Hsp72 Inhibits Apoptosis Upstream of the Mitochondria and Not through Interactions with Apaf-1

Hsp72 protects cells against apoptosis in response to various stresses. By simultaneously measuring cytochrome c localization and nuclear morphology in mouse embryo fibroblasts, we have shown that Hsp72 blocks cytochrome c release from mitochondria in response to cytotoxic stress and that permeabilization of the outer mitochondrial membrane is the critical point in deciding the fate of the cell. Hsp72 did not inhibit apoptosis in mouse embryo fibroblasts once cytochrome c had been released from the mitochondria. Recent reports have claimed that Hsp72 can prevent caspase activation by inhibiting the oligomerization of Apaf-1 in the presence of cytochrome c and dATP. We now show that this apparent function of recombinant Hsp72 is due to the presence of salt in the Hsp72 preparation and that the same response can be achieved by the addition of heat-denatured Hsp72 in the same high salt buffer or by the high salt buffer alone. Hsp72 expressed in a range of different cell lines had no inhibitory effect on cytochrome c-stimulated caspase activity of cytosolic extracts. We conclude that the protective effect of Hsp72 occurs upstream of the mitochondria and not through the inhibition of the apoptosome. Hsp72 protects cells against apoptosis in response to various stresses. By simultaneously measuring cytochrome c localization and nuclear morphology in mouse embryo fibroblasts, we have shown that Hsp72 blocks cytochrome c release from mitochondria in response to cytotoxic stress and that permeabilization of the outer mitochondrial membrane is the critical point in deciding the fate of the cell. Hsp72 did not inhibit apoptosis in mouse embryo fibroblasts once cytochrome c had been released from the mitochondria. Recent reports have claimed that Hsp72 can prevent caspase activation by inhibiting the oligomerization of Apaf-1 in the presence of cytochrome c and dATP. We now show that this apparent function of recombinant Hsp72 is due to the presence of salt in the Hsp72 preparation and that the same response can be achieved by the addition of heat-denatured Hsp72 in the same high salt buffer or by the high salt buffer alone. Hsp72 expressed in a range of different cell lines had no inhibitory effect on cytochrome c-stimulated caspase activity of cytosolic extracts. We conclude that the protective effect of Hsp72 occurs upstream of the mitochondria and not through the inhibition of the apoptosome. Hsp72 belongs to an ancient family of proteins that play a role in many different cellular processes, including the folding of newly synthesized proteins, protein trafficking across cellular membranes, and the assembly/disassembly of protein complexes (1Hartl F.U. Martin J. Curr. Opin. Struct. Biol. 1995; 5: 92-102Crossref PubMed Scopus (154) Google Scholar, 2Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3607) Google Scholar). Less well understood is the ability of Hsp72 to protect cells from cell death. Hsp72 has been shown to protect cells from necrosis and is capable of protecting and repairing important cell metabolic processes (3Gabai V.L. Kabakov A.E. FEBS Lett. 1993; 327: 247-250Crossref PubMed Scopus (55) Google Scholar, 4Kabakov A.E. Gabai V.L. Exp. Cell Res. 1995; 217: 15-21Crossref PubMed Scopus (48) Google Scholar, 5Yaglom J.A. Ekhterae D. Gabai V.L. Sherman M.Y. J. Biol. Chem. 2003; 278: 50483-50496Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 6Li L. Shen G. Li G.C. Exp. Cell Res. 1995; 217: 460-468Crossref PubMed Scopus (22) Google Scholar). It has also been shown to inhibit apoptosis, whereby cells actively engineer their own destruction in response to extracellular signals and intracellular damage (7Samali A. Robertson J.D. Peterson E. Manero F. van Zeijl L. Paul C. Cotgreave I.A. Arrigo A.P. Orrenius S. Cell Stress Chaperones. 2001; 6: 49-58Crossref PubMed Scopus (149) Google Scholar, 8Buzzard K.A. Giaccia A.J. Killender M. Anderson R.L. J. Biol. Chem. 1998; 273: 17147-17153Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 9Gabai V.L. Meriin A.B. Mosser D.D. Caron A.W. Rits S. Shifrin V.I. Sherman M.Y. J. Biol. Chem. 1997; 272: 18033-18037Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 10Mosser D.D. Caron A.W. Bourget L. Denis-Larose C. Massie B. Mol. Cell. Biol. 1997; 17: 5317-5327Crossref PubMed Scopus (872) Google Scholar, 11Jaattela M. Wissing D. Kokholm K. Kallunki T. Egeblad M. EMBO J. 1998; 17: 6124-6134Crossref PubMed Scopus (620) Google Scholar). In contrast to necrosis, which is caused by metabolic failure leading to cell swelling and the eventual rupture of the cell membrane, apoptosis requires active cell metabolism to degrade the nuclear DNA, divide the cell into smaller membrane-bound vesicles, and mark these for phagocytosis by surrounding healthy cells (12Adams J.M. Genes Dev. 2003; 17: 2481-2495Crossref PubMed Scopus (680) Google Scholar, 13Lawen A. Bioessays. 2003; 25: 888-896Crossref PubMed Scopus (355) Google Scholar). Apoptosis commonly involves the activation of caspases, a family of intracellular proteases normally present as zymogens (14Wolf B.B. Green D.R. J. Biol. Chem. 1999; 274: 20049-20052Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar). These caspases commit a cell to apoptosis by activating proteins directly involved in the apoptotic process, or inactivating anti-apoptotic proteins and those responsible for normal homeostasis and repair. Apoptosis through extracellular signaling (the extrinsic pathway) and in response to intracellular damage (the intrinsic pathway) are both dependent on caspase activation but differ in how that activation is achieved. In the case of the extrinsic pathway, membrane-bound receptors are oligomerized in response to ligand binding and directly activate the initiator caspases, caspase-8 and -10 (15Muzio M. Stockwell B.R. Stennicke H.R. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1998; 273: 2926-2930Abstract Full Text Full Text PDF PubMed Scopus (885) Google Scholar). These initiator caspases are then responsible for activating the effector caspases 3, 6, and 7 (16Sun X.M. MacFarlane M. Zhuang J. Wolf B.B. Green D.R. Cohen G.M. J. Biol. Chem. 1999; 274: 5053-5060Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar). In some cells (known as type I cells) this is sufficient to induce apoptosis. In type II cells death is dependent on the release of cytochrome c and other pro-apoptotic factors from the mitochondria in response to caspase-8 activity (17Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2633) Google Scholar). This mitochondrial pathway is the same pathway activated in the cell in response to intracellular damage. Cytochrome c is released from the intermembrane space of the mitochondria and is bound by Apaf-1 along to a protein as the K. C. G. X.M. Cohen G.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Martin Green D.R. D.D. EMBO J. 1997; PubMed Scopus Google Scholar, D. M. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). This then and which is responsible for activating the effector caspases in a to that achieved by caspase-8 D. M. 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Massie B. Mol. Cell. Biol. PubMed Scopus Google Scholar, Exp. Cell Res. PubMed Scopus Google Scholar). This inhibition of cytochrome c release be achieved by the ability of Hsp72 to prevent in response to stress T. K. S. M. Cell PubMed Scopus Google Scholar). other reports have shown that Hsp72 to inhibit cytochrome c release but by inhibiting the activation of in J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google or by of the caspase and cytosolic activity M. Wissing D. Kokholm K. Kallunki T. Egeblad M. EMBO J. 1998; 17: 6124-6134Crossref PubMed Scopus (620) Google Scholar). It has also been that Hsp72 inhibit apoptosis by the protein mouse embryo necrosis cell pathway V.L. Meriin A.B. Mosser D.D. Caron A.W. Rits S. Shifrin V.I. Sherman M.Y. J. Biol. Chem. 1997; 272: 18033-18037Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, A.B. J.A. Gabai V.L. L. S. Mosser D.D. Sherman M.Y. Mol. Cell. 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Cell activated by and cytochrome c for on a and buffer to membrane and the as Cell as for to and for to including mitochondria. from the and by Cytochrome c by c release a of the of and J.A. Cell 2003; PubMed Scopus Google Scholar). and for on in and in permeabilization of cells by by cells an c an by cells to on a on a a and to a of Hsp72 Apoptosis in to and but to shown K.A. Giaccia A.J. Killender M. Anderson R.L. J. Biol. Chem. 1998; 273: 17147-17153Abstract Full Text Full Text PDF PubMed Scopus (268) Google Hsp72 protects mouse embryo fibroblasts from apoptosis by and Hsp72 did not protect from and K.A. Giaccia A.J. Killender M. Anderson R.L. J. Biol. Chem. 1998; 273: 17147-17153Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). of Hsp72 in lines Hsp72 and is the normal has been shown that Hsp72 in these cells is that K.A. Giaccia A.J. Killender M. Anderson R.L. J. Biol. 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In did a to in the presence of which through the extrinsic signaling pathway that Hsp72 apoptosis in response to caspase activity in to Hsp72 also by inhibiting caspase activity to be in These that caspase activity is for apoptosis and that Hsp72 by inhibiting the ability of the cell to activate the caspase Hsp72 Cytochrome c of in an in has been that Hsp72 directly inhibiting ability to activate in response to cytochrome c and by inhibiting the of the or by the binding of to this A. L. Cell. Biol. PubMed Scopus Google Scholar, Wolf B.B. K. Mosser D.D. A. T. Cohen G.M. Green D.R. Cell. Biol. PubMed Scopus Google Scholar). that cell from be to caspase activation in response to cytochrome c and from as shown in and the addition of cytochrome c and as not activity in from This a range of cytochrome c and that caspase activity to the in the cell lines from and cells cells not In these cell lines Hsp72 is protective against various apoptotic stresses. J. K. C. J. and L. of expressed Hsp72 to inhibit caspase activation in cell in response to cytochrome c and that Hsp72 not inhibit caspase activation through the apoptosome. of Hsp72 to in Cell in the of Cytochrome c and to the of inhibition of activity in from cells not be from the A. L. Cell. Biol. PubMed Scopus Google Scholar, Wolf B.B. K. Mosser D.D. A. T. Cohen G.M. Green D.R. Cell. Biol. PubMed Scopus Google Scholar). this from cells and cytochrome in the presence or of the same recombinant Hsp72 as in the Hsp72 to inhibit caspase activation and Hsp72 from Hsp72 in of a buffer for the caspase activation addition of recombinant Hsp72 apparent an of an buffer to that in the Hsp72 in or not caspase activation to a as the recombinant Hsp72 that had been by the ability to inhibit caspase activation and recombinant Hsp72 against a to inhibit caspase activation in also inhibitory in the same buffer of Hsp72 by ability to a binding of D.R. S. A. 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Hsp72 inhibiting be that Hsp72 to in a Apaf-1 or the the and of complexes by an and the proteins on a binding of the an in Hsp72 the ability of this to It is that the addition of or inhibit the binding of Hsp72 to Apaf-1 or to the is to inhibit binding as shown in the addition of cytochrome c to sufficient to induce some by not these no to the of Hsp72 Hsp72 to Hsp72 and Apaf-1 not be by Hsp72 the of Cytochrome c in to a of to cytochrome c release from to reports that the ability of Hsp72 to inhibit apoptosis of cytochrome c release M. Wissing D. Kokholm K. Kallunki T. Egeblad M. EMBO J. 1998; 17: 6124-6134Crossref PubMed Scopus (620) Google Scholar, Exp. Cell Res. PubMed Scopus Google Scholar). of the cells a in cytochrome c release in cells but of cytochrome c release by cytochrome c release a by and J.A. Cell 2003; PubMed Scopus Google a different permeabilization of the membrane by the release of proteins from the mitochondrial proteins are the mitochondria. cells are then in and cytochrome c is by shown in of and cells a that an c this to be to the cytochrome c not In and a of cells cytochrome c Cytochrome c release by but a of cells had cytochrome c release and apoptosis not not a of cells cytochrome c due to the of cell membrane apoptosis. Hsp72 a of cytochrome c cells the cytochrome c release and apoptosis, the cells and by a and cytochrome c present in a of a mitochondrial In cells that had been different cell apparent cells to cells or a and nuclear morphology apoptosis and a of cytochrome In of cells the cells a normal and cytochrome c or apoptotic cells and cytochrome c not cells that had not been by cytochrome c that the to the mitochondria not also and and for cytochrome c In the of an apoptotic nuclear morphology the of cytochrome c from the the different the of cells Cytochrome c the in the of in to has been that Hsp72 cytochrome c release D. S. T. J. J. 2003; PubMed Scopus Google Scholar, D.D. Caron A.W. Bourget L. Meriin A.B. Sherman M.Y. Massie B. Mol. Cell. Biol. PubMed Scopus Google Scholar, Exp. Cell Res. 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In this the cells to to the that caspases for apoptotic signaling to inhibit cytochrome c to inhibit cytochrome c release in response to not Hsp72 Apoptosis by has been that Hsp72 protects cells from stress by repairing damage to the protein through we show that Hsp72 can inhibit apoptosis by and not protein damage. and proteins induce the of proteins in is as the response S. PubMed Google Scholar). to and the of Hsp72 as a of Hsp72 to be by but not by the other role of Hsp72 in protecting cells from apoptosis has been active for a of and an of the apoptotic has the by which Hsp72 apoptosis in response to cellular and extracellular signaling is of for by Hsp72 have been including cytochrome c the of the and the inhibition of V.L. Meriin A.B. Mosser D.D. Caron A.W. Rits S. Shifrin V.I. Sherman M.Y. J. Biol. Chem. 1997; 272: 18033-18037Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 11Jaattela M. Wissing D. Kokholm K. Kallunki T. Egeblad M. 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This is responsible for activating the initiator which in and the effector the that the ability of Hsp72 to protect from apoptosis is due to the inhibition of caspase This also that Hsp72 inhibit the in activation of caspases by the addition of cytochrome c and dATP. in the of we that this not the case and that cytochrome caspase activity as in from cells Hsp72 cell lines in these from and that this failure of Hsp72 to inhibit caspase activation is a this is in to that by Li J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google cells for we In an to Hsp72 and cell to but Hsp72 to the or of In this capable of complexes Hsp72 and a binding for the ability of Hsp72 to inhibit cell death in Hsp72 shown to a for cells in response to the caspase to cell a of apoptosis This failure of to protect cells from as to apoptosis, has been in and cells C. M. Mol. 2001; PubMed Google Scholar, C. K. A. FEBS Lett. 1998; PubMed Scopus Google as well as in the fibroblasts in this In to a and inhibit cytochrome c release in through the inhibition of the caspase in the extrinsic signaling extrinsic pathway to caspase activation as a of the intrinsic pathway caspase activation is a have been reports of activity in the intrinsic apoptotic pathway upstream of mitochondrial permeabilization A. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, S. J. Cell Biol. PubMed Scopus Google reports have shown that is not an of intrinsic apoptosis J. S. J. Cell Biol. PubMed Scopus Google Scholar, M. J.M. A. J. Cell Biol. PubMed Scopus Google Scholar). the of to inhibit cytochrome c release in response to that caspases not play an important role in apoptosis in cytochrome c release has in apoptosis the inhibition of by is to inhibition of the and not the cell that Hsp72 is to the inhibition of the by Hsp72 have for the of a cell. this J. S. J. Cell Biol. PubMed Scopus Google have that the of Apaf-1 from cells did not against or cytotoxic the inhibition of apoptosis. for measuring nuclear apoptosis and cytochrome c release to the fate of cells to a of apoptotic of this is the ability to simultaneously cells for a apoptotic upstream of function c and a apoptotic dependent on caspase activation It that cells that an apoptotic nuclear morphology had also cytochrome c cytochrome c release of caspase not by the caspase which nuclear Hsp72 to be an of cytochrome c release in response to and as D. S. T. J. J. 2003; PubMed Scopus Google Scholar, D.D. Caron A.W. Bourget L. Meriin A.B. Sherman M.Y. Massie B. Mol. Cell. Biol. PubMed Scopus Google Scholar, Exp. Cell Res. PubMed Scopus Google Scholar, V.L. K. Mosser D.D. Sherman M.Y. Mol. Cell. Biol. PubMed Scopus Google Scholar). 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This effect in from both and from cells that in the It that the effect of recombinant Hsp72 different to that of Hsp72 that had been expressed that the addition of the buffer in which Hsp72 sufficient to inhibit caspase Hsp72 in in is from and is in a buffer of the are in E. J. 2001; PubMed Google Scholar, K. C. X.M. Cohen G.M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google salt is a of It also that Hsp72 that had been in a salt buffer a ability to inhibit caspase activity and that the Hsp72 preparation from to protective function as we are in own be that Li J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google have that Hsp72 in a salt buffer capable of inhibiting caspase activation in an in Hsp72 in an buffer had no the we have from in a we their cytochrome c release has been by of cells and by this on and cells to that cytochrome c release but not by different the cells and and the cytochrome c by from the of cells mitochondrial the of measuring cell can be is to and not be to in cytochrome c release this fate of cell an apoptotic is on a and this is the same that be for the of apoptosis. that Hsp72 by inhibiting cytochrome c by directly on the mitochondria or some no that Hsp72 apoptosis in once cytochrome c release had is that inhibition of the to This a to the of the anti-apoptotic function of the apoptotic pathway leading to cytochrome c release is and the by which Hsp72 this pathway It is that repairing protein damage a role in inhibiting apoptosis, signaling through extracellular receptors not protein damage and as not induce as shown in is that the ability of Hsp72 to inhibit apoptosis is and from ability to inhibit necrosis D. 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