Different Subcellular Distribution of Caspase-3 and Caspase-7 following Fas-induced Apoptosis in Mouse Liver
1998; Elsevier BV; Volume: 273; Issue: 18 Linguagem: Inglês
10.1074/jbc.273.18.10815
ISSN1083-351X
AutoresJulia M. Chandler, Gerald M. Cohen, Marion MacFarlane,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoCaspases plays a key role in the execution phase of apoptosis. "Initiator" caspases, such as caspase-8, activate "effector" caspases, such as caspase-3 and -7, which subsequently cleave cellular substrates thereby precipitating the dramatic morphological changes of apoptosis. Following treatment of mice with an agonistic anti-Fas antibody to induce massive hepatocyte apoptosis, we now demonstrate a distinct subcellular localization of the effector caspases-3 and -7. Active caspase-3 is confined primarily to the cytosol, whereas active caspase-7 is associated almost exclusively with the mitochondrial and microsomal fractions. These data suggest that caspases-3 and -7 exert their primary functions in different cellular compartments and offer a possible explanation of the presence of caspase homologs with overlapping substrate specificities. Translocation and activation of caspase-7 to the endoplasmic reticulum correlates with the proteolytic cleavage of the endoplasmic reticular-specific substrate, sterol regulatory element-binding protein 1. Liver damage, induction of apoptosis, activation and translocation of caspase-7, and proteolysis of sterol regulatory element-binding protein 1 are all blocked by the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.fmk). Our data demonstrate for the first time the differential subcellular compartmentalization of specific effector caspases following the induction of apoptosis in vivo. Caspases plays a key role in the execution phase of apoptosis. "Initiator" caspases, such as caspase-8, activate "effector" caspases, such as caspase-3 and -7, which subsequently cleave cellular substrates thereby precipitating the dramatic morphological changes of apoptosis. Following treatment of mice with an agonistic anti-Fas antibody to induce massive hepatocyte apoptosis, we now demonstrate a distinct subcellular localization of the effector caspases-3 and -7. Active caspase-3 is confined primarily to the cytosol, whereas active caspase-7 is associated almost exclusively with the mitochondrial and microsomal fractions. These data suggest that caspases-3 and -7 exert their primary functions in different cellular compartments and offer a possible explanation of the presence of caspase homologs with overlapping substrate specificities. Translocation and activation of caspase-7 to the endoplasmic reticulum correlates with the proteolytic cleavage of the endoplasmic reticular-specific substrate, sterol regulatory element-binding protein 1. Liver damage, induction of apoptosis, activation and translocation of caspase-7, and proteolysis of sterol regulatory element-binding protein 1 are all blocked by the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.fmk). Our data demonstrate for the first time the differential subcellular compartmentalization of specific effector caspases following the induction of apoptosis in vivo. Apoptosis is a crucial mechanism by which multicellular organisms control cell numbers and ensure the removal of damaged or potentially harmful cells (1Arends M.J. Wyllie A.H. Int. Rev. Exp. Pathol. 1991; 32: 223-254Crossref PubMed Scopus (1394) Google Scholar). Administration of an agonistic anti-Fas antibody results in ligation of the Fas (CD95, APO-1) receptor, extensive hepatocyte apoptosis, and liver damage (2Ogasawara J. Watanabe-Fukunaga R. Adachi M. Matsuzawa A. Kasugai T. Kitamura Y. Itoh N. Suda T. Nagata S. Nature. 1993; 364: 806-809Crossref PubMed Scopus (1815) Google Scholar). The intracellular death domain of the Fas receptor binds to FADD/MORT1, which in turn recruits and activates caspase-8 (MACH/FLICE/Mch5) through its N-terminal death effector domain (3Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2113) Google Scholar, 4Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2743) Google Scholar, 5Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (483) Google Scholar). Recombinant caspase-8 cleaves and activates all other known caspases and has been proposed to be at the apex of a hypothetical caspase cascade (3Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2113) Google Scholar, 4Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2743) Google Scholar, 5Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (483) Google Scholar). Caspases are a family of aspartate-specific cysteine proteases, which pre-exist in the cytoplasm as single chain inactive zymogens (6Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4146) Google Scholar, 7Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2187) Google Scholar). They are proteolytically processed to active heterodimeric enzymes during the execution phase of apoptosis. Caspases may be divided into "initiator" caspases with long prodomains (caspases-8, -9, and -10), which activate "effector" caspases with short prodomains (caspases-3, -6, and -7), which in turn cleave intracellular substrates, resulting in the dramatic morphological and biochemical changes of apoptosis (6Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4146) Google Scholar, 7Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2187) Google Scholar, 8Fraser A. Evan G. Cell. 1996; 85: 781-784Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar). Following Fas-induced apoptosis of cells in vitro, activation of a number of caspases, including caspases-3, -4, -6, -7, -8, and a caspase-1-like activity have all been reported (4Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2743) Google Scholar,9Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4561) Google Scholar, 10Enari M. Hug H. Nagata S. Nature. 1995; 375: 78-81Crossref PubMed Scopus (798) Google Scholar, 11Schlegel J. Peters I. Orrenius S. Miller D.K. Thornberry N.A. Yamin T.-T. Nicholson D.W. J. Biol. Chem. 1996; 271: 1841-1844Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 12Enari M. Talanian R.V. Wong W.W. Nagata S. Nature. 1996; 380: 723-726Crossref PubMed Scopus (969) Google Scholar, 13Kamada S. Washida M. Hasegawa J.-I. Kusano H. Funahashi Y. Tsujimoto Y. Oncogene. 1997; 15: 285-290Crossref PubMed Scopus (93) Google Scholar). To date, a family of at least 10 caspases have been identified, but it is not known precisely which of these caspase(s) are activated in vivo and which are responsible for the cleavage of particular substrates. Many of the caspases have overlapping substrate specificities, suggesting that there may be redundancy (6Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4146) Google Scholar, 7Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2187) Google Scholar, 14Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1852) Google Scholar). Many cellular proteins are cleaved during the execution phase of apoptosis at a DXXD motif by the effector caspases-3 and -7 (reviewed in Refs. 6Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4146) Google Scholar and 7Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2187) Google Scholar). Relatively little is known about the subcellular distribution of the caspases. Caspase-1 is found predominantly in the cytosol (15Ayala J.M. Yamin T.-T. Egger L.A. Chin J. Kostura M.J. Miller D.K. J. Immunol. 1994; 153: 2592-2599PubMed Google Scholar) although some has been localized to the external cell surface membrane (16Singer I.I. Scott S. Chin J. Bayne E.K. Limjuco G. Weidner J. Miller D.K. Chapman K. Kostura M.J. J. Exp. Med. 1995; 182: 1447-1459Crossref PubMed Scopus (140) Google Scholar). Other caspases have been considered to be cytosolic, a conclusion based largely on data with caspase-1 and on the isolation and purification of caspase-3 (17Nicholson D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulson M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3804) Google Scholar). In this study, we demonstrate for the first time the differential subcellular distribution of specific caspases during the induction of apoptosisin vivo. Following Fas-induced apoptosis in vivo, active caspase-3 is found primarily in the cytosol, whereas active caspase-7 is associated almost exclusively with the mitochondrial and microsomal fractions. Both the activation of caspase-7 in the endoplasmic reticulum and the cleavage of the endoplasmic reticular-specific substrate, sterol regulatory element-binding protein 1 (SREBP-1), 1The abbreviations used are: SREBP, sterol regulatory element-binding protein; Z-VAD.fmk, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone; Z-DEVD.afc, benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin; DEVDase, proteolytic activity to cleave Z-DEVD.afc; MOPS, 4-morpholinepropanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; PAGE, polyacrylamide gel electrophoresis. are blocked by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.fmk). These results support the hypothesis that during the execution phase of apoptosis, different caspase homologs cleave specific substrates in different cellular compartments. In this study 6–8-week-old (20 g) Balb/c males were used. All mice were bred in the Biomedical Sciences Department of the University of Leicester. Mice were injected either with 10 μg of purified hamster monoclonal antibody to mouse Fas (JO2) (PharMingen, Los Angeles, CA) (2Ogasawara J. Watanabe-Fukunaga R. Adachi M. Matsuzawa A. Kasugai T. Kitamura Y. Itoh N. Suda T. Nagata S. Nature. 1993; 364: 806-809Crossref PubMed Scopus (1815) Google Scholar) in 160 μl of 0.9% (w/v) saline, 12.5% (v/v) Me2SO, or 160 μl of 0.9% (w/v) saline, 12.5% (v/v) Me2SO (controls). Where indicated, mice were injected with JO2 antibody (10 μg) in 80 μl of 0.9% (w/v) saline followed 5 min later by Z-VAD.fmk (500 μg) (Enzyme Systems Ltd., Dublin, CA) in 80 μl of 0.9% (w/v) saline, 25% (v/v) Me2SO. Animals were sacrificed at the indicated times by cervical dislocation. Livers were removed and fixed in 10% formaldehyde in buffered saline. Representative sections of the left lateral, median, and posterior lobes were stained with hematoxylin and eosin and examined for apoptosis. Following removal of the livers, excess hair and blood were removed by washing several times in buffer A (0.3 m mannitol, 5 mm MOPS, 1 mmEGTA, 4 mm KH2PO4). The livers were then chopped up and homogenized using a dounce homogenizer in 5 ml of buffer A supplemented with protease inhibitors (20 μg/ml leupeptin, 10 μg/ml pepstatin A, 10 μg/ml aprotinin, 2 mmphenylmethylsulfonyl fluoride). The crude homogenates were centrifuged at 650 × g for 10 min at 4 °C and the resultant supernatant centrifuged at 10,000 × g for 15 min at 4 °C to sediment the mitochondria. The mitochondria were washed in supplemented buffer A and pelleted. The microsomal and cytosolic fractions were obtained following centrifugation of the 10,000 ×g supernatant fraction at 100,000 × g for 45 min at 4 °C. Purity of the mitochondrial, cytosolic, and microsomal fractions was assessed by Western blotting using antibodies to cytochrome c oxidase subunit IV (Molecular Probes, Eugene, OR), glutathione S-transferase π (18Manson M.M. Ball H.W.L. Barrett M.C. Clark H.L. Judah D.J. Williamson G. Neal G.E. Carcinogenesis. 1997; 18: 1729-1738Crossref PubMed Scopus (204) Google Scholar) (kindly provided by Dr. M. Manson, Medical Research Council Toxicology Unit) and SREBP-1. Cytochrome c oxidase subunit IV is located on the inner mitochondrial membrane (19Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Nakashima R. Yaono R. Yoshikawa S. Science. 1996; 272: 1136-1144Crossref PubMed Scopus (1936) Google Scholar), glutathioneS-transferase π is a cytosolic enzyme (20Hayes J.D. Pulford D.J. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3267) Google Scholar), and SREBP-1 is located in the endoplasmic reticulum (21Wang X. Sato R. Brown M.S. Hua X. Goldstein J.L. Cell. 1994; 77: 53-62Abstract Full Text PDF PubMed Scopus (859) Google Scholar). Densitometric analysis revealed that 77, 9, and 14% of total glutathioneS-transferase and 5, 93, and 2% of total cytochromec oxidase subunit IV were detected in the cytosolic, mitochondrial, and microsomal fractions, respectively. SREBP-1 was found almost exclusively in the endoplasmic reticulum (Fig. 4 and data not shown). SDS-PAGE and Western blotting were carried out on liver fractions as described previously (22MacFarlane M. Cain K. Sun X.-M. Alnemri E.S. Cohen G.M. J. Cell Biol. 1997; 137: 469-479Crossref PubMed Scopus (129) Google Scholar). The membranes were probed using a rabbit polyclonal antibody to the p17 subunit of caspase-3 (kindly provided by Merck Frosst, Quebec, Canada) (11Schlegel J. Peters I. Orrenius S. Miller D.K. Thornberry N.A. Yamin T.-T. Nicholson D.W. J. Biol. Chem. 1996; 271: 1841-1844Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 23Boulakia C.A. Chen G. Ng F.W.H. Teodoro J.G. Branton P.E. Nicholson D.W. Poirier G.G. Shore G.C. Oncogene. 1996; 12: 529-535PubMed Google Scholar), a rabbit polyclonal antibody to the p17 fragment of caspase-7 (22MacFarlane M. Cain K. Sun X.-M. Alnemri E.S. Cohen G.M. J. Cell Biol. 1997; 137: 469-479Crossref PubMed Scopus (129) Google Scholar), and a rabbit polyclonal antibody to amino acids 470–479 of human SREBP-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The proteolytic activity of the liver fractions was measured using a continuous fluorometric assay with benzyloxycarbonyl-Asp-Glu-Val-Asp- 7-amino-4-trifluoromethylcoumarin (Z-DEVD.afc) (Enzyme Systems Products, Dublin, CA) as substrate as described previously (22MacFarlane M. Cain K. Sun X.-M. Alnemri E.S. Cohen G.M. J. Cell Biol. 1997; 137: 469-479Crossref PubMed Scopus (129) Google Scholar). Cleavage of Z-DEVD.afc releases the fluorescent moiety, 7-amino-4-trifluoromethylcoumarin, allowing the quantitative analysis of the proteolytic activities of caspases-3 and -7 (referred to as DEVDase). The agonistic Fas receptor antibody JO2 induced extensive liver damage and hemorrhage in Balb/c mice, with >60% of hepatocytes showing apoptotic morphology after 4 h (Fig. 1 B) (2Ogasawara J. Watanabe-Fukunaga R. Adachi M. Matsuzawa A. Kasugai T. Kitamura Y. Itoh N. Suda T. Nagata S. Nature. 1993; 364: 806-809Crossref PubMed Scopus (1815) Google Scholar). The caspase inhibitor, Z-VAD.fmk (50 μmol/kg) blocked Fas-induced liver damage and hemorrhage and dramatically reduced hepatocyte apoptosis to <1% (Fig. 1 C). Almost complete protection was still observed 24 h after exposure to the agonistic antibody (data not shown). The protection conferred by Z-VAD.fmk suggested a critical role for the activation of caspases in Fas-induced apoptosis in vivo, consistent with previous studies (24Rodriguez I. Matsuura K. Ody C. Nagata S. Vassalli P. J. Exp. Med. 1996; 184: 2067-2072Crossref PubMed Scopus (271) Google Scholar, 25Rouquet N. Pagès J.-C. Molina T. Briand P. Joulin V. Curr. Biol. 1996; 6: 1192-1195Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). To date, very few studies on Fas-induced apoptosis have been carried out in vivo, most being in vitro (9Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4561) Google Scholar, 10Enari M. Hug H. Nagata S. Nature. 1995; 375: 78-81Crossref PubMed Scopus (798) Google Scholar, 11Schlegel J. Peters I. Orrenius S. Miller D.K. Thornberry N.A. Yamin T.-T. Nicholson D.W. J. Biol. Chem. 1996; 271: 1841-1844Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 12Enari M. Talanian R.V. Wong W.W. Nagata S. Nature. 1996; 380: 723-726Crossref PubMed Scopus (969) Google Scholar, 13Kamada S. Washida M. Hasegawa J.-I. Kusano H. Funahashi Y. Tsujimoto Y. Oncogene. 1997; 15: 285-290Crossref PubMed Scopus (93) Google Scholar). In order to confirm the involvement of caspases in Fas-mediated apoptosisin vivo, their activation was assessed by measuring DEVDase in crude liver homogenates from control and treated mice. This activity is primarily a measure of caspase-3 and caspase-7 activities, although there may be a minor contribution from other caspases (14Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1852) Google Scholar, 17Nicholson D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulson M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3804) Google Scholar, 26Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2279) Google Scholar, 27Fernandes-Alnemri T. Takahashi A. Armstrong R. Krebs J. Fritz L. Tomaselli K.J. Wang L. Yu Z. Croce C.M. Salvesen G. Earnshaw W.C. Litwack G. Alnemri E.S. Cancer Res. 1995; 55: 6045-6052PubMed Google Scholar). Treatment with the agonistic Fas antibody resulted in a marked increase in total liver DEVDase (270 nmol/min) after 4 h in comparison with that in controls (80 nmol/min). These results demonstrated the activation of the effector caspase-3 and/or caspase-7 in Fas-induced apoptosis in vivo in agreement with in vitro studies, which have shown the activation of these caspases following treatment of cells with Fas or tumor necrosis factor (11Schlegel J. Peters I. Orrenius S. Miller D.K. Thornberry N.A. Yamin T.-T. Nicholson D.W. J. Biol. Chem. 1996; 271: 1841-1844Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 28Duan H. Chinnaiyan A.M. Hudson P.L. Wing J.P. He W.-W. Dixit V.M. J. Biol. Chem. 1996; 271: 1621-1625Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar,29Chinnaiyan A.M. Orth K. O'Rourke K. Duan H. Poirier G.G. Dixit V.M. J. Biol. Chem. 1996; 271: 4573-4576Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar). In order to further dissect the role of caspases in Fas-induced apoptosis in vivo, we examined DEVDase and caspase processing in subcellular liver fractions prepared from control and Fas-treated mice. Fas treatment induced 11-, 21-, and 23-fold increases in total DEVDase in cytosolic, microsomal, and mitochondrial fractions, respectively. In all cases, Z-VAD.fmk markedly inhibited the increases in DEVDase. Thus Z-VAD.fmk blocked apoptosis either by directly inhibiting the activity of caspases-3 and -7 or by inhibiting an upstream caspase, such as caspase-8. Caspase-3 is generally present in control cells as an inactive p32 zymogen (11Schlegel J. Peters I. Orrenius S. Miller D.K. Thornberry N.A. Yamin T.-T. Nicholson D.W. J. Biol. Chem. 1996; 271: 1841-1844Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 22MacFarlane M. Cain K. Sun X.-M. Alnemri E.S. Cohen G.M. J. Cell Biol. 1997; 137: 469-479Crossref PubMed Scopus (129) Google Scholar, 29Chinnaiyan A.M. Orth K. O'Rourke K. Duan H. Poirier G.G. Dixit V.M. J. Biol. Chem. 1996; 271: 4573-4576Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar). On induction of apoptosis, it is initially processed at Asp-175 between the large and small subunits, yielding a p20 subunit, which is further processed at Asp-9 and Asp-28 to yield p19 and p17 large subunits, respectively (17Nicholson D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulson M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3804) Google Scholar, 30Fernandes-Alnemri T. Armstrong R.C. Krebs J. Srinivasula S.M. Wang L. Bullrich F. Fritz L.C. Trapani J.A. Tomaselli K.J. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7464-7469Crossref PubMed Scopus (694) Google Scholar). In control mice, procaspase-3 was present in the cytosolic fraction (Fig. 2, lane 3) with none detected in the mitochondrial or microsomal fractions (Fig. 2, lanes 1 and 6). Following treatment with the Fas antibody, complete processing of procaspase-3 together with the appearance of its catalytically active p17 subunit was observed in the cytosolic fraction (Fig. 2, lane 4). In addition, immunologically reactive fragments of ∼29 kDa (Fig. 2, lanes 3–7) and ∼25 kDa (Fig. 2, lane 4) were also observed. The ∼p29 fragment was observed in all the cytosolic and microsomal fractions (Fig. 2,lanes 3–7). While the identity of this fragment is not known, it has been proposed to be due to processing of caspase-3 following cleavage at Asp-28, yielding a zymogen, which is not further processed to active caspase-3 (23Boulakia C.A. Chen G. Ng F.W.H. Teodoro J.G. Branton P.E. Nicholson D.W. Poirier G.G. Shore G.C. Oncogene. 1996; 12: 529-535PubMed Google Scholar). Treatment with Z-VAD.fmk resulted in almost complete inhibition of the processing of procaspase-3, formation of the p17 large subunit, and the p25 fragment (Fig. 2,lane 5). Inhibition of caspase-3 processing was also accompanied by the appearance of a very small amount of an ∼p19 fragment (Fig. 2, lane 5), which may be attributed either to irreversible binding of Z-VAD.fmk to the p17 subunit or to partial blocking of the processing of the large subunit (22MacFarlane M. Cain K. Sun X.-M. Alnemri E.S. Cohen G.M. J. Cell Biol. 1997; 137: 469-479Crossref PubMed Scopus (129) Google Scholar, 31Polverino A.J. Patterson S.D. J. Biol. Chem. 1997; 272: 7013-7021Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The p17 subunit was detected primarily in the cytosolic fraction of livers from Fas-treated mice (Fig. 2, lane 4) with little if any being present in any other fraction (Fig. 2). These data demonstrate that following Fas induction of apoptosis, active caspase-3 is located primarily in the cytosol. Caspase-7 exists as an inactive p35 zymogen in control cells (22MacFarlane M. Cain K. Sun X.-M. Alnemri E.S. Cohen G.M. J. Cell Biol. 1997; 137: 469-479Crossref PubMed Scopus (129) Google Scholar, 28Duan H. Chinnaiyan A.M. Hudson P.L. Wing J.P. He W.-W. Dixit V.M. J. Biol. Chem. 1996; 271: 1621-1625Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 32Chandler J.M. Alnemri E.S. Cohen G.M. MacFarlane M. Biochem. J. 1997; 322: 19-23Crossref PubMed Scopus (25) Google Scholar). On induction of apoptosis it is activated by initial processing at Asp 198 between the large and small subunits followed by cleavage at Asp-23 to yield the catalytically active p19 large subunit (22MacFarlane M. Cain K. Sun X.-M. Alnemri E.S. Cohen G.M. J. Cell Biol. 1997; 137: 469-479Crossref PubMed Scopus (129) Google Scholar, 27Fernandes-Alnemri T. Takahashi A. Armstrong R. Krebs J. Fritz L. Tomaselli K.J. Wang L. Yu Z. Croce C.M. Salvesen G. Earnshaw W.C. Litwack G. Alnemri E.S. Cancer Res. 1995; 55: 6045-6052PubMed Google Scholar, 28Duan H. Chinnaiyan A.M. Hudson P.L. Wing J.P. He W.-W. Dixit V.M. J. Biol. Chem. 1996; 271: 1621-1625Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). In livers from control mice, caspase-7 was present as an unprocessed p35 proform in both the cytosolic and microsomal fractions with no detectable p19 subunit (Fig. 3, lanes 4 and 7). While caspase-7 has previously been detected in the cytoplasm of Jurkat cells (28Duan H. Chinnaiyan A.M. Hudson P.L. Wing J.P. He W.-W. Dixit V.M. J. Biol. Chem. 1996; 271: 1621-1625Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar), this is the first time it has also been recognized to have a microsomal location. No detectable procaspase-7 or p19 subunit was present in the mitochondrial fraction from control mouse livers (Fig. 3, lane 1). Following treatment with the Fas antibody, complete processing of procaspase-7 was observed in both the cytosolic and microsomal fractions (Fig. 3, lanes 5 and 8). Although complete processing of caspase-7 was observed in the cytosolic fraction, little if any p19 subunit was detected (Fig. 3, lane 5). However, the p19 catalytically active large subunit of caspase-7 was clearly detected in both the mitochondrial and microsomal fractions (Fig. 3, lanes 2 and 8). It was very unlikely that the p19 fragment in the mitochondrial fraction was due to microsomal contamination, because the endoplasmic reticular protein SREBP-1 was located exclusively in the microsomal fraction (Fig. 4 and data not shown). In addition, the uncharacterized p29 band, detected using the caspase-3 antibody, in the microsomal fraction from control or Fas-treated livers (Fig. 2,lanes 6 and 7) was not present in the mitochondrial fraction (Fig. 2, lanes 1 and 2). The amount of the large p19 subunit of caspase-7 in the microsomal fraction following Fas-induced apoptosis was greater than the amount of procaspase-7 in control liver microsomes (Fig. 3, compare lanes 7 and 8). These results suggested that caspase-7 was translocated from the cytosol to the microsomes following its catalytic activation by an initiator caspase. The data clearly demonstrate that following Fas induction of apoptosis in mouse liver, caspase-7 is completely processed to its catalytically active p19 subunit, which is found primarily in the mitochondrial and microsomal fractions with little if any remaining in the cytosol. Z-VAD.fmk completely inhibited the Fas-induced cleavage of procaspase-7 as well as the formation of the p19 subunit in all subcellular fractions (Fig. 3, lanes 3, 6, and 9). Z-VAD.fmk also blocked the appearance of an uncharacterized ∼p32 fragment in the microsomal fraction (Fig. 3, lane 9). Taken together with the caspase-3 results, our data suggest that Z-VAD.fmk blocks the processing of both caspase-3 and caspase-7. Although it is not known precisely which caspase activates caspase-3 and caspase-7 during Fas-induced apoptosis, caspase-8 has been considered the most likely candidate (3Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. 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This complex results in the activation of caspase-9, which in turn cleaves and activates caspase-3 (34Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6261) Google Scholar). The mechanism of activation of procaspase-7 is not known, it may be due to activation by caspase-8 (5Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (483) Google Scholar) or to a mechanism involving Apaf-1 and procaspase-9 (34Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6261) Google Scholar), but it does not appear to be due to a direct activation by caspase-3 (35Hirata H. Takahashi A. Kobayashi S. Yonehara S. Sawai H. Okazaki T. Yamamoto K. Sasada M. J. Exp. Med. 1998; 187: 587-600Crossref PubMed Scopus (398) Google Scholar). Although activation of caspase-8 is clearly a very early event following Fas-induced apoptosis, it is not yet clear how this is related to the activation of caspases following mitochondrial damage with the subsequent release of cytochrome c and the activation of procaspase-9. Therefore, Z-VAD.fmk may exert its action by blocking the caspase cascade initiated by both caspase-8 and caspase-9. Further support for the hypothesis that different effector caspases are responsible for the enzymic activity in different subcellular compartments was provided by comparing the Western blot data with DEVDase. DEVDase in cells undergoing apoptosis is believed to be primarily due to activation of caspase-3 and caspase-7 (14Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. 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Although the total DEVDase loaded onto the polyacrylamide gel from the cytosolic fraction (20 pmol/min) (Fig. 3, lane 5) was greater than that from either the mitochondrial or microsomal fractions (9 and 15 pmol/min) (Fig. 3, lanes 2 and 8, respectively), the antibody to caspase-7 only detected the p19 large subunit in the mitochondrial and microsomal fractions (Fig. 3,lanes 2 and 8). This suggested that a caspase other than caspase-7 was primarily responsible for DEVDase in the cytosolic fraction. Most probably this was caspase-3, based on the data demonstrating that the p17 catalytically active large subunit of caspase-3 was primarily located in the cytosol (Fig. 2). Thus our results strongly suggest that the major DEVDase in the microsomal and mitochondrial fractions is due to caspase-7, while in the cytosolic fraction it is due to caspase-3. Taken together, our results suggest that following its activation, caspase-7 is translocated to the microsomal and mitochondrial fractions, where it is responsible for the cleavage of specific substrates in these distinct subcellular compartments. A recent study using an affinity label also noted differences in the pattern of active caspases in the nuclei and cytosol between two cell lines (36Martins L.M. Mesner P.W. Kottke T.J. Basi G.S. Sinha S. Tung J.S. Svingen P.A. Madden B.J. Takahashi A. McCormick D.J. Earnshaw W.C. Kaufmann S.H. Blood. 1997; 90: 4283-4296Crossref PubMed Google Scholar). Their results together with the present study raise the question about how different active caspases may be targeted to different subcellular localizations. Many previous studies have highlighted overlapping substrate specificities of caspases-3 and -7. For example, combinatorial studies using tetrapeptide substrates assigned virtually indistinguishable substrate specificities to caspases-3 and -7 (14Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1852) Google Scholar), and both enzymes effectively cleave poly(ADP-ribose) polymerase (17Nicholson D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulson M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3804) Google Scholar,26Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2279) Google Scholar, 27Fernandes-Alnemri T. Takahashi A. Armstrong R. Krebs J. Fritz L. Tomaselli K.J. Wang L. Yu Z. Croce C.M. Salvesen G. Earnshaw W.C. Litwack G. Alnemri E.S. Cancer Res. 1995; 55: 6045-6052PubMed Google Scholar). It has often been suggested that there is a redundancy for certain caspases, which may be due to the important biological function of this system in removing damaged or unwanted cells (6Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4146) Google Scholar, 7Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2187) Google Scholar). However results with caspase-3 knockout mice, which exhibit normal apoptosis in most tissues except neuronal cells, indicate that some caspases may function in a tissue selective manner (37Kuida K. Zheng T.S. Na S. Kuan C.-Y. Yang D. Karasuyama H. Rakic P. Flavell R.A. Nature. 1996; 384: 368-372Crossref PubMed Scopus (1713) Google Scholar). Alternatively our data suggest that, at least in some tissues, caspases with overlapping substrate specificity may exert their functions in different cellular compartments, where they catalyze the cleavage of specific substrates. To explore this possibility, we examined the fate of SREBP-1, one of the few endoplasmic reticulum-associated proteins known to be cleaved in apoptosis (21Wang X. Sato R. Brown M.S. Hua X. Goldstein J.L. Cell. 1994; 77: 53-62Abstract Full Text PDF PubMed Scopus (859) Google Scholar, 38Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO J. 1996; 15: 1012-1020Crossref PubMed Scopus (296) Google Scholar, 39Pai J.-T. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5437-5442Crossref PubMed Scopus (65) Google Scholar). SREBPs belong to the basic-helix-loop-helix-leucine zipper family of transcription factors and are involved in the regulation of sterol metabolism (21Wang X. Sato R. Brown M.S. Hua X. Goldstein J.L. Cell. 1994; 77: 53-62Abstract Full Text PDF PubMed Scopus (859) Google Scholar). On the induction of apoptosis both SREBP-1 and SREBP-2 are cleaved by the hamster homologs of caspases-3 and -7 (38Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO J. 1996; 15: 1012-1020Crossref PubMed Scopus (296) Google Scholar, 39Pai J.-T. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5437-5442Crossref PubMed Scopus (65) Google Scholar). In livers from control mice, SREBP-1 was exclusively associated with the microsomal fraction with none being detectable in the cytosolic or mitochondrial fractions (Fig. 4, compare lanes 1 and2, and data not shown). Complete loss of the ∼125-kDa SREBP-1 was observed in liver microsomes obtained from mice treated 4 h earlier with the agonistic Fas antibody (Fig. 4, lane 3). The antibody to SREBP-1 detected the intact but not the cleaved molecule. The Fas-induced cleavage of SREBP-1 was largely prevented by Z-VAD.fmk (Fig. 4, lane 4). As active caspase-7 and SREBP-1 share the same subcellular localization, it is possible that in vivo caspase-7 is responsible for the Fas-induced cleavage of SREBP-1. However based on our present data and the contiguous nature of the cytosol and endoplasmic reticulum, we cannot totally exclude the possibility that SREBP-1 may also be cleaved, at least in part, by caspase-3. We have clearly shown that following Fas-induced apoptosis in vivo, active caspase-3 was located primarily in the cytosol, whereas active caspase-7 was associated with both the mitochondrial and microsomal fractions. Our data represent the first example of the differential subcellular distribution of specific caspases in anin vivo model of apoptosis. We thank Drs. K. Cain, P. Carthew, and B. Nolan for their helpful advice.
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