Endoplasmic Reticulum Stress-induced Apoptosis

Abstract

Endoplasmic reticulum (ER) stress-induced apoptosis has been implicated in the development of multiple diseases. However, the in vivo signaling pathways are still not fully understood. In this report, through the use of genetically deficient mouse embryo fibroblasts (MEFs) and their matched wild-type controls, we have demonstrated that the mitochondrial apoptotic pathway mediated by Apaf-1 is an integral part of ER stress-induced apoptosis and that ER stress activates different caspases through Apaf-1-dependent and -independent mechanisms. In search of the molecular link between ER stress and the mitochondrial apoptotic pathway, we have discovered that in MEFs, ER stress selectively activates BH3-only proteins PUMA and NOXA at the transcript level through the tumor suppressor gene p53. In p53-/- MEFs, ER stress-induced apoptosis is partially suppressed. The p53-independent apoptotic pathway may be mediated by C/EBP homologous protein (CHOP) and caspase-12, as their activation is intact in p53-/- MEFs. In multiple MEF lines, p53 is primarily nuclear and its level is elevated upon ER stress. To establish the role of NOXA and PUMA in ER stress-induced apoptosis, we have shown that, in MEFs deficient in NOXA or PUMA, ER stress-induced apoptosis is reduced. Reversibly, overexpression of NOXA or PUMA induces apoptosis as evidenced by the activation of BAK and caspase-7. Our results provide new evidence that, in MEFs, in addition to PUMA, p53 and NOXA are novel components of the ER stress-induced apoptotic pathway, and both contribute to ER stress-induced apoptosis. Endoplasmic reticulum (ER) stress-induced apoptosis has been implicated in the development of multiple diseases. However, the in vivo signaling pathways are still not fully understood. In this report, through the use of genetically deficient mouse embryo fibroblasts (MEFs) and their matched wild-type controls, we have demonstrated that the mitochondrial apoptotic pathway mediated by Apaf-1 is an integral part of ER stress-induced apoptosis and that ER stress activates different caspases through Apaf-1-dependent and -independent mechanisms. In search of the molecular link between ER stress and the mitochondrial apoptotic pathway, we have discovered that in MEFs, ER stress selectively activates BH3-only proteins PUMA and NOXA at the transcript level through the tumor suppressor gene p53. In p53-/- MEFs, ER stress-induced apoptosis is partially suppressed. The p53-independent apoptotic pathway may be mediated by C/EBP homologous protein (CHOP) and caspase-12, as their activation is intact in p53-/- MEFs. In multiple MEF lines, p53 is primarily nuclear and its level is elevated upon ER stress. To establish the role of NOXA and PUMA in ER stress-induced apoptosis, we have shown that, in MEFs deficient in NOXA or PUMA, ER stress-induced apoptosis is reduced. Reversibly, overexpression of NOXA or PUMA induces apoptosis as evidenced by the activation of BAK and caspase-7. Our results provide new evidence that, in MEFs, in addition to PUMA, p53 and NOXA are novel components of the ER stress-induced apoptotic pathway, and both contribute to ER stress-induced apoptosis. The endoplasmic reticulum (ER) 2The abbreviations used are: ER, endoplasmic reticulum; BFA, brefeldin A; Etop, etoposide; FITC, fluorescein isothiocyanate; MEFs, mouse embryo fibroblasts; PBS, phosphate-buffered saline; PI, propidium iodide; TG, thapsigargin; Tun, tunicamycin; FACS, fluorescence-activated cell sorter; WT, wild-type; CMV, cytomegalovirus; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PUMA, p53-up-regulated modulator of apoptosis; CHOP, C/EBP homologous protein; BH3, BCL-2 homology domain 3.2The abbreviations used are: ER, endoplasmic reticulum; BFA, brefeldin A; Etop, etoposide; FITC, fluorescein isothiocyanate; MEFs, mouse embryo fibroblasts; PBS, phosphate-buffered saline; PI, propidium iodide; TG, thapsigargin; Tun, tunicamycin; FACS, fluorescence-activated cell sorter; WT, wild-type; CMV, cytomegalovirus; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PUMA, p53-up-regulated modulator of apoptosis; CHOP, C/EBP homologous protein; BH3, BCL-2 homology domain 3. is the site for synthesis, folding, modification, and trafficking of secretory and cell-surface proteins. As a major intracellular calcium storage compartment, the ER also plays a critical role toward maintenance of cellular calcium homeostasis. Disruption of these physiological functions by ER stress has been implicated in a wide variety of human diseases, including Alzheimer disease, Parkinson disease, neuronal damage by ischemia, prion disease, cystic fibrosis, and diabetes mellitus (1Kaufman R.J. J. Clin. Investig. 2002; 110: 1389-1398Crossref PubMed Scopus (1072) Google Scholar). ER stress could also be elicited in the cell culture system by pharmacological agents including tunicamycin (Tun), a protein N-glycosylation inhibitor; brefeldin A (BFA), which blocks protein transport from ER to Golgi; and thapsigargin (TG), which blocks ER uptake of calcium by inhibiting the sarcoplasmic/endoplasmic Ca2+-ATPase (SERCA) (2Lee A.S. Trends Biochem. Sci. 2001; 26: 504-510Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar). A variety of ER stresses result in unfolded protein accumulation, which triggers the unfolded protein response (1Kaufman R.J. J. Clin. Investig. 2002; 110: 1389-1398Crossref PubMed Scopus (1072) Google Scholar, 3Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1912) Google Scholar). For survival, the cells induce ER chaperone proteins to alleviate protein aggregation, transiently attenuate translation, and activate the proteasome machinery to degrade misfolded proteins. Nonetheless, under severe and prolonged ER stress, unfolded protein response activates unique pathways that lead to cell death through apoptosis (4Ferri K.F. Kroemer G. Nat. Cell Biol. 2001; 3: E255-E263Crossref PubMed Scopus (1284) Google Scholar). Currently, several pathways have been directly implicated in ER stress-induced apoptosis. For example, the transcription factor CHOP/GADD153 is induced by ER stress at the transcript level, which sensitizes cells to ER stress through down-regulating BCL-2 and activating GADD34 and ERO1α, an ER oxidase (5Marciniak S.J. Yun C.Y. Oyadomari S. Novoa I. Zhang Y. Jungreis R. Nagata K. Harding H.P. Ron D. Genes Dev. 2004; 18: 3066-3077Crossref PubMed Scopus (1441) Google Scholar, 6McCullough K.D. Martindale J.L. Klotz L.O. Aw T.Y. Holbrook N.J. Mol. Cell. Biol. 2001; 21: 1249-1259Crossref PubMed Scopus (1547) Google Scholar). ER stress also activates the ER transmembrane protein kinases type I ER membrane protein kinase (IRE1) and PKR-like ER kinase (PERK), which have been implicated in the activation of the pro-apoptotic c-Jun NH2-terminal kinase (JNK) (7Harding H.P. Zhang Y. Ron D. Nature. 1999; 397: 271-274Crossref PubMed Scopus (2457) Google Scholar, 8Urano F. Wang X. Bertolotti A. Zhang Y. Chung P. Harding H.P. Ron D. Science. 2000; 287: 664-666Crossref PubMed Scopus (2256) Google Scholar). Furthermore, ER stress leads to proteolytic cleavage of caspase-12 (C-12) in mouse and caspase-4 (C-4) in human, both of which localize to the cytoplasmic side of the ER membrane (9Hitomi J. Katayama T. Eguchi Y. Kudo T. Taniguchi M. Koyama Y. Manabe T. Yamagishi S. Bando Y. Imaizumi K. Tsujimoto Y. Tohyama M. J. Cell Biol. 2004; 165: 347-356Crossref PubMed Scopus (726) Google Scholar, 10Nakagawa T. Zhu H. Morishima N. Li E. Xu J. Yankner B.A. Yuan J. Nature. 2000; 403: 98-103Crossref PubMed Scopus (2912) Google Scholar). Evidence is also emerging that there is cross-talk between the ER and the mitochondria. The mitochondria-initiated apoptotic pathway is a major one in mammalian cells and is tightly regulated by BCL-2 family proteins. BAX and BAK are pro-apoptotic members activated by a variety of apoptotic stimuli, leading to oligomerization and insertion into the mitochondrial outer membrane to release cytochrome c (4Ferri K.F. Kroemer G. Nat. Cell Biol. 2001; 3: E255-E263Crossref PubMed Scopus (1284) Google Scholar, 11Griffiths G.J. Dubrez L. Morgan C.P. Jones N.A. Whitehouse J. Corfe B.M. Dive C. Hickman J.A. J. Cell Biol. 1999; 144: 903-914Crossref PubMed Scopus (393) Google Scholar, 12Gross A. Jockel J. Wei M.C. Korsmeyer S.J. EMBO J. 1998; 17: 3878-3885Crossref PubMed Scopus (961) Google Scholar). Cytochrome c binds to Apaf-1 and caspase-9 (C-9), resulting in the activation of and the activation of and and cell evidence that also localize to the ER and are activated in response to ER stress, leading to calcium and (C-12) activation L. T. Korsmeyer S.J. Science. PubMed Scopus Google Scholar, Li C. G. T. Yuan J. J. Cell Biol. PubMed Scopus Google Scholar). In MEFs deficient in BAX or ER cleavage is be by the of However, activate apoptosis from both the and the ER, to be the mitochondria-initiated apoptotic pathways are ER stress-induced apoptosis in this the the molecular between ER stress and of the mitochondrial apoptotic In mitochondria-initiated apoptosis, the activation of members of the BH3-only BCL-2 family which are of apoptotic cell of proteins the which is used for to the members of the BCL-2 family and for apoptosis A. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, H. A. Cell 2002; PubMed Scopus Google Scholar). this of a is under one PUMA, has been as by ER stress in human cells and may contribute to ER stress-induced apoptosis in human cells C. D. A. T. J. Cell Biol. PubMed Scopus Google Scholar). PUMA is both a and of apoptosis. In this report, through the use of genetically deficient MEFs and their matched wild-type controls, we the of ER activation and apoptosis the in In search of of ER stress activation of in addition to PUMA, we NOXA as a novel BH3-only protein that is activated by ER stress at the transcript that the of and by ER stress in MEFs is the tumor suppressor gene p53 and both proteins contribute to ER stress-induced apoptosis. In MEFs, p53 is primarily and its level is elevated in MEFs upon ER stress. ER stress-induced apoptosis is partially in p53-/- MEFs, the activation of and the of intact in p53-/- both and -independent Our results provide new evidence that, in addition to PUMA, p53 and NOXA are novel components of the ER stress-induced apoptotic The of these novel in MEFs to results cell is Cell wild-type and MEFs N. C. J.A. Wang X. J. Dev. Biol. 2000; PubMed Scopus Google by X. Wang of and MEFs E. Y. C. Wang J. J. J. T. J.L. Cell. Full Text Full Text PDF PubMed Scopus Google by G. P. MEFs A. L. F. G. A. Science. PubMed Scopus Google to G. by A. and of and p53-/- MEFs M. F. S. N.J. Cell Scopus Google by D. for and The fibroblasts and cells in and cells to by from MEFs and the and The directly into the and by For the For a and as a and the and The into to PUMA K. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google by K. H. for and of protein by Cell Cell mouse NOXA p53 mouse for MEF mouse for for mouse for human and mouse ER stress TG, Tun, and from from of BAK of the of BAK as Li C. G. T. Yuan J. J. Cell Biol. PubMed Scopus Google Scholar). cells or a of the cells used for The of the cells in in phosphate-buffered for The cells and or in in for the cells fluorescein for The cells to by apoptosis of MEFs by and propidium as C. P. R.J. A.S. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). In cells or in The of the to the the cells and The apoptosis of cells by For cell apoptosis by and cell and cells in PBS, and in for at The cells in for The cells and for at for at at The cells by the of in which a of cells for as apoptotic from cells the the of to which as M. S. P. Li M. A.S. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). by from MEFs and the and and and and The into and by The for and from and and The for human M. 2002; 21: PubMed Scopus Google a from G. C. and the for human M. Holbrook N.J. Mol. Cell. Biol. 13: PubMed Scopus Google a from N. Holbrook for and from as M. S. P. Li M. A.S. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The by a as the The of by the level by that of the that is as cell from or cells in and of for the at for at of protein of the by or and to as C. P. R.J. A.S. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). by in a and to or for or as C. K. P. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the cells in PBS, in for for in at the at and by The cells in propidium and a Apaf-1 an of ER Apaf-1 binds to cytochrome c from the and to the and activates and caspases the activation of Apaf-1 as a major for apoptosis. To the of the mitochondrial apoptotic in ER stress-induced apoptosis, and matched MEFs or ER stress TG, Tun, and of ER stress the of apoptotic cells by and propidium For the cells also Etop, a to apoptosis through the mitochondrial pathway G. 2000; Google Scholar). As shown in there in the of apoptotic cells in MEFs under as these results that apoptotic pathways contribute to cell death the of ER stress that, severe ER stress and for a of Apaf-1 could be To the of cells of TG, Tun, and The of apoptosis by and cell the of to Our results that, under severe ER stress for prolonged of MEFs deficient in Apaf-1 to apoptosis apoptotic as cells for ER stress The for provide evidence that Apaf-1 is an integral part of ER stress-induced cell death in the is also that the of apoptosis in MEFs is not that pathways of Apaf-1 are in to ER stress-induced apoptosis. ER through Apaf-1-dependent and -independent the of Apaf-1 activation ER stress, cell from and their matched MEFs ER stress Tun, and and Etop, under as for and to As shown in for activated by TG, Tun, and BFA, as by the of the of For its in cells the in cells or As a activated both and In MEFs, the activation of both and In activation by and BFA, as evidenced by the of its of Apaf-1 in MEFs, activated and its cleavage The of the ER stress by their to the level of the ER chaperone protein and the of the protein by results that ER stress activates primarily by mitochondrial pathways of activation by ER stress, as and BFA, is and may contribute to apoptosis in MEF The BH3-only PUMA and NOXA at the upon ER BCL-2 family proteins are of as are for the mitochondrial apoptotic the mammalian BH3-only the transcript of and could be induced by a variety of as as and in MEFs TG, Tun, and by The of as a and by of the are shown in A and and the the and in Our results that and transcript not by ER stress or that the and The level of in MEFs not In and by under ER stress and for at The of the is as K. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Zhang L. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). As a their also activated in MEFs and are of p53 and are critical of the apoptotic induced by p53. To the ER stress of and is of and in p53-/- and matched MEFs ER stress Tun, or as a ER stress by activation and by of the are shown in and the results are in that, in MEFs, of by ER stress as the level in p53-/- MEFs. For by TG, Tun, and also a level of activation could be at As a of and in p53-/- MEFs. results that, in MEFs, both and by ER stress is of ER in p53-/- of and by ER stress is p53 in MEFs and p53 has been shown to have a signaling role at the in the of apoptosis P. M. Nat. Cell Biol. 2004; PubMed Scopus Google we the of p53 in ER stress-induced apoptosis by the matched of and p53-/- MEFs. used as a As shown in apoptosis induced by for by in p53-/- MEFs result the of apoptotic cell death a results p53 as a novel of the ER stress-induced apoptotic signaling pathway in MEFs. Nonetheless, the of apoptosis in the p53 cells that pathways are in ER stress-induced apoptosis. ER p53 in MEFs and and of ER stress-induced apoptosis in p53-/- MEFs, we p53 ER stress-induced In MEFs, by of stress in p53 protein As a the level of elevated by ER stress, and the p53 level induced by The of p53 by ER stress is not to this MEF in the matched of and the p53-/- MEFs, p53 by and in the cells The PUMA protein in multiple to K. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Zhang L. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The is which is by and is the of this In MEFs, PUMA protein induced by and a of PUMA in the p53-/- MEFs, p53 that, PUMA by ER stress is mediated by p53 in MEFs, may also p53-independent at a level also to NOXA protein by However, the in MEFs the not the matched of and the p53-/- MEFs, we and activation by ER stress is As shown in and induced and as demonstrated by the of and the of in both and p53-/- MEFs. results that ER stress of and activation in may in part for the p53-independent apoptosis in MEFs. that ER stress results in cytoplasmic of p53 in cell of leading to L. S. D. M. C. Y. A. Genes Dev. 2004; 18: PubMed Scopus Google Scholar). In the of p53 in the MEFs by we that p53 is primarily nuclear the the there in p53 at under to of The MEFs for nuclear of p53 As the p53-/- MEFs not p53 in MEFs in or we the of PUMA by ER stress in MEFs. is that, ER stress results in a is elevated H.P. M. F. Novoa I. Ron D. Cell Dev. Biol. 2002; 18: PubMed Scopus Google Scholar). As shown in the protein level elevated in MEFs ER Tun, for and by the PUMA protein level also elevated under stress MEFs from or of also in cells results that of PUMA by ER stress is of the of MEFs and is of the Apaf-1 To PUMA is for ER stress-induced apoptosis in MEFs, MEFs and its matched to ER or to apoptosis. The of PUMA in the MEFs by As shown in and apoptosis induced by or by in MEFs as the of the matched of and MEFs also that apoptosis by and in and cells both PUMA and NOXA contribute to ER stress-induced apoptosis. of NOXA BAK and and NOXA is induced and for ER stress-induced apoptosis in MEFs, a novel of the ER stress apoptotic signaling To establish the pro-apoptotic role of we a protein in which to the of NOXA E. R. H. J. T. T. T. Taniguchi T. N. Science. 2000; PubMed Scopus Google Scholar). into its by as as of NOXA in the of BAK that by a of BAK activation of NOXA also activated and induced apoptosis, as by overexpression of human PUMA activated and induced apoptosis and of NOXA and PUMA induced apoptosis of NOXA or PUMA is that prolonged ER stress lead to cell apoptosis. novel pathways have been that cells cell death that be by the unfolded protein these the in vivo molecular ER stress-induced apoptosis are Furthermore, is from cell or and results from in to cellular mechanisms. In the as we matched of genetically deficient MEFs to the and of molecular components implicated in the of ER stress-induced apoptosis. In the we have a new a BH3-only protein which in PUMA to induce the apoptotic that the tumor suppressor gene p53 is a novel of the ER stress apoptotic a for the between the ER mitochondrial and nuclear signaling pathways leading to ER stress-induced apoptosis is in we the mitochondrial apoptotic pathway plays an role in ER stress-induced cell A the the mouse embryo cell and the that ER activation and cell death could to Apaf-1 S. H. M. G. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the use of a matched of and MEFs and the of the of cell death to different ER stress at different we provide evidence that the mitochondrial as by plays a major role in ER cell death the of stress as MEFs ER stress-induced activation of and and cell However, there are pathways of Apaf-1 that for the cell death in MEFs. of may one pathway, we that is upon ER stress in both wild-type and MEFs. Our results also that, under in vivo activation of in MEFs, activation of or by ER stress still in these in MEFs, that in vivo upon activates which in activates and as shown in N. K. H. T. Y. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). also that could be activated by in E. S. G. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). However, is to be the major for in in MEFs, there is activation of in is by and Nonetheless, one for the is cell as a that and MEFs in the of in response to Li R. E. EMBO J. 2004; PubMed Scopus Google Scholar). The role of in ER stress-induced cell death has been in multiple both gene and suppressor protein T. Zhu H. Morishima N. Li E. Xu J. Yankner B.A. Yuan J. Nature. 2000; 403: 98-103Crossref PubMed Scopus (2912) Google Scholar, Li C. G. T. Yuan J. J. Cell Biol. PubMed Scopus Google Scholar, N. K. K. T. E. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google there is still the of and in its M. M. G. M. S. 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PubMed Scopus Google Scholar). activation upon ER stress, resulting in ER calcium and the are the of that are by ER that ER stress selectively induces BH3-only proteins PUMA and NOXA at the transcript level and that both and MEFs stress-induced apoptosis. PUMA and NOXA are to mitochondrial and oligomerization of BAK and resulting in mitochondrial apoptosis Wei M.C. S. T. Korsmeyer S.J. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google and ER stress has been to cytochrome c release from the J. L. L. S. T. I. C. 2000; PubMed Scopus Google Scholar). has been shown that overexpression of PUMA or NOXA induces apoptosis K. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Zhang L. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, E. R. H. J. T. T. T. Taniguchi T. N. Science. 2000; PubMed Scopus Google Scholar, S. Wang G. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). 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D. M. C. Y. A. Genes Dev. 2004; 18: PubMed Scopus Google Scholar). In of MEFs that p53 primarily nuclear to of the protein level of p53 or a the of ER stress in the p53 level elevated by of the p53 level in MEFs and cell and ER stress by that, PUMA level is induced by ER stress in of the cell lines, p53 level elevated in MEFs and to a in and the p53 level not elevated in there ER stress not for these the of different cell to the and of p53 upon ER stress and that the the of stress The of PUMA p53 also cell In cell may have multiple pathways to induce PUMA ER stress, a that human cells p53 induce PUMA C. D. A. T. J. Cell Biol. PubMed Scopus Google Scholar, J. C. R.J. Zhu L. T. Sci. S. A. 2001; PubMed Scopus Google and that cells of p53 of PUMA by ER stress. and A. S. In that the ER is emerging as a new site for the of cell death Evidence is that ER stress-induced apoptosis is an factor in to a variety of diseases, in diseases, diabetes and the molecular ER stress-induced apoptosis provide for and may provide for X. G. P. D. A. and G. for MEFs, G. C. N. and K. H. for or and of and H. for the