BAX inhibitor-1 regulates autophagy by controlling the IRE1α branch of the unfolded protein response
2011; Springer Nature; Volume: 30; Issue: 21 Linguagem: Inglês
10.1038/emboj.2011.318
ISSN1460-2075
AutoresKaren Castillo, Diego Rojas‐Rivera, Fernanda Lisbona, Benjamı́n Caballero, Melissa Nassif, Felipe A. Court, Sebastian Schuck, Consuelo Ibar, Peter Walter, Jimena Sierralta, Álvaro Glavic, Claudio Hetz,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle16 September 2011free access BAX inhibitor-1 regulates autophagy by controlling the IRE1α branch of the unfolded protein response Karen Castillo Karen Castillo Center for Molecular Studies of the Cell, Department of Cellular and Molecular Biology, Institute of Biomedical Sciences, University of Chile, Santiago, Chile Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile Search for more papers by this author Diego Rojas-Rivera Diego Rojas-Rivera Center for Molecular Studies of the Cell, Department of Cellular and Molecular Biology, Institute of Biomedical Sciences, University of Chile, Santiago, Chile Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile Search for more papers by this author Fernanda Lisbona Fernanda Lisbona Center for Molecular Studies of the Cell, Department of Cellular and Molecular Biology, Institute of Biomedical Sciences, University of Chile, Santiago, Chile Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile Search for more papers by this author Benjamín Caballero Benjamín Caballero Center for Molecular Studies of the Cell, Department of Cellular and Molecular Biology, Institute of Biomedical Sciences, University of Chile, Santiago, Chile Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile Search for more papers by this author Melissa Nassif Melissa Nassif Center for Molecular Studies of the Cell, Department of Cellular and Molecular Biology, Institute of Biomedical Sciences, University of Chile, Santiago, Chile Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile Search for more papers by this author Felipe A Court Felipe A Court Millennium Nucleus for Regenerative Biology, Department of Physiological Sciences, Faculty of Biology, P. Catholic University of Chile, Santiago, Chile Search for more papers by this author Sebastian Schuck Sebastian Schuck Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA, USA Search for more papers by this author Consuelo Ibar Consuelo Ibar Center for Genome Regulation, Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile Search for more papers by this author Peter Walter Peter Walter Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA, USA Search for more papers by this author Jimena Sierralta Jimena Sierralta Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile Search for more papers by this author Alvaro Glavic Alvaro Glavic Center for Genome Regulation, Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile Search for more papers by this author Claudio Hetz Corresponding Author Claudio Hetz Center for Molecular Studies of the Cell, Department of Cellular and Molecular Biology, Institute of Biomedical Sciences, University of Chile, Santiago, Chile Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA, USA Search for more papers by this author Karen Castillo Karen Castillo Center for Molecular Studies of the Cell, Department of Cellular and Molecular Biology, Institute of Biomedical Sciences, University of Chile, Santiago, Chile Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile Search for more papers by this author Diego Rojas-Rivera Diego Rojas-Rivera Center for Molecular Studies of the Cell, Department of Cellular and Molecular Biology, Institute of Biomedical Sciences, University of Chile, Santiago, Chile Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile Search for more papers by this author Fernanda Lisbona Fernanda Lisbona Center for Molecular Studies of the Cell, Department of Cellular and Molecular Biology, Institute of Biomedical Sciences, University of Chile, Santiago, Chile Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile Search for more papers by this author Benjamín Caballero Benjamín Caballero Center for Molecular Studies of the Cell, Department of Cellular and Molecular Biology, Institute of Biomedical Sciences, University of Chile, Santiago, Chile Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile Search for more papers by this author Melissa Nassif Melissa Nassif Center for Molecular Studies of the Cell, Department of Cellular and Molecular Biology, Institute of Biomedical Sciences, University of Chile, Santiago, Chile Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile Search for more papers by this author Felipe A Court Felipe A Court Millennium Nucleus for Regenerative Biology, Department of Physiological Sciences, Faculty of Biology, P. Catholic University of Chile, Santiago, Chile Search for more papers by this author Sebastian Schuck Sebastian Schuck Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA, USA Search for more papers by this author Consuelo Ibar Consuelo Ibar Center for Genome Regulation, Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile Search for more papers by this author Peter Walter Peter Walter Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA, USA Search for more papers by this author Jimena Sierralta Jimena Sierralta Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile Search for more papers by this author Alvaro Glavic Alvaro Glavic Center for Genome Regulation, Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile Search for more papers by this author Claudio Hetz Corresponding Author Claudio Hetz Center for Molecular Studies of the Cell, Department of Cellular and Molecular Biology, Institute of Biomedical Sciences, University of Chile, Santiago, Chile Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA, USA Search for more papers by this author Author Information Karen Castillo1,2, Diego Rojas-Rivera1,2, Fernanda Lisbona1,2, Benjamín Caballero1,2, Melissa Nassif1,2, Felipe A Court3, Sebastian Schuck4, Consuelo Ibar5, Peter Walter4, Jimena Sierralta2, Alvaro Glavic5 and Claudio Hetz 1,2,6 1Center for Molecular Studies of the Cell, Department of Cellular and Molecular Biology, Institute of Biomedical Sciences, University of Chile, Santiago, Chile 2Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile 3Millennium Nucleus for Regenerative Biology, Department of Physiological Sciences, Faculty of Biology, P. Catholic University of Chile, Santiago, Chile 4Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA, USA 5Center for Genome Regulation, Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile 6Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA, USA *Corresponding author. Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115, USA or Independencia 1027, Faculty of Medicine, Institute of Biomedical Sciences, University of Chile, Santiago, Chile. Tel.: +56 2 9786506; Fax: +56 2 9786871; E-mail: [email protected] or [email protected] The EMBO Journal (2011)30:4465-4478https://doi.org/10.1038/emboj.2011.318 Correction(s) for this article BAX inhibitor-1 regulates autophagy by controlling the IRE1α branch of the unfolded protein response02 November 2021 BAX inhibitor-1 regulates autophagy by controlling the IRE1α branch of the unfolded protein response01 June 2017 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Both autophagy and apoptosis are tightly regulated processes playing a central role in tissue homeostasis. Bax inhibitor 1 (BI-1) is a highly conserved protein with a dual role in apoptosis and endoplasmic reticulum (ER) stress signalling through the regulation of the ER stress sensor inositol requiring kinase 1 α (IRE1α). Here, we describe a novel function of BI-1 in the modulation of autophagy. BI-1-deficient cells presented a faster and stronger induction of autophagy, increasing LC3 flux and autophagosome formation. These effects were associated with enhanced cell survival under nutrient deprivation. Repression of autophagy by BI-1 was dependent on cJun-N terminal kinase (JNK) and IRE1α expression, possibly due to a displacement of TNF-receptor associated factor-2 (TRAF2) from IRE1α. Targeting BI-1 expression in flies altered autophagy fluxes and salivary gland degradation. BI-1 deficiency increased flies survival under fasting conditions. Increased expression of autophagy indicators was observed in the liver and kidney of bi-1-deficient mice. In summary, we identify a novel function of BI-1 in multicellular organisms, and suggest a critical role of BI-1 as a stress integrator that modulates autophagy levels and other interconnected homeostatic processes. Introduction Macroautophagy, here referred to as autophagy, is a highly conserved and regulated process involved in the catabolism of cytoplasmic components that are recycled to maintain energy production and macromolecule synthesis. Autophagy involves the encapsulation of cargoes into double-membrane vesicles (autophagosome), which fuse with lysosomes forming the autolysosomes where cargoes are degraded (Levine and Kroemer, 2008). Under nutrient starvation, autophagy maintains energy homeostasis, but it also regulates tissue remodelling during development, and catalyses the removal of harmful or superfluous cellular organelles, aggregation-prone proteins, and intracellular pathogens (Mizushima et al, 2008; He and Klionsky, 2009). Furthermore, emerging evidence indicates that altered autophagy also contributes to a number of inflammatory and neurodegenerative diseases, in addition to cancer and diabetes (Levine and Kroemer, 2008; Wong and Cuervo, 2010). Autophagy-related (ATG) genes regulate different sequential steps in the autophagy process (Maiuri et al, 2007b; He and Klionsky, 2009), starting with the formation of a protein kinase-autophagy regulatory complex and a lipid kinase-signalling complex. ATG proteins, including a class III PI-3-kinase complex, are involved in vesicle nucleation, which is positively regulated by Beclin-1 (He and Levine, 2010). A key event in autophagosome formation is the conversion of microtubule-associated protein 1 light chain 3 (LC3-I) into the LC3-II form through its lipidation. Monitoring LC3 flux through the autophagolysosomal pathway is the current gold standard to monitor autophagy activity (Klionsky et al, 2008; Tanida et al, 2008). A complex inter-relationship operates between autophagy and apoptosis signalling pathways, where the BCL-2 protein family plays a central role in mediating the cross talk of both processes (Hetz and Glimcher, 2008; Wei et al, 2008b; Pattingre et al, 2009; Chen and Debnath, 2010). For example, the anti-apoptotic BCL-2 and BCL-XL proteins negatively regulate autophagy by binding to and inhibiting Beclin-1 (Pattingre et al, 2005). Pro-apoptotic BH3-only proteins (Maiuri et al, 2007a) or BCL-2 phosphorylation by cJun-N terminal kinase (JNK; Pattingre et al, 2009) antagonize BECLIN-1/BCL-2 interactions, enhancing autophagy. This autophagy regulatory network is proposed to operate at the endoplasmic reticulum (ER) membrane (Pattingre et al, 2005). Although the BCL-2 family of proteins has an essential role in apoptosis and autophagy in mammals and C. elegans, the function of these proteins in other model organisms where autophagy is physiologically relevant, such as fly and yeast, is not clear. This is based on the fact that BCL-2 family homologues have not been described in yeast and that the two BCL-2 family members identified in Drosophila melanogaster (BCL-2/Buffy and BAX/Debcl) have unclear roles in programmed cell death (Sevrioukov et al, 2007; Galindo et al, 2009). Furthermore, their apoptosis activities are restricted to specific conditions (Quinn et al, 2003; Wu et al, 2010). Interestingly, a genetic screening using a fly cell line identified Debcl and Buffy as possible pro-autophagy regulators (Hou et al, 2008). Bax inhibitor-1 (BI-1), also known as transmembrane BAX inhibitor motif containing 6 (TMBIM6), is a highly conserved cell death regulator and its sequence is present in mammals, insects, plants, yeasts, viruses, and other species (Chae et al, 2003; Huckelhoven, 2004; Reimers et al, 2008). BI-1 is an ER-located protein containing six transmembrane regions with anti-apoptotic functions, involved in the suppression of intrinsic cell death mediated by ER calcium release (Xu and Reed, 1998; Xu et al, 2008), ER stress (Chae et al, 2004; Lisbona et al, 2009), and ischaemia (Bailly-Maitre et al, 2006; Dohm et al, 2006; Krajewska et al, 2011). At the mechanistic level, BI-1 has been shown to influence the steady state of ER calcium levels (Westphalen et al, 2005; Kim et al, 2008; Xu et al, 2008; Ahn et al, 2010). BI-1 inhibits the activity of the ER stress sensor inositol requiring kinase 1 α (IRE1α) by a direct interaction (Lisbona et al, 2009; Bailly-Maitre et al, 2010). Interestingly, ER stress is a particularly efficient stimulus for autophagy (Hoyer-Hansen and Jaattela, 2007). ER stress is caused by the accumulation of incorrectly folded proteins in the ER lumen, engaging an adaptive reaction known as the unfolded protein response (UPR) (Hetz and Glimcher, 2009). In mammals, the UPR signals through the activation of three transmembrane proteins where IRE1α is the most conserved stress sensor (Ron and Walter, 2007). IRE1α is a kinase/endoribonuclease that, upon activation, initiates the splicing of the mRNA encoding the transcription factor X-Box-binding protein 1 (XBP-1), converting it into a potent activator of UPR target genes (Hetz and Glimcher, 2009). IRE1α also regulates autophagy levels during ER stress by binding to the adaptor protein TNF-receptor associated factor-2 (TRAF2), followed by the downstream activation of JNK (Ogata et al, 2006; Ding et al, 2007). XBP-1 levels could also modulate autophagy in mammals and fly cells (Arsham and Neufeld, 2009; Hetz et al, 2009). In this study, we have identified a new function of the evolutionary conserved protein BI-1 in the control of autophagy. Our results indicate that BI-1 negatively modulates the kinetics and amplitude of autophagy fluxes in cells undergoing nutrient starvation. BI-1 expression controlled autophagy by regulating JNK activation, possibly due to a mechanism involving IRE1α and TRAF2. This study may give additional clues about the physiological integration between apoptosis and autophagy in different species. Results BI-1 deficiency leads to accumulation of acidic vesicles and autophagosomes To explore the possible impact of BI-1 on the lysosomal pathway, we first visualized the content of acidic compartments using lysotracker staining in BI-1 wild-type (WT) and deficient (KO) mouse embryonic fibroblasts (MEFs). We also monitor ER morphology expressing a Cytochrome b5–EGFP fusion protein. Unexpectedly, augmented number of acidic vesicles with larger size was observed in BI-1-deficient cells cultured in standard media (Figure 1A). In contrast, the distribution pattern of the ER network in BI-1 WT and KO cells was similar under this condition, suggesting a specific effect on acidic compartments (Figure 1A). Figure 1.Increased accumulation of autophagosomes and lysosomes in BI-1-deficient cells. (A) BI-1 WT and KO MEFs cells were stably transduced with retroviruses expressing cytochrome b5–GFP to visualize the ER (green). Then cells were stained with lysotracker (red) and observed with a confocal microscope. Nucleus was stained with Hoechst (blue). Scale bar: 30 μm. (B) BI-1 WT and KO MEFs cells stimulated with EBSS for 3 h to induce autophagy. Acidic vesicles were visualized with a confocal microscope after lysotracker staining. Data represent the results of three independent experiments. Scale bar: 50 μm. (C) (a) Epifluorescence imaging of BI-1 KO cells stained with lysotracker; (b) electron micrograph of the same field of cells shown in (a); (c–e), magnification of lysotracker-positive vesicles in BI-1 KO cells exposed to EBSS for 3 h and visualized with a fluorescent microscope (c) and the same field subsequently imaged by EM (d). Overlapping images are presented (e). Left panel: analysis of vesicular structures by EM with morphologies resembling early (f), intermediate (g) and late (h) autophagy vesicles. (D) Left panel: the distribution of endogenous LC3 was monitored by immunofluorescence and confocal microscopy in BI-1 WT and KO MEFs cells at basal conditions (NT) or after exposure to EBSS for 3 h. Scale bar: left 15 μm and right 10 μm. Right panel: quantification of the number cells containing three or more LC3-positive vesicles (N=160 cells). Mean and standard deviation are presented (N=4). Student's t-test was used to analyse statistical significance, **P<0.001 and *P<0.01. (E) BI-1 WT and KO cells were transiently transfected with an expression vector for a monomeric-tandem LC3–RFP–GFP construct. After 24 h, cells were exposed to EBSS for 3 h. Autophagy fluxes were monitored in living cells by visualizing the distribution of LC3-positive dots in the red and green channels using a confocal microscope. Scale bar: 10 μm. Right panel: quantification of the ratio between red and yellow dots per cell is presented. Mean and standard error of the analysis of 15 cells are shown. Download figure Download PowerPoint Recent reports indicate that autophagy involves enhanced lysosome biogenesis (Settembre et al, 2011) and redistribution (Korolchuk et al, 2011). We exposed cells to nutrient starvation to induce autophagy by incubations in Earle's Balanced Salt Solution (EBSS) or serum/glucose-free RPMI media. These treatments led to a stronger redistribution and accumulation of large acidic vesicles in BI-1 KO cells when compared with WT control cells (Figure 1B), an effect reverted by reconstituting cells with a BI-1–GFP expression vector (Supplementary Figure S1A). Then, we analysed the morphology of these acidic vesicles by electron microscopy (EM) using the combined colocalization between fluorescence and EM images. A low magnification of BI-1 KO MEFs cells subjected to nutrient starvation and stained with lysotracker is shown in Figure 1C (panels a–e) (see also Supplementary Figure S1B and C). Ultrastructural EM analysis of the larger lysotracker-positive vacuoles revealed the appearance of vesicular structures with distinct characteristics, including multivesicular membranes (Figure 1C, panel f), lysosomes with intracellular content (Figure 1C, panel g), and late stage autophagy vesicles which accumulated in clusters (Figure 1C, panel h), which is an indirect indicative of enhanced autophagy (Korolchuk et al, 2011). To directly monitor the possible impact of BI-1 on autophagosome formation, we then visualized the presence of LC3-positive vesicles in cells cultured in standard media or after nutrient starvation. Increased accumulation of LC3-positive dots, that also present dots of higher size, was observed in BI-1 KO cells compared with control cells in both conditions (Figure 1D). BI-1 deficiency enhances the kinetic and amplitude of autophagy flux The accumulation of autophagosomes in BI-1-deficient cells could have two paradoxical interpretations: increased autophagy activity or impaired fusion of autophagosome vesicles, decreasing their flow through the autophagy pathway. Thus, we performed LC3-II flux assays using inhibitors of lysosomal activity and western blot analysis. First of all, we monitored in kinetic experiments the levels of LC3-II in BI-1 WT and KO cells under resting or nutrient starvation conditions (Figure 2A). Quantification revealed a faster induction of LC3-II in BI-1-deficient cells (0–1 h) in relation to a loading control after stimulation with EBSS or with serum/glucose-free RPMI media (Figure 2B). In addition, an ∼2.5-fold increase in LC3-II levels was observed after 3 h of nutrient starvation compared with control cells (Figure 2B). Although, a slight upregulation of LC3-I form was observed, no changes on lc3 mRNA levels were detected in BI-1-deficient cells by real-time PCR compared with control cells (Supplementary Figure S1D). Figure 2.BI-1 deficiency enhances autophagy flux. (A) BI-1 WT and KO MEFs were treated with EBSS (left panel) or glucose/serum-free RPMI media (right panel) for the indicated time points. Then, levels of LC3 were determined by western blot analysis. LC3-I and LC3-II forms are indicated. Hsp90 levels were assessed as loading control. (B) Quantification of LC3-II levels relative to Hsp90 expression was performed in several experiments performed as presented in (A). (C) Cells were pre-treated with a cocktail of lysosomal inhibitors (200 nM bafilomycin A1, 10 μg/ml pepstatin, and E64d; left panel) or 10 mM 3-methyladenine (3-MA; right panel) for 12 h and then exposed to starvation. LC3 levels monitored by western blot (D) and quantification of LC3-II levels relative to Hsp90 were performed in the experimental conditions described in (C). (E) Basal autophagy flux was monitored in cells treated with a cocktail of lysosomal inhibitors (Lys. Inh.) in the presence of normal cell culture media. Right panel: quantification of independent experiments is presented. (F) BI-1 KO cells were stably transduced with retroviruses expressing BI-1–GFP or empty vector, and then levels of LC3-II were assessed over time by western blot analysis after exposure to EBSS. Right panel: as control, the levels of BI-1–GFP were monitored by western blot. Hsp90 levels were used as loading control. In (B, D and E) mean and standard deviation are presented. Two-way ANOVA was applied to analyse statistical significance. In parenthesis, the number of independent experiments for each time point is indicated. Student's t-test was also used in (E) to analyse the statistical significance between each time point (*P<0.001). In (B, D and E), normalization was performed as a ratio with the LC3-II/Hsp90 normalized levels from non-treated BI-1 WT cells. Download figure Download PowerPoint We then measured LC3-II flux through the autophagy pathway by exposing cells to nutrient deprivation in the presence or absence of a cocktail of lysosomal inhibitors (200 nM bafilomycin A1, 10 μg/ml pepstatin and 10 μg/ml E64d). A further accumulation of LC3-II in BI-1-deficient cells was observed when lysosomal activity was inhibited (Figure 2C and D), indicating enhanced autophagy activity in the absence of BI-1. In addition, blocking PI3K by the treatment with 10 mM 3-methyladenine (3-MA) abrogated the accumulation of LC3-II in cells undergoing nutrient starvation (Figure 2C and D). Taken together, these data suggest that BI-1 negatively controls the magnitude and the kinetic of autophagy in response to starvation. As shown in Figure 2A, we also observed a slight accumulation of LC3-II in BI-1 KO cells grown in normal cell culture media. This prompts us to perform an LC3 flux assay in the presence of nutrients. A higher accumulation of LC3-II was observed in BI-1 KO cells compared with control cells after inhibition of lysosomal activity under this condition (Figure 2E), implying enhanced basal autophagy levels in cells lacking BI-1. Finally, as additional control, we reconstituted BI-1 KO cells using retroviruses to express BI-1WT–GFP, which as expected reduced LC3-II levels in these cells after nutrient starvation (Figure 2F). To examine autophagy flux in the absence of lysosomal inhibitors, we transiently expressed a tandem monomeric LC3–RFP–GFP construct (Klionsky et al, 2008) in BI-1 WT and KO MEFs. Using this dynamic autophagy sensor, we detected a large accumulation of LC3-red dots in BI-1 KO cells undergoing nutrient starvation, which is indicative of a flux of LC3 from autophagosomes (colocalization RFP and GFP) to autophagolysosomes (quenched GFP signal by acidic lysosomal environment) (Figure 1E). Colocalization of EM images with fluorescent images confirmed the presence of autophagolysosomes vesicles in BI-1 KO cells (Supplementary Figure S1E). Additionally, we monitored the flux of the autophagy substrate p62/SQSTM1 during the starvation period. Although a slight increase in basal p62/SQSTM1 expression was observed in BI-1 KO cells by western blot analysis, enhanced p62/SQSTM1 degradation was detected under conditions of nutrient starvation over time (Supplementary Figure S2A, B and D), and confirmed by immunofluorescence analysis of p62/SQSTM1 distribution (Supplementary Figure S2C). BI-1 deficiency improves cell survival under nutrient starvation conditions To functionally address the cellular consequences of augmented autophagy in BI-1-deficient cells, we determined the rate of cell survival under nutrient starvation using several complementary assays. We first monitored relative cell number with the MTS assay over time. After exposing cells to EBSS, we observed enhanced viability of BI-1 KO cells compared with control cells (Figure 3A, left panel). In sharp contrast, BI-1-deficient cells were more susceptible to ER stress-induced apoptosis triggered by different concentrations of tunicamycin (Figure 3A, right panel), consistent with previous reports describing a downstream regulation of the apoptosis machinery (Chae et al, 2004; Bailly-Maitre et al, 2006). Figure 3.BI-1 deficiency increases cell survival under nutrient starvation conditions. (A) Left panel: BI-1 WT and KO MEFs cells were incubated in EBSS, and then cell viability was monitored using the MTS assay. Right panel: a similar experiment was performed after treating cells with the indicated concentration of tunicamycin for 24 h. Mean and standard deviation are presented of triplicates representative of three independent experiments. (B) BI-1 WT and KO cells were treated with three different starvation stimuli for 6 and 24 h. Cell death was determined after propidium iodide (PI) staining and FACS analysis. In addition, cells were treated with 100 ng/ml tunicamycin (Tm) for 24 h. Mean and standard deviation are presented of one experiment performed in triplicates. (C) Cells were exposed to EBSS for 6 h or 1 μg/ml Tm for 2 h, and then replated in normal cell culture media. After 5 days, cell viability was monitored by staining with crystal violet. Data are representative of three independent experiments. (D) BI-1 WT MEFs were stably transduced with lentiviral expression vectors to deliver an shRNA against bi-1 mRNA or control mRNA (luciferase shRNA). Cell survival was measured after treatment of cells as described in (B). Mean and standard deviation of an experiment made by triplicate, representative of two independent experiments. (E) BI-1 KO cells were stably transduced with retroviruses expressing BI-1–GFP or empty vector, and then exposed to EBSS for indicated time points. Cell viability was monitored after PI staining by FACS. Mean and standard deviation are presented of triplicates representative of two independent experiments. Download figure Download PowerPoint We then quantified the levels of cell death using propidium iodide (PI) staining and FACS analysis. By stimulating with three different conditions of nutrient starvation, we detected a dramatic protection of BI-1 KO cells after 6 h of treatment (Figure 3B). These effects were observed even after prolonged starvation (24 h of treatment, Figure 3B). Again, treatment of cells with the ER stress agent tunicamycin led to enhanced cell death of BI-1-deficient cells when compared with control WT cells (Figure 3B). Then, we monitored the ability of cells to adapt to nutrient starvation using transient exposure to the stimuli and a replating assay. Using this method, we observed a higher ability of BI-1-deficient cells to survive under nutrient starvation conditions (Figure 3C), which contrasted with their high susceptibility to tunicamycin treatment. Finally, to confirm these results, we knocked down the expression of BI-1 in WT cells using shRNA and stable lentiviral transduction. This strategy leads to a decrease of ∼75% of the bi-1 mRNA levels monitored by real-time PCR (not shown) as described before (Lisbona et al, 2009). Targeting bi-1 partially protected cells against nutrient starvation, and slightly enhanced the susceptibility to tunicamycin toxicity (Figure 3D). We also stably overexpressed BI-1–GFP using retroviral transduction in BI-1 KO MEFs and then monitored the susceptibility of these cells to nutrient starvation (Figure 3E). An enhanced susceptibility to cell death was observed in BI-1 expressing cells after exposure to nutrient starvation (Figure 3E). Taken together, these data suggest a direct correlation between the effect of BI-1 expression on cell survival under nutrie
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