Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis

Resistance to stress-induced apoptosis was examined in cells in which the expression of hsp70 was either constitutively elevated or inducible by a tetracycline-regulated transactivator. Heat-induced apoptosis was blocked in hsp70-expressing cells, and this was associated with reduced cleavage of the common death substrate protein poly(ADP-ribose) polymerase (PARP). Heat-induced cell death was correlated with the activation of the stress-activated protein kinase SAPK/JNK (c-Jun N-terminal kinase). Activation of SAPK/JNK was strongly inhibited in cells in which hsp70 was induced to a high level, indicating that hsp70 is able to block apoptosis by inhibiting signaling events upstream of SAPK/JNK activation. In contrast, SAPK/JNK activation was not inhibited by heat shock in cells with constitutively elevated levels of hsp70. Cells that constitutively overexpress hsp70 resist apoptosis induced by ceramide, a lipid signaling molecule that is generated by apoptosis-inducing treatments and is linked to SAPK/JNK activation. Similar to heat stress, resistance to ceramide-induced apoptosis occurs in spite of strong SAPK/JNK activation. Therefore, hsp70 is also able to inhibit apoptosis at some point downstream of SAPK/JNK activation. Since PARP cleavage is prevented in both cell lines, these results suggest that hsp70 is able to prevent the effector steps of apoptotic cell death. Processing of the CED-3-related protease caspase-3 (CPP32/Yama/apopain) is inhibited in hsp70-expressing cells; however, the activity of the mature enzyme is not affected by hsp70 in vitro. Caspase processing may represent a critical heat-sensitive target leading to cell death that is inhibited by the chaperoning function of hsp70. The inhibition of SAPK/JNK signaling and apoptotic protease effector steps by hsp70 likely contributes to the resistance to stress-induced apoptosis seen in transiently induced thermotolerance.

[1]  D. Vaux,et al.  The molecular biology of apoptosis. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[2]  L. Zon,et al.  The stress-activated protein kinase pathway mediates cell death following injury induced by cis-platinum, UV irradiation or heat , 1996, Current Biology.

[3]  J. Mankovich,et al.  Inhibition of ICE family proteases by baculovirus antiapoptotic protein p35. , 1995, Science.

[4]  E. Coligan.  Current protocols in immunology , 1991 .

[5]  Patrick R. Griffin,et al.  Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis , 1995, Nature.

[6]  Y. Tsujimoto.  Stress-resistance conferred by high level of bcl-2 alpha protein in human B lymphoblastoid cell. , 1989, Oncogene.

[7]  Junying Yuan,et al.  Human ICE/CED-3 Protease Nomenclature , 1996, Cell.

[8]  David Stokoe,et al.  Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins , 1992, FEBS letters.

[9]  O. Bensaude,et al.  MAP kinase activation during heat shock in quiescent and exponentially growing mammalian cells , 1993, FEBS letters.

[10]  J. Avruch,et al.  Protein kinase cascades activated by stress and inflammatory cytokines , 1996, BioEssays : news and reviews in molecular, cellular and developmental biology.

[11]  Y. Hannun.  Functions of Ceramide in Coordinating Cellular Responses to Stress , 1996, Science.

[12]  J. Woodgett,et al.  The stress-activated protein kinase subfamily of c-Jun kinases , 1994, Nature.

[13]  Y. Hannun,et al.  Programmed cell death induced by ceramide. , 1993, Science.

[14]  E. White,et al.  Life, death, and the pursuit of apoptosis. , 1996, Genes & development.

[15]  L. Zon,et al.  Signal Transduction Pathways Regulated by Mitogen-activated/Extracellular Response Kinase Kinase Kinase Induce Cell Death (*) , 1996, The Journal of Biological Chemistry.

[16]  David Wallach,et al.  Involvement of MACH, a Novel MORT1/FADD-Interacting Protease, in Fas/APO-1- and TNF Receptor–Induced Cell Death , 1996, Cell.

[17]  G. Gores,et al.  The role of proteases during apoptosis , 1996, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[18]  R. Morimoto,et al.  The human heat shock protein hsp70 interacts with HSF, the transcription factor that regulates heat shock gene expression. , 1992, Genes & development.

[19]  V. Adler,et al.  UV Irradiation and Heat Shock Mediate JNK Activation via Alternate Pathways (*) , 1995, The Journal of Biological Chemistry.

[20]  Dick D. Mosser,et al.  Hsp70 Prevents Activation of Stress Kinases , 1997, The Journal of Biological Chemistry.

[21]  K. Schulze-Osthoff,et al.  Small Stress Proteins as Novel Regulators of Apoptosis , 1996, The Journal of Biological Chemistry.

[22]  M. Jäättelä,et al.  Heat-shock proteins protect cells from monocyte cytotoxicity: possible mechanism of self-protection , 1993, The Journal of experimental medicine.

[23]  Shai Shaham,et al.  The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1β-converting enzyme , 1993, Cell.

[24]  M. Werner.  Stopping death cold. , 1996, Structure.

[25]  C. Borner,et al.  Bcl-2 interrupts the ceramide-mediated pathway of cell death. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[26]  M. Whyte,et al.  ICE/CED-3 proteasesin apoptosis. , 1996, Trends in Cell Biology.

[27]  Jong-il Kim,et al.  T Cell Lymphoma in Transgenic Mice Expressing the HumanHsp70Gene , 1996 .

[28]  G. Li,et al.  Thermal response of rat fibroblasts stably transfected with the human 70-kDa heat shock protein-encoding gene. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[29]  A. Chinnaiyan,et al.  Molecular Ordering of the Cell Death Pathway , 1996, The Journal of Biological Chemistry.

[30]  D. Parsell.  Heat shock proteins and stress tolerance. , 1994 .

[31]  D. Brenner,et al.  Ceramide Activates the Stress-activated Protein Kinases (*) , 1995, The Journal of Biological Chemistry.

[32]  T. Tan,et al.  Persistent Activation of c-Jun N-terminal Kinase 1 (JNK1) in γ Radiation-induced Apoptosis (*) , 1996, The Journal of Biological Chemistry.

[33]  D R Ciocca,et al.  Heat shock protein hsp70 in patients with axillary lymph node-negative breast cancer: prognostic implications. , 1993, Journal of the National Cancer Institute.

[34]  M. Gossen,et al.  Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. , 1994, Molecular and cellular biology.

[35]  S. Mercille,et al.  Induction of apoptosis in nutrient‐deprived cultures of hybridoma and myeloma cells , 1994, Biotechnology and bioengineering.

[36]  Y. Hannun,et al.  prICE: a downstream target for ceramide-induced apoptosis and for the inhibitory action of Bcl-2. , 1996, The Biochemical journal.

[37]  L. Bourget,et al.  Use of a dicistronic expression cassette encoding the green fluorescent protein for the screening and selection of cells expressing inducible gene products. , 1997, BioTechniques.

[38]  Z. Werb,et al.  Correlation between synthesis of heat shock proteins and development of thermotolerance in Chinese hamster fibroblasts. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[39]  Y. Lazebnik,et al.  Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE , 1994, Nature.

[40]  M. Clarke,et al.  The Bcl-2 family of proteins: regulators of cell death and survival. , 1994, Trends in cell biology.

[41]  N. Miyasaka,et al.  Ceramide induces apoptosis via CPP32 activation , 1996, FEBS letters.

[42]  J. Buchner.  The biology of heat shock proteins and molecular chaperones: edited by R.I. Morimoto, A. Tisslères and C. Georgopoulos, Cold Spring Harbor Laboratory Press, 1994. $97.00 (vii + 610 pages) ISBN 0 87969 427 0 , 1994 .

[43]  A. Abe,et al.  Ceramide formation during heat shock: a potential mediator of alpha B-crystallin transcription. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[44]  W. Telford,et al.  Quantification of apoptotic events in pure and heterogeneous populations of cells using the flow cytometer. , 1995, Methods in cell biology.

[45]  M. Gossen,et al.  Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[46]  G. Natoli,et al.  Activation of SAPK/JNK by TNF Receptor 1 Through a Noncytotoxic TRAF2-Dependent Pathway , 1997, Science.

[47]  G. Li,et al.  Heat shock protein hsp70 protects cells from thermal stress even after deletion of its ATP-binding domain. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[48]  D. Green,et al.  The killer and the executioner: how apoptosis controls malignancy. , 1995, Current opinion in immunology.

[49]  H. Horvitz,et al.  Mechanisms and functions of cell death. , 1991, Annual review of cell biology.

[50]  Matthias Mann,et al.  FLICE, A Novel FADD-Homologous ICE/CED-3–like Protease, Is Recruited to the CD95 (Fas/APO-1) Death-Inducing Signaling Complex , 1996, Cell.

[51]  John Calvin Reed.  Bcl-2 and the regulation of programmed cell death , 1994, The Journal of cell biology.

[52]  D. Nicholson.  ICE/CED3-like Proteases as Therapeutic Targets for the Control of Inappropriate Apoptosis , 1996, Nature Biotechnology.

[53]  S. Spiegel,et al.  Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate , 1996, Nature.

[54]  A. Culhane,et al.  Apoptosis: molecular regulation of cell death. , 1996, European journal of biochemistry.

[55]  S. Srinivasula,et al.  In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[56]  S. H. Kim,et al.  T cell lymphoma in transgenic mice expressing the human Hsp70 gene. , 1996, Biochemical and biophysical research communications.

[57]  D. Dix,et al.  Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[58]  C. Ling,et al.  Apoptosis in heat-induced cell killing: the protective role of hsp-70 and the sensitization effect of the c-myc gene. , 1996, Radiation research.

[59]  T G Cotter,et al.  Heat shock proteins increase resistance to apoptosis. , 1996, Experimental cell research.

[60]  R. Johnson,et al.  Heat shock proteins and thermotolerance; a comparison of induction kinetics. , 1982, The British journal of radiology.

[61]  Z. Oltvai,et al.  Checkpoints of dueling dimers foil death wishes , 1994, Cell.

[62]  Michael E. Greenberg,et al.  Opposing Effects of ERK and JNK-p38 MAP Kinases on Apoptosis , 1995, Science.

[63]  L. Zon,et al.  Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis , 1996, Nature.

[64]  A. Strasser,et al.  Bcl-2 and thermotolerance cooperate in cell survival. , 1995, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[65]  R. Morimoto,et al.  Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with HDJ‐1. , 1995, The EMBO journal.

[66]  N. Marceau,et al.  Synthesis and degradation of heat shock proteins during development and decay of thermotolerance. , 1982, Cancer research.

[67]  M. Jäättelä,et al.  Major heat shock protein hsp70 protects tumor cells from tumor necrosis factor cytotoxicity. , 1992, The EMBO journal.

[68]  J. Hickman,et al.  Drug-target interactions: only the first step in the commitment to a programmed cell death? , 1991, British Journal of Cancer.

[69]  J. Landry,et al.  Heat shock resistance conferred by expression of the human HSP27 gene in rodent cells , 1989, The Journal of cell biology.

[70]  Michael Karin,et al.  Dissection of TNF Receptor 1 Effector Functions: JNK Activation Is Not Linked to Apoptosis While NF-κB Activation Prevents Cell Death , 1996, Cell.

[71]  A. Anel,et al.  CPP32 inhibition prevents Fas‐induced ceramide generation and apoptosis in human cells , 1996, FEBS letters.

[72]  N. Thornberry,et al.  Apopain/CPP32 cleaves proteins that are essential for cellular repair: a fundamental principle of apoptotic death , 1996, The Journal of experimental medicine.

[73]  P. Amiri,et al.  Requirement of an ICE-Like Protease for Induction of Apoptosis and Ceramide Generation by REAPER , 1996, Science.

[74]  D. D. Mosser,et al.  The DNA-binding activity of the human heat shock transcription factor is regulated in vivo by hsp70 , 1993, Molecular and cellular biology.

[75]  Muneesh Tewari,et al.  Yama/CPP32β, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase , 1995, Cell.

[76]  R. Morimoto,et al.  Stress proteins in biology and medicine , 1991 .

[77]  S. Grant,et al.  Induction of apoptotic DNA damage and cell death by activation of the sphingomyelin pathway. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[78]  F. Hartl.  Molecular chaperones in cellular protein folding , 1996, Nature.

[79]  S. Lindquist,et al.  18 Heat Shock Proteins and Stress Tolerance , 1994 .

[80]  Roger Y. Tsien,et al.  Improved green fluorescence , 1995, Nature.

[81]  Sharad Kumar,et al.  ICE-like proteases in apoptosis. , 1995, Trends in biochemical sciences.

[82]  D. D. Mosser,et al.  Induced thermotolerance to apoptosis in a human T lymphocyte cell line , 1992, Journal of cellular physiology.