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• W2012166934 abstract "Endoplasmic reticulum (ER) stress caused by misfolded proteins or cytotoxic drugs can kill cells and although activation of this pathway has been implicated in the etiology of certain degenerative disorders its mechanism remains unresolved. Bim, a proapoptotic BH3-only member of the Bcl-2 family is required for initiation of apoptosis induced by cytokine deprivation or certain stress stimuli. Its proapoptotic activity can be regulated by several transcriptional or posttranslational mechanisms, such as ERK-mediated phosphorylation, promoting its ubiquitination and proteasomal degradation. We found that Bim is essential for ER stress-induced apoptosis in a diverse range of cell types both in culture and within the whole animal. ER stress activates Bim through two novel pathways, involving protein phosphatase 2A-mediated dephosphorylation, which prevents its ubiquitination and proteasomal degradation and CHOP-C/EBPα-mediated direct transcriptional induction. These results define the molecular mechanisms of ER stress-induced apoptosis and identify targets for therapeutic intervention in ER stress-related diseases. Endoplasmic reticulum (ER) stress caused by misfolded proteins or cytotoxic drugs can kill cells and although activation of this pathway has been implicated in the etiology of certain degenerative disorders its mechanism remains unresolved. Bim, a proapoptotic BH3-only member of the Bcl-2 family is required for initiation of apoptosis induced by cytokine deprivation or certain stress stimuli. Its proapoptotic activity can be regulated by several transcriptional or posttranslational mechanisms, such as ERK-mediated phosphorylation, promoting its ubiquitination and proteasomal degradation. We found that Bim is essential for ER stress-induced apoptosis in a diverse range of cell types both in culture and within the whole animal. ER stress activates Bim through two novel pathways, involving protein phosphatase 2A-mediated dephosphorylation, which prevents its ubiquitination and proteasomal degradation and CHOP-C/EBPα-mediated direct transcriptional induction. These results define the molecular mechanisms of ER stress-induced apoptosis and identify targets for therapeutic intervention in ER stress-related diseases. Eukaryotic cells have specific signaling pathways and effecter mechanisms to deal with the temporal and developmental variations in endoplasmic reticulum (ER) load (Ron, 2002Ron D. Translational control in the endoplasmic reticulum stress response.J. Clin. Invest. 2002; 110: 1383-1388Crossref PubMed Scopus (684) Google Scholar). The upstream signal that activates these pathways is referred to as ER stress and is defined as an imbalance between the load of resident and transit proteins in the ER and the organelle's ability to process that load. ER stress can be provoked by a variety of patho-physiological conditions, such as ischemia, viral infection, or mutations that impair resident/secretory protein folding (Ron, 2002Ron D. Translational control in the endoplasmic reticulum stress response.J. Clin. Invest. 2002; 110: 1383-1388Crossref PubMed Scopus (684) Google Scholar) and has been implicated as an initiator or contributing factor in a range of diseases, including liver cirrhosis or type 2 diabetes (Rutishauser and Spiess, 2002Rutishauser J. Spiess M. Endoplasmic reticulum storage diseases.Swiss Med. Wkly. 2002; 132: 211-222PubMed Google Scholar). The ER stress response can promote cellular repair and sustained survival by reducing the load of unfolded proteins through global attenuation of protein synthesis and/or upregulation of chaperones, enzymes, and structural components of the ER (Kaufman, 2002Kaufman R.J. Orchestrating the unfolded protein response in health and disease.J. Clin. Invest. 2002; 110: 1389-1398Crossref PubMed Scopus (1032) Google Scholar). When ER stress is overwhelming, cells undergo apoptosis, and although this was initially reported to be mediated by caspase-12 (Nakagawa et al., 2000Nakagawa T. Zhu H. Morishima N. Li E. Xu J. Yankner B.A. Yuan J. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-β.Nature. 2000; 403: 98-103Crossref PubMed Scopus (2832) Google Scholar), this has been challenged (Saleh et al., 2006Saleh M. Mathison J.C. Wolinski M.K. Bensinger S.J. Fitzgerald P. Droin N. Ulevitch R.J. Green D.R. Nicholson D.W. Enhanced bacterial clearance and sepsis resistance in caspase-12-deficient mice.Nature. 2006; 440: 1064-1068Crossref PubMed Scopus (256) Google Scholar), and the mechanism is therefore still unclear. BH3-only proteins constitute a subgroup of proapoptotic members of the Bcl-2 protein family that are essential for initiation of programmed cell death and stress-induced apoptosis in species as distantly related as C. elegans and mice (Huang and Strasser, 2000Huang D.C.S. Strasser A. BH3-only proteins – essential initiators of apoptotic cell death.Cell. 2000; 103: 839-842Abstract Full Text Full Text PDF PubMed Scopus (879) Google Scholar). BH3-only proteins share with each other and the Bcl-2 family at large only the short (9-16 aa) BH3 (Bcl-2 Homology) region, which is essential for their ability to kill cells and bind to prosurvival Bcl-2-like proteins (Huang and Strasser, 2000Huang D.C.S. Strasser A. BH3-only proteins – essential initiators of apoptotic cell death.Cell. 2000; 103: 839-842Abstract Full Text Full Text PDF PubMed Scopus (879) Google Scholar). Apoptosis induced by BH3-only proteins requires a second proapoptotic subgroup of the Bcl-2 protein family, the Bax/Bak-like proteins, which in healthy cells are kept in check by the prosurvival Bcl-2 family members (Wei et al., 2001Wei M.C. Zong W.X. Cheng E.H. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death.Science. 2001; 292: 727-730Crossref PubMed Scopus (3206) Google Scholar). The proapoptotic activity of BH3-only proteins can be regulated by a variety of transcriptional as well as posttranslational control mechanisms (Puthalakath and Strasser, 2002Puthalakath H. Strasser A. Keeping killers on a tight leash: transcriptional and post-translational control of the pro-apoptotic activity of BH3-only proteins.Cell Death Differ. 2002; 9: 505-512Crossref PubMed Scopus (613) Google Scholar). For example, noxa and puma/bbc3 were discovered as p53-inducible genes (Vousden and Lu, 2002Vousden K.H. Lu X. Live or let die: the cell's response to p53.Nat. Rev. Cancer. 2002; 2: 594-604Crossref PubMed Scopus (2597) Google Scholar) and are now known to be essential for DNA damage-induced apoptosis (Jeffers et al., 2003Jeffers J.R. Parganas E. Lee Y. Yang C. Wang J. Brennan J. MacLean K.H. Han J. Chittenden T. Ihle J.N. et al.Puma is an essential mediator of p53-dependent and -independent apoptotic pathways.Cancer Cell. 2003; 4: 321-328Abstract Full Text Full Text PDF PubMed Scopus (736) Google Scholar, Villunger et al., 2003Villunger A. Michalak E.M. Coultas L. Müllauer F. Böck G. Ausserlechner M.J. Adams J.M. Strasser A. p53- and drug-induced apoptotic responses mediated by BH3-only proteins Puma and Noxa.Science. 2003; 302: 1036-1038Crossref PubMed Scopus (1040) Google Scholar). Moreover, upon cytokine withdrawal bim is transcriptionally upregulated in hemopoietic cells by FOXO3A, which in healthy cells is kept in check by PI3K/Akt-mediated phosphorylation (Dijkers et al., 2000Dijkers P.F. Medema R.H. Lammers J.J. Koenderman L. Coffer P.J. Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1.Curr. Biol. 2000; 10: 1201-1204Abstract Full Text Full Text PDF PubMed Scopus (812) Google Scholar). Bim and Bmf can be regulated by their interaction with DLC1 or DLC2 dynein light chains, which causes their sequestration to the microtubular dynein motor complex or the actin-bound myosin V motor complex, respectively (Puthalakath et al., 1999Puthalakath H. Huang D.C.S. O'Reilly L.A. King S.M. Strasser A. The pro-apoptotic activity of the Bcl-2 family member Bim is regulated by interaction with the dynein motor complex.Mol. Cell. 1999; 3: 287-296Abstract Full Text Full Text PDF PubMed Scopus (891) Google Scholar, Puthalakath et al., 2001Puthalakath H. Villunger A. O'Reilly L.A. Beaumont J.G. Coultas L. Cheney R.E. Huang D.C.S. Strasser A. Bmf: a pro-apoptotic BH3-only protein regulated by interaction with the myosin V actin motor complex, activated by anoikis.Science. 2001; 293: 1829-1832Crossref PubMed Scopus (470) Google Scholar). In addition, in growth factor-stimulated cells, Bim levels are kept low by ERK-mediated phosphorylation, which targets it for ubiquitination and proteasomal degradation (Ley et al., 2005Ley R. Ewings K.E. Hadfield K. Cook S.J. Regulatory phosphorylation of Bim: sorting out the ERK from the JNK.Cell Death Differ. 2005; 12: 1008-1014Crossref PubMed Scopus (249) Google Scholar). Gene targeting studies in mice have shown that Bim is a critical regulator of lymphocyte homeostasis, serves as a barrier against autoimmune disease (Bouillet et al., 1999Bouillet P. Metcalf D. Huang D.C.S. Tarlinton D.M. Kay T.W.H. Köntgen F. Adams J.M. Strasser A. Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity.Science. 1999; 286: 1735-1738Crossref PubMed Scopus (1261) Google Scholar), and is required for deletion of autoreactive T (Bouillet et al., 2002Bouillet P. Purton J.F. Godfrey D.I. Zhang L.-C. Coultas L. Puthalakath H. Pellegrini M. Cory S. Adams J.M. Strasser A. BH3-only Bcl-2 family member Bim is required for apoptosis of autoreactive thymocytes.Nature. 2002; 415: 922-926Crossref PubMed Scopus (627) Google Scholar) and B cells (Enders et al., 2003Enders A. Bouillet P. Puthalakath H. Xu Y. Tarlinton D.M. Strasser A. Loss of the pro-apoptotic BH3-only Bcl-2 family member Bim inhibits BCR stimulation-induced apoptosis and deletion of autoreative B cells.J. Exp. Med. 2003; 198: 1119-1126Crossref PubMed Scopus (233) Google Scholar) and shutdown of T cell immune responses (Hildeman et al., 2002Hildeman D.A. Zhu Y. Mitchell T.C. Bouillet P. Strasser A. Kappler J. Marrack P. Activated T cell death in vivo mediated by pro-apoptotic Bcl-2 family member, Bim.Immunity. 2002; 16: 759-767Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). Experiments with cultured cells have shown that Bim is essential for apoptosis induced by certain Bcl-2-inhibitable stress stimuli, including cytokine deprivation or treatment with ionomycin or taxol, but not others, such as stimulation with phorbol ester (Bouillet et al., 1999Bouillet P. Metcalf D. Huang D.C.S. Tarlinton D.M. Kay T.W.H. Köntgen F. Adams J.M. Strasser A. Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity.Science. 1999; 286: 1735-1738Crossref PubMed Scopus (1261) Google Scholar). Here, we report that ER stress, induced by a broad range of cytotoxic stimuli, requires Bim for initiating apoptosis in diverse cell types, both in vitro and even in a pathological setting within the whole animal. ER stress increases Bim levels through CHOP-C/EBPα-dependent transcriptional activation and posttranslationally by protein phosphatase 2A (PP2A)-mediated dephosphorylation, which prevents its ubiquitin-dependent proteasomal degradation. This definition of the ER stress-induced apoptosis signaling pathways identifies potential targets for therapeutic intervention in diseases associated with ER stress. ER stress-induced apoptosis can be inhibited by overexpression of Bcl-2 or one of its homologs (Hitomi et al., 2004Hitomi J. Katayama T. Eguchi Y. Kudo T. Taniguchi M. Koyama Y. Manabe T. Yamagishi S. Bando Y. Imaizumi K. et al.Involvement of caspase-4 in endoplasmic reticulum stress-induced apoptosis and Abeta-induced cell death.J. Cell Biol. 2004; 165: 347-356Crossref PubMed Scopus (707) Google Scholar), indicating that this process is initiated by proapoptotic BH3-only proteins. We therefore treated MCF-7 breast carcinoma-derived cells with thapsigargin (TG), a selective inhibitor of SERCA (seroplasmic endoplasmic reticulum calcium ATPase) (Lewis, 2001Lewis R.S. Calcium signaling mechanisms in T lymphocytes.Annu. Rev. Immunol. 2001; 19: 497-521Crossref PubMed Scopus (676) Google Scholar) or, as a control, with taxol, a microtubule polymerizing agent that induces apoptosis in MCF-7 cells, and analyzed the levels of BH3-only proteins by Western blotting. Interestingly, treatment with thapsigargin but not exposure to taxol caused an increase in Bim protein levels (Figure 1A). Increased levels of bim mRNA were also seen in thapsigargin-treated MCF-7 cells (Figure 1B), but the magnitude of this increase (∼2-fold) was less than the increase seen at the protein level (∼5-fold) (compare Figures 1A and 1B). Thapsigargin-induced upregulation of Bim was also observed in mouse FDC-P1 myeloid cells (Figure 1C), embryonic fibroblasts (MEF) (Figure 1D) and thymocytes (Figure 1E). Moreover, treatment with other drugs that induce ER stress, such as tunicamycin, DTT or ionomycin, also caused a substantial increase in Bim in MCF-7 cells (Figure 1F). Induction of Bim appeared to be specific, because ER stress caused only a minor or no increase in the expression of the related BH3-only proteins Bid or Puma in FDC-P1 (Figure 1C) or MCF-7 cells (Figure 1G). Viral infection constitutes a patho-physiological stimulus that causes ER stress (Rutishauser and Spiess, 2002Rutishauser J. Spiess M. Endoplasmic reticulum storage diseases.Swiss Med. Wkly. 2002; 132: 211-222PubMed Google Scholar) and, remarkably, Bim levels rose in measles infected Vero monkey kidney epithelial cells (Figure 1H), coinciding with induction of the ER stress-induced transcription factor CHOP (see Figure S1 in the Supplemental Data available with this article online). Collectively, these results demonstrate that ER stress induced by diverse cytotoxic drugs or a patho-physiological stimulus causes an increase in bim mRNA and Bim protein levels in diverse cell types. We next used cells from Bim-deficient mice to examine whether this BH3-only protein is essential for ER stress-induced apoptosis. Upon treatment with tunicamycin or thapsigargin, bim−/− thymocytes survived significantly better than their WT counterparts (Figure 2A). Puma has been reported to be critical for ER stress-induced apoptosis of neuronal cells (Reimertz et al., 2003Reimertz C. Kogel D. Rami A. Chittenden T. Prehn J.H. Gene expression during ER stress-induced apoptosis in neurons: induction of the BH3-only protein Bbc3/PUMA and activation of the mitochondrial apoptosis pathway.J. Cell Biol. 2003; 162: 587-597Crossref PubMed Scopus (312) Google Scholar). We therefore also examined puma−/− thymocytes but found only minor resistance to tunicamycin and normal sensitivity to thapsigargin (Figure 2A) or ionomycin (E.M. and A.S., unpublished data). Because we performed many of our biochemical analyses with MCF-7 cells, we wanted to verify that ER stress-induced apoptosis was also Bim-dependent in these cells. Stable transfection with an RNA interference (RNAi) construct (Bouillet et al., 2005Bouillet P. Robati M. Bath M.L. Strasser A. Polycystic kidney disease prevented by transgenic RNA interference.Cell Death Differ. 2005; 12: 831-833Crossref PubMed Scopus (26) Google Scholar) allowed us to generate MCF-7 cells with reduced Bim expression. These Bim-deficient MCF-7 cells were significantly resistant to thapsigargin-induced killing compared to parental cells or cells expressing a control RNAi construct (Figure 2B). However, after 72 hr, a substantial fraction of the Bim-deficient cells had died, although still significantly fewer compared to control cells (Figure 2B). This is likely a consequence of the fact that although the RNAi technique efficiently reduced bim mRNA and Bim protein levels in untreated MCF-7 cells, thapsigargin treatment was still able to increase Bim levels over time (Figures 2C and 2D). Knockdown of Bim expression using stable expression of an RNAi vector also protected Vero cells from apoptosis caused by measles infection (Figures 2E and 2F). These results demonstrate that diverse cell types require Bim for ER stress-induced apoptosis elicited by a broad range of cytotoxic or patho-physiological stimuli. Because Bim is critical for ER stress-induced apoptosis, we examined the mechanisms that stimulate its proapoptotic activity. First, by using an intracellular calcium antagonist and cell surface calcium release activated channel (CRAC) blockers, we showed that calcium is not required for the ER stress-induced Bim upregulation (Figure S2). Second, in MCF-7 cells, ER stress augmented bim mRNA levels by only ∼2-fold, whereas Bim protein levels increased by ∼5-fold (compare Figures 1A and 1B), indicating that only a component of Bim protein induction is due to increased bim transcription. We therefore investigated whether ER stress can regulate Bim levels through a posttranslational mechanism. In contrast to treatment with the chemotherapeutic drug taxol (Puthalakath et al., 1999Puthalakath H. Huang D.C.S. O'Reilly L.A. King S.M. Strasser A. The pro-apoptotic activity of the Bcl-2 family member Bim is regulated by interaction with the dynein motor complex.Mol. Cell. 1999; 3: 287-296Abstract Full Text Full Text PDF PubMed Scopus (891) Google Scholar), ER stress did not result in substantial release of Bim from the dynein motor complex (Figure S3). Bim can also be regulated by ERK-mediated phosphorylation and this has been associated with protection from apoptosis in lymphocytes, fibroblasts, and osteoclasts (Ley et al., 2005Ley R. Ewings K.E. Hadfield K. Cook S.J. Regulatory phosphorylation of Bim: sorting out the ERK from the JNK.Cell Death Differ. 2005; 12: 1008-1014Crossref PubMed Scopus (249) Google Scholar). In five different cell types (MCF-7, thymocytes, FDC-P1, MEF, and Vero), Bim (particularly the BimEL isoform but also BimL) underwent a mobility shift (faster migration on SDS-PAGE) during ER stress (see Figures 1A, 1C–1F, and 3A), but this change did not occur after exposure to taxol (Figure 1A). This prompted us to examine whether ER stress causes a change in Bim phosphorylation. Treatment of protein extracts from healthy MCF-7 cells with λ phosphatase, a Ser/Thr and Tyr phosphatase, but not exposure to the tyrosine phosphatase LAR resulted in mobility shifts of BimEL and BimL that were similar to those observed in Bim from extracts of MCF-7 cells exposed to thapsigargin (Figure 3B). This indicated that ER stress causes a change in Ser/Thr phosphorylation of Bim. This was investigated in detail by separating proteins from extracts of healthy or thapsigargin-treated MCF-7 cells by 2D gel electrophoresis. BimEL and BimL from healthy MCF-7 cells each migrated on 2D gels as at least 5 spots (Figure 3C), indicating the presence of multiple differentially phosphorylated forms of these proteins. Bim immunopurified from MCF-7 cells exposed to thapsigargin or Bim immunopurified from healthy cells and then treated in vitro with λ phosphatase migrated as five spots with different mobility, with the major spot being the least phosphorylated form (migrating toward the cathode; see arrowheads in Figure 3C). Loss of Bim phosphorylation did not appear to be due to a reduction in ERK activity, because Western blot analysis of extracts from MCF-7 cells undergoing ER stress-induced apoptosis found no decrease in ERK phosphorylation (Figure S4). Upon treatment of MCF-7 cells with thapsigargin, Bad, another BH3-only protein that can be regulated by phosphorylation (Puthalakath and Strasser, 2002Puthalakath H. Strasser A. Keeping killers on a tight leash: transcriptional and post-translational control of the pro-apoptotic activity of BH3-only proteins.Cell Death Differ. 2002; 9: 505-512Crossref PubMed Scopus (613) Google Scholar), did not undergo a noticeable change in phosphorylation or migration on SDS-PAGE (Figure 3A). These results demonstrate that Bim is dephosphorylated specifically in response to ER stress. Having established that Bim undergoes dephosphorylation during ER stress induced apoptosis, we wanted to identify the phosphatase responsible. Thapsigargin is known to activate calcineurin (PP2B), a phosphatase that has been implicated in cell killing and dephosphorylation of Bad during calcium flux-induced apoptosis of rat hippocampal neurons (Wang et al., 1999Wang H.-G. Pathan N. Ethell I.M. Krajewski S. Yamaguchi Y. Shibasaki F. McKeon F. Bobo T. Franke T.F. Reed J.C. Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD.Science. 1999; 284: 339-343Crossref PubMed Scopus (931) Google Scholar). However, treatment with FK506 (600 nM; sufficient for blocking calcineurin) had no effect on thapsigargin-induced Bim dephosphorylation or apoptosis in MCF-7 cells (Figure S5 and data not shown). In contrast, in extracts from MCF-7 cells treated with thapsigargin plus okadaic acid (OA) or with OA alone, hyperphosphorylated forms of both BimEL and BimL could be seen (indicated by arrowheads in Figure 4A). Although OA can inhibit both protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), it is much less effective against PP1 (Ki of 147 nM versus 0.032 nM, respectively). In MCF-7 cells, specific inhibition of PP2A has previously been achieved with 1 μM OA (Favre et al., 1997Favre B. Turowski P. Hemmings B.A. Differential inhibition and posttranslational modification of protein phosphatase 1 and 2A in MCF7 cells treated with calyculin-A, okadaic acid, and tautomycin.J. Biol. Chem. 1997; 272: 13856-13863Crossref PubMed Scopus (275) Google Scholar), and we found that as little as 50 nM OA could inhibit thapsigargin-induced Bim dephosphorylation (Figure 4A). PP2A immunoprecipitated from MCF-7 cells undergoing ER stress, but not PP2A isolated from healthy cells, could dephosphorylate immunopurified Bim in vitro (Figure 4B), demonstrating that PP2A acts directly on Bim. Interestingly, in MCF-7 cells treated with OA, we observed not only hyperphosphorylation of Bim but also a dramatic reduction in the levels of BimEL and BimL. This was particularly striking in cells treated with 100 nM OA and appeared to be specific to Bim, because the levels of Bcl-2 and HSP70 did not change (Figure 4A). Similar results were obtained with another PP2A inhibitor, calyculin A, even at concentrations as low as 1 nM (Figure S6). As a control, we performed Northern blot analysis to confirm that OA (100 nM) had no effect on thapsigargin-induced upregulation of bim mRNA in MCF-7 cells (Figure 4C). Collectively, these observations indicate that PP2A dephosphorylates Bim during ER stress-induced apoptosis and thereby increases its levels. Since phosphorylation controls ubiquitination and proteasomal degradation of Bim (Ley et al., 2005Ley R. Ewings K.E. Hadfield K. Cook S.J. Regulatory phosphorylation of Bim: sorting out the ERK from the JNK.Cell Death Differ. 2005; 12: 1008-1014Crossref PubMed Scopus (249) Google Scholar), we investigated whether ER stress-induced dephosphorylation affects Bim turnover. Addition of the proteosome inhibitor PS 341 restored Bim protein levels in MCF-7 (Figure 4D) and FDC-P1 cells (data not shown) treated with thapsigargin plus OA. Moreover, protein half-life analysis demonstrated that Bim turnover was more rapid in healthy MCF-7 cells compared to cells undergoing ER stress (Figure 4E). This indicates that dephosphorylation may inhibit ubiquitin-dependent proteasomal degradation thereby causing Bim accumulation during ER stress. Indeed, immunoprecipitation with anti-Bim antibodies followed by Western blotting using antibodies to Bim or ubiquitin demonstrated that a significant fraction of Bim was ubiquitinated in healthy MCF-7 cells but no ubiquitinated Bim was seen in immunoprecipitates from thapsigargin-treated cells (Figure 4F). The relationship between phosphorylation and ubiquitination of Bim was further examined by exposing immunopurified Bim to a reaction system that supports ubiquitination in vitro. In this assay, only phosphorylated Bim immunopurified from healthy MCF-7 cells could be ubiquitinated, whereas no such modification was observed with Bim purified from cells exposed to thapsigargin (which is mostly dephosphorylated; Figure 4G, bottom left panel) or with λ phosphatase-treated Bim immunopurified from healthy MCF-7 cells (Figure 4G, bottom right panel). These results demonstrate that during ER stress, Bim protein levels rise because PP2A-mediated dephosphorylation renders it refractory to ubiquitination and proteasomal degradation. An ∼2-fold increase in bim transcript levels was observed during ER stress induced apoptosis (see Figures 1B and 4C). This could be attributed to a transcriptional increase rather than a change in mRNA stability, because treatment of MCF-7 cells with actinomycin D blocked thapsigargin-induced accumulation of bim mRNA levels (Figure S7). It has previously been shown that bim is transcriptionally induced by FOXO3A in response to IL-3 withdrawal in a pro-B cell line (Dijkers et al., 2000Dijkers P.F. Medema R.H. Lammers J.J. Koenderman L. Coffer P.J. Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1.Curr. Biol. 2000; 10: 1201-1204Abstract Full Text Full Text PDF PubMed Scopus (812) Google Scholar). In thapsigargin-treated MCF-7 cells, FOXO3A and FOXO1 did, however, not undergo dephosphorylation prior to the time of Bim induction (in fact, FOXO3A appeared to be degraded; see Figure S8), indicating that these transcription factors are not critical for ER stress-induced apoptosis. Consistent with this notion, FOXO3A-deficient thymocytes were normally sensitive to thapsigargin and tunicamycin (Figure 5A). Involvement of the known ER stress-induced transcription factor ATF6 could also be ruled out because Pefabloc, a serine protease inhibitor, which blocks ATF6 activation (Okada et al., 2003Okada T. Haze K. Nadanaka S. Yoshida H. Seidah N.G. Hirano Y. Sato R. Negishi M. Mori K. A serine protease inhibitor prevents endoplasmic reticulum stress-induced cleavage but not transport of the membrane-bound transcription factor ATF6.J. Biol. Chem. 2003; 278: 31024-31032Crossref PubMed Scopus (162) Google Scholar), had no effect on thapsigargin-induced transcriptional induction of bim (data not shown). The transcription factor CHOP is activated by ER stress and a range of other cytotoxic stimuli, including amino acid starvation, γ-irradiation or treatment with DNA damaging drugs (Marciniak et al., 2004Marciniak S.J. Yun C.Y. Oyadomari S. Novoa I. Zhang Y. Jungreis R. Nagata K. Harding H.P. Ron D. CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum.Genes Dev. 2004; 18: 3066-3077Crossref PubMed Scopus (1340) Google Scholar [and references therein]). Since many of these stimuli also upregulate Bim expression (HP, LO'R, and AS, manuscript in preparation) and since we observed concomitant induction of CHOP (plus its heterodimeric partner C/EBPα) and Bim in several cell types undergoing ER stress (see Figures 5B, S9, and S10), we investigated whether the ER stress-induced increase in Bim is mediated by CHOP. Remarkably, although thapsigargin treatment caused a readily detectable increase in bim mRNA levels in thymocytes from WT mice, this was not observed in chop−/− thymocytes (Figure 5C). We therefore investigated whether CHOP can directly upregulate bim transcription. Sequence analysis failed to detect a CHOP binding site within the conventional bim promoter (Bouillet et al., 2001Bouillet P. Zhang L.C. Huang D.C. Webb G.C. Bottema C.D. Shore P. Eyre H.J. Sutherland G.R. Adams J.M. Gene structure alternative splicing, and chromosomal localization of pro-apoptotic Bcl-2 relative Bim.Mamm. Genome. 2001; 12: 163-168Crossref PubMed Scopus (127) Google Scholar), but a perfect CHOP-C/EBPα heterodimer binding site (TGCAAT) was found within the first intron of the mouse and human bim genes (at 447–452 nt from the translation initiation site within the second exon of mouse bim; Figure 5D). This indicated that there might be an alternate promoter within the first intron of the bim gene. Indeed, database searches revealed that many entries of bim transcripts start within the first intron (see legend to Figure S11). Semiquantitative RT-PCR and Q-PCR analysis using primers specific to noncoding exon 1, the internal transcription initiation site and a common 3′ primer binding to coding exon II revealed that both promoters are active in cells undergoing ER stress (Figure S11). To test whether CHOP-C/EBPα can activate this site within the bim gene, we generated a luciferase reporter construct containing 0.8 kb from the conventional promoter, the first noncoding exon and the first intron. Cotransfection of this reporter in combination with CHOP, C/EBPα, or both demonstrated that CHOP together with C/EBPα could activate transcription from this site (Figure 5D). Mutating this putative binding site abolished the induction by CHOP plus C/EBPα (Figure 5D). In addition, electrophoretic mobility shift assays (EMSA) showed that although CHOP or C/EBPα by themselves failed to interact with a radiolabeled probe containing the putative binding site, together these two proteins produced a marked mobility shift, which could be super-shifted with antibodies specific to CHOP or C/EBPα (Figure 5E). No binding was seen using a probe in which the critical residues had been mutated (Figure 5E) and the mobility shift of the radiolabeled WT probe could be prevented by adding a 5-fold molar excess of the cold WT probe, whereas addition of the mutant cold probe had no effect (Figure S12). These results demonstrate that during ER stress CHOP and C/" @default.
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• W2012166934 title "ER Stress Triggers Apoptosis by Activating BH3-Only Protein Bim" @default.
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