and Noxa (41–54), which have the MTD domain but harbor a disrupted BH3 domain or no BH3 domain, respectively, failed to induce apoptosis, indicating that the MTD domain is necessary but not enough to induce cell death (Fig. 3D). These results demonstrate that the killing activity of the Noxa BH3 domain is functional only when it targets to mitochondria, and this mitochondria targeting is mediated by the MTD. It is known that after engagement of death ligands to death receptors, caspase-8-activated tBid binds to cardiolipin, which exists exclusively in the mitochondrial membrane and is enriched at the inner membrane and the mitochondrial contact sites. The interaction of tBid with cardiolipin is a critical step for tBid targeting to mitochondria (10Lutter M. Fang M. Luo X. Nishijima M. Xie X. Wang X. Nat. Cell. Bio. 2000; 2: 754-761Crossref PubMed Scopus (404) Google Scholar, 11Lutter M. Perkins G.A. Wang X. BMC Cell Biol. 2001; 2: 22Crossref PubMed Scopus (126) Google Scholar). To understand the molecular mechanism by which Noxa targets to mitochondria in p53-dependent apoptosis, we examined whether Noxa also targets to cardiolipin like tBid. It is unlikely that Noxa binds to cardiolipin (data not shown), and it implies that p53-mediated mitochondrial dysfunction through Noxa might be distinct from mitochondria dysfunction triggered by death ligands that is mediated through tBid. To examine whether Noxa triggers an efflux of mitochondrial proteins in a manner distinct from tBid, we examined the inhibitory activity of CsA in a cytochrome c release assay. CsA showed no significant change in the release of cytochrome c induced by tBid, consistent with a previous report (12Kim T.H. Zhao Y. Barber M.J. Kuharsky D.K. Yin X.M. J. Biol. Chem. 2000; 275: 39474-39481Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) (Fig. 5A). On the other hand, calcium, which is a strong activator of the PT pore, was able to release cytochrome c from isolated mitochondria and, as expected, was inhibited by CsA. Surprisingly, CsA also blocked Noxa-induced cytochrome c release. In addition, Mg2+, another inhibitor of the PT pore, was also able to inhibit Noxa-induced cytochrome c release, indicating that Noxa in p53-dependent apoptosis is likely to cause efflux of mitochondrial proteins through a PT-related pore in a manner that is distinct from the tBid-induced pathway activated by death ligands. Bak-oligomerization is a key event of tBid-induced cytochrome c release. In fact, tBid is unable to release cytochrome c from Bak-deficient mitochondria (8Wei M.C. Zong W.X. Cheng E.H. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Crossref PubMed Scopus (3299) Google Scholar, 9Wei M.C. Lindsten T. Mootha V.K. Weiler S. Gross A. Ashiya M. Thompson C.B. Korsmeyer S.J. Genes Dev. 2000; 14: 2060-2071Crossref PubMed Google Scholar). A recent report showed that unlike mouse liver, rat liver mitochondria display little or no cytochrome c release in response to tBid because of either insufficient or absent Bak in their mitochondria (13Brustovetsky N. Dubinsky J.M. Antonsson B. Jemmerson R. J. Neurochem. 2003; 84: 196-207Crossref PubMed Scopus (59) Google Scholar). Therefore, we analyzed Bak-oligomerization using mouse and rat liver mitochondria to determine whether Bak-oligomerization is an important component of Noxa-induced cytochrome c release. The cross-linker bismaleimide hexane or (as a control) Me2SO has been used for Bak-oligomerization analyses. As expected, tBid induced significant Bak-oligomerization from mouse but not rat mitochondria. Noxa did not cause significant Bak-oligomerization in either case, although it was able to effect cytochrome c release (Fig. 5B). Furthermore, our results confirmed that rat liver mitochondria only release a small amount of cytochrome c in response to tBid (Fig. 5B). On the other hand, Noxa was able to release a comparable amount of cytochrome c from both rat and mouse mitochondria without These results that Bak-oligomerization is not for cytochrome c release in response to However, we not out the that Bax may be in the Noxa-induced cytochrome c release because Bax is able to for CsA and are blocking agents of the PT pore and inhibit Noxa-induced cytochrome c release (Fig. we that Noxa is likely to activate the PT pore, in mitochondrial We mitochondrial by after Noxa however, we were unable to significant (data not reported that tBid causes of the mitochondrial in the of cytochrome c L. Ashiya M. Weiler S. C.A. Korsmeyer S.J. Dev. Cell. 2002; 2: Full Text Full Text PDF PubMed Scopus Google Scholar). We hypothesized that Noxa might also cause ultra-structural changes in the mitochondria without and this change might be distinct from that induced by tBid. To examine the changes of mitochondria treated with tBid, Noxa, or were using As shown in Fig. isolated mitochondria showed an of the isolated mitochondria, the typical by J. Cell Biol. PubMed Scopus Google Scholar). tBid was used to the mitochondria, the was to have mitochondrial showing a (Fig. as by L. Ashiya M. Weiler S. C.A. Korsmeyer S.J. Dev. Cell. 2002; 2: Full Text Full Text PDF PubMed Scopus Google Scholar). mitochondria, however, showed an and and small with were dispersed (Fig. These changes are of “swollen” mitochondria as by It is likely that these changes without mitochondrial because no significant changes in mitochondrial were Thus, Noxa seems to cause changes in the mitochondrial of mitochondria without Mitochondrial dysfunction in response to FasL or tumor necrosis seems to be mediated through tBid activated by This either or to release cytochrome c and without the of PT (8Wei M.C. Zong W.X. Cheng E.H. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Crossref PubMed Scopus (3299) Google Scholar, 9Wei M.C. Lindsten T. Mootha V.K. Weiler S. Gross A. Ashiya M. Thompson C.B. Korsmeyer S.J. Genes Dev. 2000; 14: 2060-2071Crossref PubMed Google Scholar, X. Genes Dev. 2001; PubMed Scopus Google Scholar). However, Bid may not be essential for mitochondrial dysfunction in death such as because are able to membrane of mitochondria in response to these death X.M. Wang Gross A. Zhao Y. S. B. Roth K.A. Korsmeyer S.J. Nature. PubMed Scopus Google Scholar). This indicates that there is an pathway leading to mitochondrial dysfunction in death Noxa may be the protein for induction of this pathway because Noxa is transactivated by p53 in response to genotoxic and Noxa causes mitochondrial dysfunction (7Oda E. Ohki R. Murasawa H. Nemoto J. Shibue T. Yamashita T. Tokino T. Taniguchi T. Tanaka N. Science. 2000; 288: 1053-1058Crossref PubMed Scopus (1678) Google Scholar). our present results show the of cytochrome c from mitochondria induced by tBid or First, Noxa-induced cytochrome c release is not dependent Bak-oligomerization, whereas or Bak-oligomerization is for tBid-induced cytochrome c release. Although our data indicate that Noxa-induced mitochondrial dysfunction is not associated with Bak-oligomerization, we not out the of Bax in this process. The that isolated from and are resistant to apoptosis suggests that Bax might be associated with Noxa-induced mitochondrial dysfunction. However, this remains to be PT-related at least in in the Noxa-induced permeabilization of the mitochondrial outer membrane because the PT pore inhibitors CsA and are able to inhibit Noxa-induced cytochrome c release. This is further by a Noxa-induced an of the of the PT This the that p53 Noxa which leads to mitochondrial dysfunction in a distinct pathway through PT-related it suggests that there are two different pathways in the permeabilization of the mitochondrial outer membrane which are activated by different death This might provide an for response of some cancer cells to of genotoxic agents and death cancer cells, for are resistant to mainly because of the of cytochrome c however, these cells are to and the inhibitor It is that cells have the pathway of mitochondrial death signaling induced by however, the might the mitochondrial death signaling through Noxa-mediated We at the of for critical of the and branch for with the confocal microscope.