Translocation of Bim to the Endoplasmic Reticulum (ER) Mediates ER Stress Signaling for Activation of Caspase-12 during ER Stress-induced Apoptosis

Endoplasmic reticulum (ER) stress activates caspase-12 in murine cells, triggering the ER stress-specific cascade for implementation of apoptosis. In C2C12 murine myoblast cells, activation of the cascade occurs without release of cytochrome c from mitochondria, suggesting that the cascade is independent of mitochondrial damage. Stable overexpression of Bcl-xL in C2C12 cells suppressed activation of caspase-12 and apoptosis. In ER-stressed cells, but not in normal cells, Bcl-xL was co-immunoprecipitated with Bim, a pro-apoptotic member of the Bcl-2 family, suggesting that Bcl-xL sequesters Bim, thereby inhibiting the apoptotic signaling. Fractionation of C2C12 cells revealed that ER stress led to translocation of Bim from a dynein-rich compartment to the ER, while stable overexpression of Bcl-xL suppressed accumulation of Bim on the ER. Although the toxic effect of Bim had been previously observed only at the mitochondrial outer membrane, overexpression of a Bim derivative, Bim(ER), targeted at the surface of the ER led to apoptosis. A C2C12 transfectant overexpressing the caspase-12 suppressor protein was resistant to Bim(ER), suggesting that the toxic effect of Bim on the ER is dependent on activation of caspase-12. Knockdown of Bim by RNA interference provided cells resistant to ER stress. These results suggest that translocation of Bim to the ER in response to ER stress is an important step toward activation of caspase-12 and initiation of the ER stress-specific caspase cascade. Endoplasmic reticulum (ER) stress activates caspase-12 in murine cells, triggering the ER stress-specific cascade for implementation of apoptosis. In C2C12 murine myoblast cells, activation of the cascade occurs without release of cytochrome c from mitochondria, suggesting that the cascade is independent of mitochondrial damage. Stable overexpression of Bcl-xL in C2C12 cells suppressed activation of caspase-12 and apoptosis. In ER-stressed cells, but not in normal cells, Bcl-xL was co-immunoprecipitated with Bim, a pro-apoptotic member of the Bcl-2 family, suggesting that Bcl-xL sequesters Bim, thereby inhibiting the apoptotic signaling. Fractionation of C2C12 cells revealed that ER stress led to translocation of Bim from a dynein-rich compartment to the ER, while stable overexpression of Bcl-xL suppressed accumulation of Bim on the ER. Although the toxic effect of Bim had been previously observed only at the mitochondrial outer membrane, overexpression of a Bim derivative, Bim(ER), targeted at the surface of the ER led to apoptosis. A C2C12 transfectant overexpressing the caspase-12 suppressor protein was resistant to Bim(ER), suggesting that the toxic effect of Bim on the ER is dependent on activation of caspase-12. Knockdown of Bim by RNA interference provided cells resistant to ER stress. These results suggest that translocation of Bim to the ER in response to ER stress is an important step toward activation of caspase-12 and initiation of the ER stress-specific caspase cascade. The caspase protease family plays a central role in the execution of apoptosis (1Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1326Crossref PubMed Scopus (6159) Google Scholar, 2Cryns V.L. Yuan J. Genes Dev. 1998; 12: 1551-1570Crossref PubMed Scopus (1161) Google Scholar). It is broadly divided into two groups: initiator caspases, which are activated first and initiate a caspase cascade, and effector caspases, which mainly dismantle cellular structures. Caspases are synthesized as precursor proteins (procaspases). Initiator caspases undergo autoprocessing in response to apoptotic stimuli, while effector caspases are cleaved by initiator caspases for activation. There are three major apoptotic cascades triggered by specific initiator caspases: the death receptor pathway, the mitochondrial pathway, and the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; UPR, unfolded protein response; BH-3, Bcl-2 homology domain-3; RNAi, RNA interference; GFP, green fluorescent protein; MAGE-3, melanoma-associated antigen-3. pathway. These are activated by caspase-8, caspase-9, and caspase-12, respectively (3Mehmet H. Nature. 2000; 403: 29-30Crossref PubMed Scopus (200) Google Scholar, 4Ferri K.F. Kroemer G. Nat. Cell Biol. 2001; 3: E255-E263Crossref PubMed Scopus (1302) Google Scholar). ER stress is caused by the accumulation of unfolded proteins in the ER, which occurs under a variety of conditions (e.g. mutation in secretory proteins or disruption of calcium homeostasis). This type of cellular stress is receiving increased attention because it is considered a cause of pathologically relevant apoptosis (5Ron D. J. Clin. Invest. 2002; 109: 443-445Crossref PubMed Scopus (146) Google Scholar) and is especially implicated in neurodegenerative disorders (6Arridor M. Balch W.E. Nat. Med. 1999; 5: 745-751Crossref PubMed Scopus (256) Google Scholar, 7Mattson M.P. LaFerla F.M. Chan S.L. Leissring M.A. Shepel P.N. Geiger J.D. Trends Neurosci. 2000; 23: 222-229Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). ER stress activates caspase-12 on the surface of the ER, and caspase-12-deficient cells are resistant to ER stress inducers (8Nakagawa T. Zhu H. Morishima N. Li E. Xu J. Yankner B.A. Yaun J. Nature. 2000; 403: 98-103Crossref PubMed Scopus (2953) Google Scholar), indicating that caspase-12 is significant in ER stress-induced apoptosis. However, the mechanism responsible for caspase-12 activation is largely unknown, unlike in the case of both caspase-8 and caspase-9 whose activation mechanisms have been revealed at the molecular level (9Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5154) Google Scholar, 10Wang X. Genes Dev. 2001; 15: 2922-2933Crossref PubMed Scopus (94) Google Scholar). The Bcl-2 family regulates activation of caspase-9. Proteins of the Bcl-2 family have either pro-apoptotic or anti-apoptotic functions (11Tsujimoto Y. J. Cell. Physiol. 2003; 195: 158-167Crossref PubMed Scopus (453) Google Scholar, 12Scorrano L. Korsmeyer S.J. Biochem. Biophys. Res. Commun. 2003; 304: 437-444Crossref PubMed Scopus (635) Google Scholar). The pro-apoptotic members are classified into two groups based on their structure, i.e. BH-3 (Bcl-2 homology domain-3) only proteins (e.g. Bad, Bid, and Bim), and multidomain pro-apoptotic members containing BH-1 through BH-3 (e.g. Bax and Bak). BH-3 only proteins can serve as intracellular death ligands proximal to multidomain pro-apoptotic members (13Huang D.C. Strasser A. Cell. 2000; 103: 839-842Abstract Full Text Full Text PDF PubMed Scopus (901) Google Scholar, 14Puthalakath H. Strasser A. Cell Death Differ. 2002; 9: 505-512Crossref PubMed Scopus (631) Google Scholar). In response to apoptotic stimuli (e.g. death ligands, mitogen withdrawal), BH-3 only proteins translocate to mitochondria and induce conformational changes in Bax and Bak, which activate them. These activated multidomain proteins, in turn, cause an increase in mitochondrial membrane permeability leading to the release of cytochrome c from the mitochondria. The released cytochrome c, together with Apaf-1, activates caspase-9 (15Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2743) Google Scholar, 16Li P. Nijhawan D. Budihardjo I. Srinicasula S.M. Ahamad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6236) Google Scholar), resulting in the activation of downstream effector caspases, e.g. caspase-3. The lack of either Bax or Bak is not sufficient to prevent apoptosis, but double knock-out cells are highly resistant to a variety of apoptotic stimuli including ER stress (17Wei M.C. Zong W.-X. Cheng E. H.-Y. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Crossref PubMed Scopus (3354) Google Scholar). Evidently, Bax and Bak have mutually redundant functions. Anti-apoptotic members of the Bcl-2 family (e.g. Bcl-2 and Bcl-xL) exert their anti-apoptotic actions either by heterodimerization with pro-apoptotic members, especially BH-3 only proteins (18Cheng E.H. Wei M.C. Weiler S. Flavell Lindsten T. Korsmeyer S.J. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), or by mitochondrial membrane N. P. M. T. Kroemer G. PubMed Scopus Google Scholar, S. Y. Y. A. V. H. Y. S. A. 1998; PubMed Scopus Google Scholar, A.J. H. M. Thompson C.B. J. 1999; PubMed Scopus Google Scholar). Although it had been that cytochrome c released from mitochondria is for caspase-9 activation (15Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2743) Google Scholar, 16Li P. Nijhawan D. Budihardjo I. Srinicasula S.M. Ahamad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6236) Google Scholar), have that caspase-12 can activate caspase-9 without cytochrome c release in C2C12 myoblast cells under ER stress N. H. T. Y. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The caspase-9, in turn, activates a downstream caspase as caspase-3. have the of an ER stress-specific caspase cascade of caspase-12, and N. H. T. Y. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). that caspase-9 activation can in an and cytochrome S. H. M. V. G. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In ER stress cytochrome c release J. L. L. I. 2000; PubMed Scopus Google Scholar, Y. N. S. N. S. S. D. Cell. Biol. 2001; PubMed Scopus Google Scholar) by an pathway. it is that the activation of caspase-9 and downstream effector caspases can in ER stress-induced apoptosis either by caspase-12 or by the c N. H. T. Y. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). ER stress-induced caspase activation in C2C12 myoblast cells is independent of cytochrome c release and the mitochondrial pathway, the to activation of caspase-12 without have of to the of pro-apoptotic members of the Bcl-2 family in the activation results suggest that ER stress translocation of Bim, which is with in cells H. S.M. Strasser A. 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S.M. Strasser A. Cell. 1999; 3: Full Text Full Text PDF PubMed Scopus Google Scholar). The of that ER stress the release of ER ER of pro-apoptotic members of the Bcl-2 family are to the ER ER stress-induced apoptosis, especially on the of the intracellular of pro-apoptotic proteins in cells, of C2C12 cells into the membrane and membrane The of or the of was by of proteins the mitochondria, and the ER cytochrome c, and respectively the intracellular of pro-apoptotic proteins in cells, proteins in to In cells, Bim proteins in the membrane in which was The of and in the The multidomain pro-apoptotic proteins Bax and Bak mainly in the and the mitochondrial as been observed in A. J. Cell Biol. 1997; PubMed Scopus Google Scholar). These results suggest that the pro-apoptotic members of the Bcl-2 family are in either mitochondria or the of of proteins, in the ER to cells, that the of pro-apoptotic proteins, for Bim, in the ER of cells ER stress either translocation of Bim to the ER or accumulation of pro-apoptotic Bcl-2 family members on the ER, as had been by C2C12 cells with for and the ER was for The of in the ER not the and not an increase of either cytochrome c or in the ER from the apoptotic cells, suggesting that the ER was not by proteins from The of Bad, Bid, Bak, and Bax either or increased ER stress Bim a in ER and Bim in the ER the of ER stress the of Bim translocation with that of caspase-12 an ER from C2C12 with for translocation of Bim to the ER a of C2C12 cells with The of Bim on the ER with caspase-12 activation This the that the Bim translocation the apoptotic which activates caspase-12 on the surface of ER The translocation of Bim was not in the with which is with that Bcl-xL sequesters Bim released from the thereby ER stress-induced apoptosis. of Bim in the apoptotic conditions used (e.g. with for ER stress apoptosis in of C2C12 cells N. H. T. Y. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Bim translocation as as caspase-12 activation in apoptotic cells, the ER of apoptotic cells from cells, both of which had been with The ER from apoptotic cells at a level with that in the ER of cells and the both not by cytochrome c and not accumulation of Bim was observed in the ER from the apoptotic cells, but only a increase was in the ER in the cells, specific translocation of Bim to the ER in the apoptotic cells to into in the apoptotic cells, while it was largely in the cells suggesting that apoptosis is caspase-12 is activated The Bim translocation and caspase-12 activation that Bim on the ER a of caspase-12 activation. of the to the apoptotic cells and the cells, because both and at These results that activation of caspase-12 in the of the of the UPR, which not stress. of an of Bim on the role of Bim in the of apoptosis, because the of Bim on the ER with the of caspase-12 activation and In the mitochondrial pathway, BH-3 only proteins, which are to mitochondria, activate multidomain proteins and thereby mitochondrial damage. It is that Bim on the ER apoptosis by Bak and which are in the ER Bim on the ER membrane can induce caspase activation and apoptosis, an of Bim whose the from cytochrome of C2C12 cells with the apoptosis death in of the It is that apoptosis by activation of caspase-12, the ER used a stable transfectant of C2C12 that a suppressor of caspase-12. previously a melanoma-associated which activation of N. H. T. Y. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Stable overexpression of cells resistant to Bim(ER), as in These results suggest that the apoptotic by activation of caspase-12. the of BH-3 only proteins on the mitochondrial outer membrane, it is that Bim to the ER conformational changes in Bax and Bak, caspase-12 in an The of was by the stable of Bcl-xL which is with the that mitochondrial Bcl-xL can Bim, it from caspase-12. of Bim in C2C12 to ER the in the level of Bim by ER stress-induced apoptosis. C2C12 cells with either a or a A was in for of of cells either not or with for of that of cells with the apoptosis In to Bim but apoptosis in C2C12 cells These results the that Bim is a major of ER stress-induced apoptosis. that ER stress translocation of Bim to the ER. It is that Bim on the ER as a for caspase-12 activation ER stress-induced apoptosis. This is by the both translocation of Bim and the activation of caspase-12 Bim targeted to the ER apoptosis, of a caspase-12 suppressor protein apoptosis, and of Bim by provided cells resistant to ER stress. BH-3 only proteins and caspase not been for by caspase-8 X. Budihardjo I. H. Wang X. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, H. Zhu H. Xu Yuan J. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). It is that Bim activates caspase-12 on the ER It is that the pro-apoptotic of Bim in the ER is the multidomain pro-apoptotic members of the Bcl-2 family (e.g. Bax and as in the case of mitochondrial translocation of BH-3 only proteins (13Huang D.C. Strasser A. Cell. 2000; 103: 839-842Abstract Full Text Full Text PDF PubMed Scopus (901) Google Scholar, 14Puthalakath H. Strasser A. Cell Death Differ. 2002; 9: 505-512Crossref PubMed Scopus (631) Google Scholar). the of Bax and Bak in ER stress-induced apoptosis been W.-X. Li G. Lindsten T. Yuan J. Thompson C.B. J. Cell Biol. 2003; PubMed Scopus Google Scholar, Lindsten T. Ross A.J. MacGregor G.R. Thompson C.B. Genes Dev. 2001; 15: PubMed Scopus Google Scholar, L. Cheng E.H. T. Korsmeyer S.J. Science. 2003; PubMed Scopus Google Scholar). to the conformational changes and of activation of caspase-12 W.-X. Li G. Lindsten T. Yuan J. Thompson C.B. J. Cell Biol. 2003; PubMed Scopus Google Scholar), a for activation not been that Bim is a for the molecular ER stress and activation. A is activates caspase-12. It is or not the effect of on caspase-12 is results the first that translocation of Bim to the ER can ER stress-induced apoptosis. The in of Bim translocation ER stress-induced apoptosis on the lack of cytochrome c release from mitochondria in C2C12 cells under ER stress cytochrome c release in activation of the caspase family, and the cells the apoptotic Bim translocation to the ER can The molecular mechanism on the release of Bim from the is triggered by either or toxic not been H. S.M. Strasser A. Cell. 1999; 3: Full Text Full Text PDF PubMed Scopus Google Scholar). the of a for Bim translocation in both the and the pathway. of apoptosis in C2C12 cells, the was is in The of apoptotic by to the of the an at which the of the Bim or the at the with the to anti-apoptotic proteins the Bim thereby inhibiting the pro-apoptotic The of to the ER the Bim and Bim(ER), because is with the ER which is in the In to the of Bim or the of protein the of apoptosis by either or (e.g. or Bim and the are both released from the and the is to H. S.M. Strasser A. Cell. 1999; 3: Full Text Full Text PDF PubMed Scopus Google Scholar). to of the is in of the Bim It is that of Bim in a and that in the activation of In the case of the Bim protein was to to the ER, as a The of the in the of the pro-apoptotic of have that Bim is important in ER stress-induced apoptosis. However, results not that BH-3 only proteins Bim not role in ER stress-induced apoptosis. These BH-3 only proteins can in to induce apoptosis, because the BH-3 only proteins by anti-apoptotic members of the Bcl-2 family as Bcl-xL L. Korsmeyer S.J. Biochem. Biophys. Res. Commun. 2003; 304: 437-444Crossref PubMed Scopus (635) Google Scholar). observed that pro-apoptotic members of the Bcl-2 family in C2C12 cells in the ER, ER to under ER stress It is that activation is in the case the of BH-3 only proteins and Bim the level that is to anti-apoptotic proteins and sufficient to activate BH-3 only proteins on the ER the level of the pro-apoptotic or the to ER stress. Although specific to Bim caused a significant in ER stress-induced apoptosis, the was This to the of Bim in the BH-3 only proteins, or proteins that can by activate caspase-12 in a a as to cytochrome c release from mitochondria is not ER stress-induced apoptosis in C2C12 ER apoptotic stimuli (e.g. or can mitochondrial and cytochrome c release in cells that cytochrome c release can N. H. T. Y. J. 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