Apop-1, a Novel Protein Inducing Cyclophilin D-dependent but Bax/Bak-related Channel-independent Apoptosis

Abstract

In the intrinsic pathway of apoptosis, mitochondria play a crucial role by releasing cytochrome c from the intermembrane space into the cytoplasm. Cytochrome c release through Bax/Bak-dependent channels in mitochondria has been well documented. In contrast, cyclophilin D (CypD), an important component of permeability transition pore-dependent protein release, remains largely undefined, and no apoptogenic proteins that act specifically in a CypD-dependent manner have been reported to date. Here, we describe a novel and evolutionarily conserved protein, apoptogenic protein (Apop). Mouse Apop-1 expression induces apoptotic death by releasing cytochrome c from mitochondria into the cytosolic space followed by activation of caspase-9 and -3. Apop-1-induced apoptosis is not blocked by Bcl-2 or Bcl-xL, inhibitors of Bax/Bak-dependent channels, whereas it is completely blocked by cyclosporin A, an inhibitor of permeability transition pore. Cells lacking CypD were resistant to Apop-induced apoptosis. Moreover, inhibition of Apop expression prevented the cell death induced by apoptosis-inducing substances. Our findings, thus, indicate that the expression of Apop-1 induces apoptosis though CypD-dependent pathway and that Apop-1 plays roles in cell death under physiological conditions. In the intrinsic pathway of apoptosis, mitochondria play a crucial role by releasing cytochrome c from the intermembrane space into the cytoplasm. Cytochrome c release through Bax/Bak-dependent channels in mitochondria has been well documented. In contrast, cyclophilin D (CypD), an important component of permeability transition pore-dependent protein release, remains largely undefined, and no apoptogenic proteins that act specifically in a CypD-dependent manner have been reported to date. Here, we describe a novel and evolutionarily conserved protein, apoptogenic protein (Apop). Mouse Apop-1 expression induces apoptotic death by releasing cytochrome c from mitochondria into the cytosolic space followed by activation of caspase-9 and -3. Apop-1-induced apoptosis is not blocked by Bcl-2 or Bcl-xL, inhibitors of Bax/Bak-dependent channels, whereas it is completely blocked by cyclosporin A, an inhibitor of permeability transition pore. Cells lacking CypD were resistant to Apop-induced apoptosis. Moreover, inhibition of Apop expression prevented the cell death induced by apoptosis-inducing substances. Our findings, thus, indicate that the expression of Apop-1 induces apoptosis though CypD-dependent pathway and that Apop-1 plays roles in cell death under physiological conditions. Apoptosis, which is programmed cell death, is essential in normal development and is involved in many diseases and pathophysiological conditions including cancer, neurodegenerative disorders, and atherosclerosis (1Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6205) Google Scholar, 2Stoneman V.E. Bennett M.R. Clin. Sci. (Lond.). 2004; 107: 343-354Crossref PubMed Scopus (194) Google Scholar, 3Bennett M.R. Evan G.I. Schwartz S.M. J. Clin. Investig. 1995; 95: 2266-2274Crossref PubMed Scopus (610) Google Scholar). There are two major pathways for the induction of caspase-dependent apoptosis; one is an extrinsic or receptor-mediated pathway which includes Fas and tumor necrosis factor receptor, whereas the other is an intrinsic pathway in which mitochondria play a crucial role by releasing cytochrome c from the intermembrane space into the cytoplasm after mitochondrial outer membrane permeabilization (4Green D.R. Kroemer G. Science. 2004; 305: 626-629Crossref PubMed Scopus (2831) Google Scholar). Mitochondrial outer membrane permeabilization can be initiated by opening of the permeability transition (PT) 2The abbreviations used are: PT, permeability transition; ANT, adenine nucleotide translocator; CypD, cyclophilin D; BH domain, Bcl-2 homology domain; SMC, smooth muscle cell(s); P, plaque; NP, non-plaque; GFP, green fluorescent protein; tBid, truncated Bid; SNP, sodium nitroprusside; PBS, phosphate-buffered saline; z-, benzyloxycarbonyl; fmk, fluoromethyl ketone; CyA, cyclosporin A. pore, an incompletely characterized protein complex presumably consisting of a voltage-dependent anion channel, adenine nucleotide translocator (ANT), and cyclophilin D (CypD) (5Crompton M. Virji S. Ward J.M. Eur. J. Biochem. 1998; 258: 729-735Crossref PubMed Scopus (407) Google Scholar), although recent findings have shown that PT pores can form in the absence of ANT (6Halestrap A.P. Nature. 2004; 430: 98310.1038/nature02816Crossref PubMed Google Scholar, 7Kokoszka J.E. Waymire K.G. Levy S.E. Sligh J.E. Cai J. Jones D.P. MacGregor G.R. Wallace D.C. Nature. 2004; 427: 461-465Crossref PubMed Scopus (903) Google Scholar). Normally, PT pores allow materials of up to 1.5 kDa to be released from mitochondria (8Zoratti M. Szabo I. Biochim. Biophys. Acta. 1995; 1241: 139-176Crossref PubMed Scopus (2199) Google Scholar). Sustained opening of PT pores allows equilibration of ions between the matrix and cytoplasm and dissipation of the inner membrane potential (9Newmeyer D.D. Ferguson-Miller S. Cell. 2003; 112: 481-490Abstract Full Text Full Text PDF PubMed Scopus (1091) Google Scholar). However, determination of whether PT pores are independent mediators of apoptosis has been hampered by the lack of an apoptogen that acts solely through them. Mitochondrial outer membrane permeabilization is also mediated by Bcl-2 family proteins. In this type of apoptosis, Bcl-2 family proteins act directly on the outer mitochondrial membrane, and PT and the inner membrane do not play major roles, although there appear to be physical and/or functional interactions between PT and Bcl-2 family proteins. Regulation of cytochrome c release by both pro- and antiapoptotic members (10Tsujimoto Y. Shimizu S. FEBS Lett. 2000; 466: 6-10Crossref PubMed Scopus (637) Google Scholar) of the Bcl-2 family of proteins is well documented (11Borner C. Mol. Immunol. 2003; 39: 615-647Crossref PubMed Scopus (622) Google Scholar). The anti-apoptotic Bcl-2 family members, such as Bcl-2, Bcl-xL and Bcl-w, share four Bcl-2 homology (BH) domains (BH1 through BH4), although some members of this group appear not to possess a BH4 domain. The pro-apoptotic Bcl-2 family members can be classified into two subgroups based on their structure, i.e. BH3-only members and multidomain members. Bad, Bid, and Bim, which share only the BH3 domain, belong to the BH3 only protein subgroup, whereas Bax and Bak, which share three BH regions (BH1, BH2, and BH3), belong to the multidomain subgroup. The members of these two subgroups are functionally distinct. BH3-only proteins appear to function as death signal sensors (12Willis S. Day C.L. Hinds M.G. Huang D.C. J. Cell Sci. 2003; 116: 4053-4056Crossref PubMed Scopus (179) Google Scholar, 13Puthalakath H. Strasser A. Cell Death Differ. 2002; 9: 505-512Crossref PubMed Scopus (631) Google Scholar), whereas multidomain members function as gateways for many types of apoptotic signals, since cells lacking both Bax and Bak are resistant to many apoptotic stimuli (14Wei 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 (3373) Google Scholar). Beside the BH regions, many Bcl-2 family members possess a C-terminal hydrophobic domain, which is predicted to facilitate membrane localization of these proteins (15Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4820) Google Scholar, 16Petros A.M. Olejniczak E.T. Fesik S.W. Biochim. Biophys. Acta. 2004; 1644: 83-94Crossref PubMed Scopus (604) Google Scholar). After being released from the intermembrane space of mitochondria into the cytosol, cytochrome c associates with Apaf-1 and pro-caspase-9 and forms a complex termed an apoptosome, which leads to the activation of pro-caspase-9 (17Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1803) Google Scholar). Subsequently, the active form of caspase-9 cleaves and activates downstream executioner caspases, such as caspase-3 (18Rodriguez J. Lazebnik Y. Genes Dev. 1999; 13: 3179-3184Crossref PubMed Scopus (461) Google Scholar). Besides cytochrome c, Smac/DIABLO is also released from the intermembrane space of mitochondria and inhibits the activity of IAP, itself is an inhibitor of caspase-3, -7, and -9 (19Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2285) Google Scholar, 20Shi Y. Mol. Cell. 2002; 9: 459-470Abstract Full Text Full Text PDF PubMed Scopus (1465) Google Scholar, 21Wang X. Genes Dev. 2001; 15: 2922-2933Crossref PubMed Scopus (94) Google Scholar). Dysregulation of apoptosis results in deficient or excessive cell death and is implicated in many human diseases. Insufficient apoptosis may be involved in carcinogenesis, whereas excessive apoptosis is implicated in the pathogenesis of stroke, myocardial infarction, and heart failure. For example, atherosclerotic plaque tends to rupture at sites of increased macrophages and reduced numbers of vascular smooth muscle cells (SMC), suggesting that apoptosis of SMC, possibly induced by activated macrophages, promotes the rupture of atherosclerotic plaque (22Bennett M.R. Cardiovasc. Res. 1999; 41: 361-368Crossref PubMed Scopus (245) Google Scholar). In the present study, we cloned a novel apoptogenic gene from atherosclerotic SMC in culture. We named this protein apoptogenic protein (Apop) and demonstrate that it induces apoptosis through the CypD-dependent but Bax/Bak-related channel-independent mechanism. Cell Culture and Transfection—SMC cultures were established from plaque (P) and non-plaque (NP) areas of the descending thoracic aorta of ApoE-deficient mice by the explant method as described previously (23Yasuda O. Zhang S.H. Miyamoto Y. Maeda N. J. Vasc. Res. 2000; 37: 158-169Crossref PubMed Scopus (20) Google Scholar). SMC were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. HeLa cells (a human cervical carcinoma cell line) were transfected with a pUC-CAGGS vector bearing human Bcl-2 to obtain a stable transfectant (24Shimizu S. Eguchi Y. Kamiike W. Matsuda H. Tsujimoto Y. Oncogene. 1996; 12: 2251-2257PubMed Google Scholar), which was designated HeLa-Bcl-2. Empty vector was introduced to obtain control HeLa cells (designated HeLa-V). HeLa cells and transfected HeLa cells were maintained in RPMI1640 containing 10% fetal calf serum. Hepatocytes were isolated by in situ collagenase perfusion from wild-type and CypD-deficient mice and cultured on collagen-coated dishes (Iwaki). CypD-deficient mice were generated in collaboration with Lexicon Genetics Inc, and the hepatocytes were kindly provided by Dr. Shimizu of Osaka University. Human umbilical vein endothelial cells were cultured in endothelial basal medium 2 (Sanko Junyaku) supplemented with 2% fetal calf serum. Differential Display—Total RNA isolated from P and NP cultures was analyzed by differential display essentially as described by Liang and Pardee (25Liang P. Pardee A.B. Science. 1992; 257: 967-971Crossref PubMed Scopus (4707) Google Scholar) with some modifications. First-strand cDNA synthesis and PCR reaction were performed as previously described (23Yasuda O. Zhang S.H. Miyamoto Y. Maeda N. J. Vasc. Res. 2000; 37: 158-169Crossref PubMed Scopus (20) Google Scholar). The DNA fragment differentially expressed by P and NP cultures was excised from the gel, reamplified by PCR, and cloned into a pGEM-T vector (Promega) for sequencing. Quantitative Real-time Reverse Transcription-PCR—RNA was isolated from mouse tissues using ISOGEN (Nippon to the and to cDNA by gene expression was used to the Quantitative PCR was performed using and analyzed on a The PCR and fluorescent for Apop-1 were as For the and were as of were after to cDNA was cloned from cultured SMC by using and DNA The Apop-1 cDNA was with to Apop-1 and green fluorescent protein protein expression Apop-1 cDNA including a was into for of the Apop-1 expression DNA human truncated and Bcl-xL were into the pUC-CAGGS expression vector H. J. PubMed Scopus Google Scholar) and and used as expression vector and For of the of was with and and the DNA fragment including the Apop was into in Cell with and DNA of expression for tBid, Bcl-xL, or vector was performed using and to the DNA was introduced into SMC using a for smooth muscle cells to the For of cells with cyclosporin HeLa cells were cultured and with this after of or from 2 of with For Apop inhibition SMC cultures were transfected with control or and at for Cells were with medium containing or sodium and cultured for an were with and with in for at After with and with in for the cells were with or c at and with was to the cells and for For of cells were with and to in for After with PBS, cells were under a mitochondrial was isolated using as described previously M. Shimizu S. T. M. Matsuda H. Tsujimoto Y. Res. 1999; Google Scholar). after with phosphate-buffered the cultured cells were and with for at The cytosolic and were by and with and of were to The protein was a sodium gel, and proteins were to a membrane The membrane was blocked with containing and and with c and at The membrane was with (Promega) for and the were by a method was by protein using as a and -9 in cells cultured for after were using a to the Cell both and cells were and with and in for Cells were in consisting of A, and and DNA was by cell on the channel, and was to and of cells were for In the Apop expression inhibition cell was using an was performed by the are expressed as of a of atherosclerotic SMC, we SMC cultured from atherosclerotic plaque (P) with from non-plaque (NP) areas of aorta of mice (23Yasuda O. Zhang S.H. Miyamoto Y. Maeda N. J. Vasc. Res. 2000; 37: 158-169Crossref PubMed Scopus (20) Google Scholar). Differential display using P and NP cell from three that in of cDNA fragment was one of these and was in P in NP in two but in P and NP in the The nucleotide of the fragment was to in the mouse The of is to that of a cDNA of function in the of the mouse that these two from an Apop cDNA on mouse Our we designated and of and of and Reverse that both Apop-1 and were expressed in mouse expression of the Apop Real-time PCR of expression of Apop gene in tissues Apop-1 expression was in whereas was expressed at the in of is of including with whereas C-terminal is with and and no with the of The regions of the mouse Apop proteins have in with the mitochondrial including being in and the of two of and of of an for of proteins M.G. P. Eur. J. Biochem. 1996; PubMed Scopus Google Scholar) a of mitochondrial localization of Apop-1 and predicted the of a mitochondrial Y. G. PubMed Scopus Google Scholar). Apop is evolutionarily and of it can be in and However, the of these proteins have not been and no to domains to other gene were that there are two for and two for in the mouse Apop-1 these one and one are conserved In to localization signal and are present in an Apop in C. The C-terminal of which is in and also potential localization in to mitochondrial Mitochondrial the mitochondrial localization of Apop-1 predicted from the of SMC were transfected with an expression vector of Apop-1 to at was in the and a to that of mitochondria was with a mitochondrial in with an to the cytosolic and mitochondrial proteins were from cells after of for cell with the results of was in the mitochondrial suggesting that Apop-1 is in mitochondria Apop-1-induced Cell the function of this novel protein, we Apop-1 and expression and and introduced into cultured wild-type mouse cells Apop-1 or after of cells transfected with also Cells of apoptosis Lazebnik Y. Science. 1998; 281: PubMed Scopus Google Scholar), i.e. cell and on with of DNA in HeLa cells was on of cells Human umbilical vein endothelial cells also after the of Apop-1 expression vector Apop-1-induced medium of SMC to SMC death in that Apop-1 not the expression of apoptogenic such as Fas was by the inhibition of cell death by a which was at and at that Apop-1-induced death is mediated by the activation of with this the activity of a in the apoptosis was in cells with that in control activation was completely by a inhibitor whether activation of the characterized is involved in Apop-1-induced apoptosis, we the activity of and that it also was in cells with a caspase-9 activation as well as apoptosis findings indicate that Apop-1-induced apoptosis is mediated by an essential component of the apoptosome, which apoptosis. Apop-1 Cytochrome whether cytochrome c, a in the apoptotic is released from SMC were transfected with control vector or and cytochrome c was with a of cytochrome c a in control mitochondrial localization In contrast, SMC transfected with a of suggesting release of cytochrome c from the mitochondria into the the of cytochrome c in both mitochondrial and cytosolic of the transfected cells to the of cytochrome c was at after with a that can the mitochondrial pathway of apoptosis, was used as a control and induced of cytochrome c from mitochondria to the Bcl-2 to Apop-1-induced is from the members of the Bcl-2 family in that it no BH has a mitochondrial localization signal at the and not have the C-terminal domain, which many Bcl-2 family proteins whether Apop-1-induced apoptosis Bcl-2 family we whether Bcl-2, an anti-apoptotic Bcl-2 family can apoptosis induced by and a vector with tBid, a pro-apoptotic Bcl-2 family induced apoptosis in control HeLa cells However, although the of was to apoptosis of cells that Bcl-2 the apoptosis induced by Apop-1 was not by expression of Bcl-2 Apop-1 induced of apoptosis in and cells in a whereas apoptosis was completely in cells Bcl-xL is anti-apoptotic of the Bcl-2 results were Bcl-xL expression vector was with or Apop-1 expression was not expression vector was introduced into HeLa cells with Bcl-xL expression whereas apoptosis was Apop-1 expression vector was with Bcl-xL Quantitative of apoptosis that of HeLa cells with the Bcl-xL expression vector and the expression vector from apoptosis in a Bcl-xL In contrast, Bcl-xL expression no on Apop-1-induced apoptosis findings indicate that Apop-1-induced apoptosis is independent of Bcl-2 and Bcl-xL, which cytochrome c release from of D Apop-1-induced is a mitochondrial of the cyclophilin family N. A. Virji S. Ward J.M. M. Eur. J. Biochem. 1999; PubMed Scopus Google Scholar) that of A. S. G. Sci. S. A. 1995; PubMed Scopus Google Scholar, A. Eur. J. Biochem. PubMed Scopus Google Scholar). The specifically to and inhibits their activity J. V. P. J. Mol. Biol. 1998; PubMed Scopus Google Scholar). CypD is to be an of the mitochondrial PT (5Crompton M. Virji S. Ward J.M. Eur. J. Biochem. 1998; 258: 729-735Crossref PubMed Scopus (407) Google Scholar), and with ANT presumably promotes in to inner membrane pores M. H. A. Biochem. J. Google Scholar, D.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). whether Apop-1 of mitochondrial proteins in CypD-dependent we the of on the release of cytochrome c by shown in reduced the release of cytochrome c by that Apop-1 induces CypD-dependent release of cytochrome that the apoptosis induced by Apop-1 was by CyA, which was to apoptosis to basal In contrast, not apoptosis, which is mediated by activated Bid, a pro-apoptotic Bcl-2 family A.B. S.W. J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). thus, acts through a CypD-dependent from apoptosis members. D Apop-1-induced apoptosis of we introduced Apop-1 into of hepatocytes of CypD-deficient mice T. Shimizu S. T. O. H. H. T. Tsujimoto Y. Nature. PubMed Scopus Google Scholar). after cells with and apoptotic was in wild-type cells but not in CypD-deficient cells In contrast, induced death of both wild-type and CypD-deficient The of CypD-deficient cells that were apoptotic after with not from that for whereas increased apoptotic death of CypD-deficient cells to a with that for wild-type cells findings indicate that Apop-1 induces apoptosis in a CypD-dependent that is from the Bax/Bak-dependent apoptosis mediated by of Apop Cell Death by the physiological function of we the of inhibition of Apop gene expression using an The of the was by of green expressed by the signal was not by the control We this by The expression of was prevented by of Cells were transfected with or control vector and cultured in the or absence of apoptosis-inducing of Apop gene expression using prevented the cell death induced by or is with the results in that Apop-1 induces cell of Apop gene expression using not completely the death induced by that death is mediated by or Apop protein cells to findings indicate that Apop plays roles in cell death under physiological conditions. We have a novel protein, that induces CypD-dependent apoptosis expressed using an expression Apop is conserved evolutionarily and other family members, it plays a crucial function in the of cells that has of Apop induces apoptotic cell death of cultured SMC, and inhibition of Apop expression cell death by apoptosis-inducing findings indicate that Apop is with cell death and that control of Apop activity be important for cell that mitochondria can release intermembrane space including cytochrome c and through two (4Green D.R. Kroemer G. Science. 2004; 305: 626-629Crossref PubMed Scopus (2831) Google Scholar, D.D. Ferguson-Miller S. Cell. 2003; 112: 481-490Abstract Full Text Full Text PDF PubMed Scopus (1091) Google Scholar, Y. J. Cell. 2003; PubMed Scopus Google Scholar). permeabilization of the outer membrane mediated by Bcl-2 family members, whereas the other induction of the mitochondrial PT, which in to the release of from is reported that Bcl-2 family cell death not on CypD and that CypD and mitochondrial are for of and cell death H. M.R. J. Nature. PubMed Scopus Google Scholar). results are with that Apop-1-induced CypD-dependent cell death is not by anti-apoptotic Bcl-2 family members and demonstrate that Bcl-2 cell death is a pathway from that on CypD is a component of PT pores with voltage-dependent anion and ANT, although the physiological role of ANT in these pores is (6Halestrap A.P. Nature. 2004; 430: 98310.1038/nature02816Crossref PubMed Google Scholar, 7Kokoszka J.E. Waymire K.G. Levy S.E. Sligh J.E. Cai J. Jones D.P. MacGregor G.R. Wallace D.C. Nature. 2004; 427: 461-465Crossref PubMed Scopus (903) Google Scholar). that Apop-1 acts on PT pores and induces apoptosis. The of Apop-1 that it is a protein suggesting that Apop-1 is in the matrix or in the intermembrane space of The by which Apop induces CypD-dependent release of cytochrome c We that Apop-1-induced apoptosis was not prevented by anti-apoptotic Bcl-2 family members, whereas it was by or CypD We also that Apop-1 is a CypD-dependent apoptogenic protein that not act on Bax/Bak-dependent Bcl-2 family members, Apop-1 BH domains a C-terminal domain. it a mitochondrial localization signal at the Apop-1 thus, a novel pro-apoptotic protein that is not a of the Bcl-2 is at present the only protein to act specifically in a CypD-dependent and may be for of the apoptotic although for both Apop-1 and are present in tissues of the of of Apop is to be The C-terminal of Apop-1 from the of and the that are conserved the Apop present in other to that share homology with the although it is Apop-1 may not have of the normal of although we have not CypD-dependent apoptosis by it is that induces apoptosis by the as Apop-1 and that CypD-dependent apoptosis is mediated by the of The of the for the function of Apop may be with that the of at the of Apop-1 not the apoptogenic activity of We also that Apop-1 was isolated expression of it was in atherosclerotic SMC in SMC by differential death of SMC is an important the development of atherosclerotic in SMC to apoptosis also to the of which is to rupture and 1992; Scholar). there is increased SMC apoptosis in plaque M. H. A. Biochem. J. Google Scholar, G. H. Cardiovasc. Res. 1999; 41: PubMed Scopus Google Scholar, Vasc. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) in with with with stable and apoptotic macrophages are with sites of plaque rupture J. A.P. N. A. Y. J. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of a between plaque rupture and apoptosis in an was with the that induction of SMC apoptosis in of mouse induces plaque rupture and of plaque 2002; PubMed Scopus Google Scholar). have the on atherosclerosis of and which are used for The results are atherosclerosis in mice J. Google Scholar), whereas a recent that blocked atherosclerosis and plaque in ApoE-deficient mice I. S. J. PubMed Scopus Google Scholar). are both the by which on atherosclerosis be complex by apoptosis. we in the present that Apop-1 expression induces apoptosis of SMC and that apoptosis induced by be important to the of expression of Apop in atherosclerotic plaque in and to the of the Apop proteins in atherosclerosis and other pathophysiological conditions. We are to S. Shimizu for