Heat Shock Protein 70 Inhibits Apoptosis Downstream of Cytochrome c Release and Upstream of Caspase-3 Activation

Heat shock protein 70 (HSP70) has been shown to act as an inhibitor of apoptosis. We have also observed an inhibitory effect of HSP70 on apoptotic cell death both in preheated U937 and stably transfected HSP70-overexpressing U937 (U937/HSP70) cells. However, the molecular mechanism whereby HSP70 prevents apoptosis still remains to be solved. To address this issue, we investigated the effect of HSP70 on apoptotic processes in an in vitro system. Caspase-3 cleavage and DNA fragmentation were detected in cytosolic fractions from normal cells upon addition of dATP, but not from preheated U937 or U937/hsp70 cells. Moreover, the addition of purified recombinant HSP70 to normal cytosolic fractions prevented caspase-3 cleavage and DNA fragmentation, suggesting that HSP70 prevents apoptosis upstream of caspase-3 processing. Because cytochrome c was still released from mitochondria into the cytosol by lethal heat shock despite prevention of caspase-3 activation and cell death in both preheated U937 and U937/hsp70 cells, it was evident that HSP70 acts downstream of cytochrome c release. Results obtained in vitrowith purified deletion mutants of HSP70 showed that the carboxyl one-third region (from amino acids 438 to 641) including the peptide-binding domain and the carboxyl-terminal EEVD sequence was essential to prevent caspase-3 processing. From these results, we conclude that HSP70 acts as a strong suppressor of apoptosis acting downstream of cytochrome c release and upstream of caspase-3 activation. Heat shock protein 70 (HSP70) has been shown to act as an inhibitor of apoptosis. We have also observed an inhibitory effect of HSP70 on apoptotic cell death both in preheated U937 and stably transfected HSP70-overexpressing U937 (U937/HSP70) cells. However, the molecular mechanism whereby HSP70 prevents apoptosis still remains to be solved. To address this issue, we investigated the effect of HSP70 on apoptotic processes in an in vitro system. Caspase-3 cleavage and DNA fragmentation were detected in cytosolic fractions from normal cells upon addition of dATP, but not from preheated U937 or U937/hsp70 cells. Moreover, the addition of purified recombinant HSP70 to normal cytosolic fractions prevented caspase-3 cleavage and DNA fragmentation, suggesting that HSP70 prevents apoptosis upstream of caspase-3 processing. Because cytochrome c was still released from mitochondria into the cytosol by lethal heat shock despite prevention of caspase-3 activation and cell death in both preheated U937 and U937/hsp70 cells, it was evident that HSP70 acts downstream of cytochrome c release. Results obtained in vitrowith purified deletion mutants of HSP70 showed that the carboxyl one-third region (from amino acids 438 to 641) including the peptide-binding domain and the carboxyl-terminal EEVD sequence was essential to prevent caspase-3 processing. From these results, we conclude that HSP70 acts as a strong suppressor of apoptosis acting downstream of cytochrome c release and upstream of caspase-3 activation. inhibitor of apoptosis heat shock protein stress-activated protein kinase/c-Jun N-terminal kinase cytokine response modifier A dithiothreitol poly(ADP-ribose)polymerase propidium iodide phosphate-buffered saline p-nitroanilide 1,4-piperazinediethanesulfonic acid Apoptosis, which is characterized by cell shrinkage, membrane blebbing, nuclear breakdown, and DNA fragmentation, is indispensable for embryo development, tissue homeostasis, and regulation of the immune system (1Steller H. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2431) Google Scholar, 2White E. 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The that HSP70 can inhibit apoptosis downstream of cytochrome c release, but upstream of caspase-3 and that the carboxyl-terminal region containing the peptide-binding domain is to inhibit caspase-3 activation. The were from Molecular and heart cytochrome c were obtained from The was obtained from The or to were obtained from The molecular for were obtained from for was from for were from the c and cytochrome were from and Molecular and were obtained from and Molecular and were from U937 cells were in with and in a containing cells were with and in a at the heat shock to HSP70 was for and lethal heat shock were for or To a cell of and J.C. Cell. 1998; PubMed Scopus Google were transfected into the cell by C. R. Howard Science. PubMed Scopus Google Scholar). cells were and the cells were in the containing The cells were as a and for The viral was as T. Nature. PubMed Scopus Google Scholar). the viral was from the of cell and through a The viral with was with cells in a and for at The cells were then with a was to the to the transfected cells. The was for which the cells were as a and the cells were for the of cell cells were with and with Molecular in for at The cells were then with a Caspase-3 was to the The cells were with of cell on for the was for caspase-3 of protein was to of cell and with of and of at for were at in a The was as C. Kim N. J. R. Wang X.D. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google with cells were with and in of A with inhibitors on for the cells were by in a The cell was by for at and the was at in a was at and vitro apoptosis were to the of Liu C. Kim N. J. R. Wang X.D. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). from mice were with and in and by of a The were through of at for in a at The were in and at and at in The human HSP70 was into deletion mutants of HSP70 were and into as and recombinant were into were at to an of and proteins were induced at with for HSP70 and its deletion proteins were purified through a by the fractions of cell proteins were A. To the of DNA into in the in vitro apoptosis of of cytosolic and were at for in the of A of and was to the and at DNA was obtained by on a and UV DNA fragmentation was as G. S. Nature. 1996; 381: PubMed Scopus Google with cells were with cells of for and with cells were to lethal heat shock for different and to for were in a and were in a S. The cell were with and on were at for the were to a and the were in and and for The from was by The DNA fragmentation was as DNA fragmentation a lethal heat shock cells were to and cytochrome was at were at for at and the cell were with and with of A containing on for the cells were with of a the was at the were at for at The were at as cytosolic The were with with and on were at for The fractions and the cytosolic fractions were for with an c of caspase-3 in the in vitro system was as of from preheated or HSP70-overexpressing cells were with in the of or and for at the of the were and of of were with for were to and the proteins were to The were with for at and then with for at The were with and then for at with G. was the To in an cell cell was from cells with lethal heat shock, and then with as We the effect of on heat in U937 cells. A heat shock treatment that not apoptosis not was to HSP70 the heat the of HSP70 in the preheated cells was to that of the control cells DNA fragmentation was observed in cells to lethal heat shock, the preheated cells, in to the control cells, showed a of to DNA fragmentation DNA fragmentation in preheated cells was HSP70 was induced by a heat To which in the apoptotic is by we an in vitro apoptosis system on the of to apoptosis in an containing cytochrome c C. Kim N. J. R. Wang X.D. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). from control and preheated cells were with and The of apoptosis activation was by the processing of and the fragmentation of the of into the and of caspase-3 was with the for this which in the from control cells, was in the from preheated cells fragmentation of the DNA was in the from preheated cells, DNA fragmentation in the from control cells Because the of cells to heat shock can the of different heat shock proteins A. Cell. Res. 1996; PubMed Scopus Google Scholar), we HSP70-overexpressing cells to address the of HSP70 inhibits apoptosis in the in vitro system. The of HSP70 in U937/hsp70 cells was in control cells and was to that of preheated cells the of DNA fragmentation was observed in U937/hsp70 cells to lethal heat shock the U937/hsp70 cells also showed a of to DNA fragmentation in to the cells to obtained with preheated cells. and the of vitro apoptosis from U937/hsp70 cells. In to caspase-3 activation and DNA fragmentation were also prevented in cytosolic of U937/hsp70 cells as shown in preheated cells. result that HSP70 blocks caspase-3 cleavage in heat apoptosis. function of HSP70 is a because we have observed in a different cell these cells HSP70 by heat shock or also showed an of caspase-3 and DNA fragmentation in response to a lethal heat shock with control cells not this the that HSP70 is a inhibitor of apoptosis acting upstream of caspase-3 activation. a of the of HSP70 to function as an inhibitor of apoptosis, the of recombinant HSP70 on caspase-3 activation and DNA fragmentation were HSP70 protein was purified a system. containing recombinant HSP70 and cytosolic from control U937 cells were vitro and for caspase-3 activation. recombinant HSP70 caspase-3 cleavage in with of DNA fragmentation was also observed in containing recombinant HSP70 but not in containing that HSP70 prevents the activation of with apoptosis in vitro system. c acts as an important molecule at the of apoptosis release from mitochondria to the activation of which then procaspase-3 into its in apoptosis. To address the of lethal heat shock initiates apoptosis cytochrome c release, the effect of heat shock on cytochrome c release was U937 cells were with lethal heat shock, cytochrome c release was by c was in cytosol lethal heat shock Because cells also showed cytochrome c release by lethal heat shock not this that cytochrome c participates in the executioner phase of apoptotic cell death cascade in response to heat we that HSP70 blocks apoptosis at upstream of caspase-3 we HSP70 cytochromec release, which is upstream in the apoptosis To address this the of cytosolic cytochromec was in preheated U937 and U937/hsp70 cells with lethal heat shown in HSP70 effect on the release of cytochrome c in lethal heat cells. Because cytochrome c an membrane protein R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4277) Google Scholar), was not detected in the cytosolic it was that was of mitochondria in the cytosolic To the of the caspase-3 and in preheated U937 and U937/hsp70 cells the cytochromec in the we observed caspase-3 and in lethal cells cytochrome c release by lethal heat shock, the of procaspase-3 and were in both preheated U937 and cells in to normal U937 and cells, the caspase-3 capable of was in the Caspase-3 in both preheated U937 and cells upon to lethal heat shock were To HSP70 the cells from caspase-3 was cytochrome c release, cell was also observed in the of cell and were by in control cells and in preheated U937 cells that were in lethal heat shock In the of preheated cells, the of or cells or was in to the The of apoptotic cell death in U937/hsp70 cells was also with that of cells by shown in U937/hsp70 cells showed apoptosis cells. of the in were in a with preheated U937 or U937/hsp70 cells, of the of HSP70 to cytochrome HSP70 with caspase-3 activation and cell To which of HSP70 might be in its deletion of HSP70 were The in the HSP70 proteins and shown in A. HSP70 and proteins were purified as proteins a and the purified proteins were by The purified recombinant proteins were to an in system to the of the HSP70 proteins on caspase-3 activation. and proteins prevented caspase-3 cleavage as the HSP70 protein In and proteins the to inhibit caspase-3 activation that the carboxyl one-third region of HSP70 acids including the peptide-binding domain and EEVD is to prevent the activation of caspase-3 in HSPs molecular that essential for the and assembly of The proteins and from to HSP70 from and to of HSP70 to cells has been shown to be a of of apoptosis D.D. Martin L.H. J. Cell. Physiol. 1992; 151: 561-570Crossref PubMed Scopus (142) Google Scholar, 30Mailhos C. Howard M.K. Latchman D.S. Neuroscience. 1993; 55: 621-627Crossref PubMed Scopus (154) Google Scholar, 31Jäättelä M. Wissing D. Bauer P.A. Li G.C. EMBO J. 1992; 11: PubMed Scopus Google Scholar, C. A. M. T. J. Clin. Invest. 1995; 95: PubMed Scopus Google Scholar, A. Cell. 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