Research ArticleStress Response

IRE1 prevents endoplasmic reticulum membrane permeabilization and cell death under pathological conditions

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Science Signaling  23 Jun 2015:
Vol. 8, Issue 382, pp. ra62
DOI: 10.1126/scisignal.aaa0341
  • Fig. 1 ER stress induces ER membrane permeabilization.

    (A) Immunoblot analysis of GRP94 and GRP78 (ER luminal), VAPB (ER membrane), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (cytosolic) in cytosolic and membrane fractions of wild-type (WT) and DKO MEFs treated with tunicamycin (TM) or thapsigargin (TG) or untreated (Untx). (B) Left: Immunoblot analysis of GRP94 and GRP78 (ER luminal), VAPB (ER membrane), and GAPDH (cytosolic) in cytosolic and membrane fractions of WT, DKO MEFs, and DKO MEFs rescued with WT-Bak (DKO + Bak) treated with tunicamycin or untreated. Right: Quantification of cytosolic GRP78 in WT, DKO MEFs, and DKO MEFs rescued with Bak (DKO + Bak) treated with tunicamycin or untreated. (C) Immunoblot analysis of GRP94, GRP78, calreticulin (CRT) and protein disulfide isomerase (PDI) (ER lumen), IRE1α and VAPB (ER membrane), and GAPDH in cytosolic and membrane fractions of WT MEFs treated with or without tunicamycin. (D) Upper: Immunoblot analysis of GAPDH and hemagglutinin (HA)–tagged A1AT-NHK mutant expressed in NSC34 cells cultured with or without kifunensine (Kif.) in the presence of cycloheximide (CHX). Lower: Quantitation of A1AT-NHK in immunoblots. (E) Immunoblot analysis of GRP94, GRP78, calreticulin, VAPB, and GAPDH in cytosolic or membrane fraction of WT MEFs treated with tunicamycin with or without kifunensine. White arrowhead, nonspecific signal. (F) Immunoblot analysis of ERDj5 and GAPDH in INS1 832/13 cells transfected with small interfering RNA (siRNA) scramble control (si-Cont) or siRNA against ERDj5 (si-ERDj5). (G) Immunoblot analysis of A1AT-NHK and GAPDH in INS1 832/13 cells transfected with siRNA scramble control or siRNA against ERDj5, treated with cycloheximide or untreated. (H) Quantitation of A1AT-NHK shown in (G). Statistical significance was calculated by Student’s t test. (I) Immunoblot analysis of GRP78, calreticulin, and GAPDH in cytosol of INS1 832/13 cells transfected with siRNA scramble control or siRNA against ERDj5, treated with thapsigargin or untreated. (J) Quantification of cytosolic GRP78 and calreticulin shown in (I). (K) Immunoblot analysis of A1AT-NHK, IRE1α, and GAPDH in whole-cell lysates (WCL), cytosol, or membrane fractions of NSC34 cells overexpressing A1AT-NHK mutant. An arrow shows glycosylated A1AT-NHK, and an arrowhead shows A1AT-NHK deglycosylated by endoglycosidase H (Endo-H). (L) Immunoprecipitation (IP) of cytosolic and membrane fractions of WT MEFs treated with tunicamycin or untreated with anti-GRP78 antibody, followed by immunoblot analysis with anti-ubiquitin (Ub) and anti-GRP78 antibodies. Left panels show input for immunoprecipitation. n = at least 3 biological replicates for (A) to (L). Representative blots are shown. Unless otherwise stated, statistical significance was calculated by one-way analysis of variance (ANOVA) followed by Tukey’s test. *P < 0.05; **P < 0.01; n.s., not significant. Error bars show SD.

  • Fig. 2 IRE1 signaling suppresses ER membrane permeabilization.

    (A) Left: Immunoblot analysis of GRP94, GRP78, calreticulin, VAPB, and GAPDH in cytosolic and membrane fractions of WT, Atf6α knockout (Atf6−/−), Ire1α knockout (Ire1α−/−), and Perk knockout MEFs (Perk−/−) treated with thapsigargin or untreated. Right: Quantification of cytosolic GRP78 in WT, Atf6−/−, Ire1α−/−, and Perk−/− MEFs treated with thapsigargin or untreated. (B) Immunoblot analysis of GRP94 and GRP78 (ER luminal), VAPB and IRE1α (ER membrane), and GAPDH (cytosolic) in cytosolic and membrane fractions of WT, Ire1α−/−, and Ire1α−/− rescued with WT-IRE1α (Ire1α−/−res.), treated with thapsigargin or untreated. (C) Left: Immunoblot analysis of GRP94, GRP78 and calreticulin (ER luminal), VAPB and IRE1α (ER membrane), and GAPDH (cytosolic) in cytosolic and membrane fractions of INS1 832/13 cells transiently transfected with scrambled control siRNA or siRNA against IRE1α (si-IRE1α). Right: Quantification of cytosolic GRP78 and calreticulin. (D) Immunoblot analysis of GRP94, GRP78, calreticulin, VAPB, GAPDH, and IRE1α in the cytosolic or membrane fractions of INS1 832/13 cells transduced with lacZ or WT IRE1α, treated with thapsigargin or untreated. (E) Quantitation of cytosolic GRP78 and calreticulin. (F) Immunoblot analysis of the proapoptotic proteins Bax and Bak, the antiapoptotic proteins Bcl-2 and Bcl-xL, and the proapoptotic BH3-only proteins PUMA, BimEL, Bad, Bid, and Bnip3 in Ire1α+/+ and Ire1α−/− MEFs treated with tunicamycin, thapsigargin, or untreated. n = at least 3 biological replicates for (A) to (F). Representative blots are shown. Statistical significance was calculated by one-way ANOVA followed by Tukey’s test. *P < 0.05; **P < 0.01. Error bars show SD.

  • Fig. 3 Bnip3 induces ER membrane permeabilization.

    (A) Left: Immunoblot analysis of GRP94, VAPB, and GAPDH in cytosolic and membrane fractions of Ire1α+/+ and Ire1α knockout (Ire1α−/−) MEFs transduced with adenovirus expressing lacZ or Bnip3 and then treated with thapsigargin or untreated. Right: Quantitation of cytosolic GRP94. (B) Immunoblot analysis of GRP94, VAPB, and GAPDH in cytosolic and membrane fractions of Ire1α−/− MEFs transduced with adenovirus expressing lacZ or Bnip3 at indicated multiplicity of infection (MOI). (C) Coimmunoprecipitation analysis of endogenous Bnip3 and Bcl-2 in Ire1α+/+ and Ire1α−/− MEFs treated with tunicamycin or untreated. Arrows indicate monomer, black arrowheads indicate dimer, and white arrowheads indicate oligomer. (D) Examples of mitochondria and ER fractions of Ire1α−/− MEFs treated with thapsigargin or untreated. Tomm20 and protein disulfide isomerase were used as mitochondrial and ER markers. (E) Gel filtration analysis of endogenous Bak in the ER fractions of Ire1α−/− MEFs transduced with adenovirus expressing lacZ or Bnip3 treated with or without thapsigargin. M.W., molecular weight. (F) Immunoprecipitation of activated Bak using anti–Bak-NT (N-terminus) antibody from the ER fraction of Ire1α−/− MEFs transduced with adenovirus expressing lacZ or Bnip3 treated with thapsigargin or untreated. (G) Immunoblot analysis of GRP78, calreticulin, VAPB, and GAPDH in cytosolic and membrane fractions of WT, DKO MEFs (DKO), and DKO MEFs rescued with Bak (DKO + Bak), transduced with adenovirus expressing lacZ or Bnip3, and then treated with tunicamycin. (H) Left: Immunoblot analysis of GRP94, calreticulin, and GAPDH in cytosolic fractions of Ire1α−/− MEFs stably expressing shRNA directed against luciferase (shLuc) or Bnip3 treated with thapsigargin or untreated. Right: Quantitation of cytosolic GRP94 and calreticulin. n = at least 3 biological replicates for (A) to (H). Representative blots are shown. n = 3 biological replicates. Statistical significance was calculated by one-way ANOVA followed by Tukey’s test. *P < 0.05. Error bars show SD.

  • Fig. 4 Impaired IRE1 signaling leads to accumulation of Bnip3.

    (A) Left: Immunoblot analysis of GRP94, GRP78, VAPB, phosphorylated JNK (p-JNK), JNK, and GAPDH in cytosolic and membrane fractions of INS1 832/13 cells untreated and/or treated with tunicamycin, STF-083010 (STF), or SP600125 (SP) as indicated. Right: Quantitation of GRP78 in the cytosolic fractions of INS1 832/13 cells untreated and/or treated with tunicamycin, STF-083010, or SP600125 as indicated. (B) Left: Immunoblot analysis of GRP94, calreticulin, GAPDH, and IRE1α in cytosolic and membrane fractions of Ire1α−/− MEFs transduced with adenovirus encoding lacZ, WT IRE1α, K599A-IRE1α, or K907A-IRE1α, treated with thapsigargin or untreated. Right: Quantification of cytosolic calreticulin. (C) Immunoblot analysis of GRP94, GRP78, calreticulin, VAPB, and GAPDH in cytosolic and membrane fractions of MEFs containing floxed TRAF2 alleles, transduced with adenovirus encoding lacZ or Cre recombinase (Cre), treated with tunicamycin or untreated. Statistical significance was determined by one-way ANOVA followed by Tukey’s test. (D) Quantitative PCR of Bnip3 of Ire1α+/+ and Ire1α−/− MEFs treated with tunicamycin or untreated. Gene expression was normalized to β-actin mRNA. (E) Left: Immunogold labeling of Bnip3 in human embryonic kidney (HEK) 293 cells. mito, mitochondria. Right: Immunogold double labeling of Bnip3 and LC3 in HEK293 cells. Bnip3 was labeled with 18-nm gold particles, and LC3 was labeled with 12-nm gold particles. Imaging was performed in two independent experiments. (F) Ire1α+/+ and Ire1α−/− MEFs transduced with adenovirus expressing GFP-LC3 treated with tunicamycin or untreated. Scale bars, 20 μm. Imaging was performed in three independent experiments. (G) Immunoblot analysis of LC3 and GAPDH of Ire1α+/+ and Ire1α−/− MEFs, treated with tunicamycin or untreated. (H) Cycloheximide chase of endogenous Bnip3 in WT, Ire1α knockout (Ire1α−/−), and Atg5 knockout (Atg5−/−) MEFs treated with tunicamycin and cycloheximide. (I) Mean intensities of Bnip3 in WT, Ire1α−/−, and Atg5−/− MEFs treated with tunicamycin and cycloheximide were plotted on a semilog graph. Statistical significance was calculated by one-way ANOVA followed by Dunnett’s test. (J) Immunoblot analysis of Bnip3 and GAPDH in WT MEFs in the absence or presence of rapamycin (Rapa). n = at least 3 biological replicates for (A) to (D) and (G) to (J). Representative blots and images are shown. Unless otherwise indicated, statistical significance was calculated by one-way ANOVA followed by Tukey’s test. *P < 0.05; **P < 0.01. Error bars show SD. The scale bar in the electron microscopic images indicates 100 nm and the scale bars in the confocal images indicate 20 μm.

  • Fig. 5 IRE1 inhibits the initial step of cell death.

    (A) Live-cell imaging of MERO-GFP (excitation, 488 nm) in Ire1α knockout (Ire1α−/−) MEFs treated with tunicamycin (upper panels). The ratio images of 488/405 are displayed in false colors (lower panels). (B) Ratio traces of Ire1α knockout (Ire1α−/−) MEFs treated with tunicamycin. n = 12 cells imaged over three independent experiments. (C) Mitochondrial membrane potential (ΔΨm) as measured by MitoProbe dye in WT MEFs expressing MERO-GFP treated with thapsigargin or untreated. Statistical significance was calculated by one-way ANOVA followed by Tukey’s test. (D) Dual time-lapse imaging of TMRM and MERO-GFP (for example, 488 nm) in Ire1α−/− MEFs treated with tunicamycin for indicated times. Yellow dashed lines indicate the shape of the cell. (E) Time-lapse tracing of the MERO-GFP ratio and TMRM in Ire1α−/− MEFs treated with tunicamycin for the indicated times. (F) Traces of TMRM intensity in Ire1α−/− MEFs treated with tunicamycin during ER membrane permeabilization (EMP). The time point when the MERO-GFP started leaking from ER was set as T = 0. n = 10 cells from three independent experiments. The Wilcoxon signed-rank test was performed to compare the signal intensities at 0, 20, and 40 min to those at −20 min. None of these comparisons were significantly different (P > 0.05). (G) Left: Immunoblot analysis of GRP78, calreticulin, cytochrome C (cyto. C), and GAPDH in cytosol fractions of Ire1α−/− MEFs treated with tunicamycin for the indicated periods. Middle and right: Quantification of cytosolic GRP78 and cytochrome C. Statistical significance was calculated by one-way ANOVA followed by Dunnett’s test. (H) Dual live-cell imaging of MERO-GFP ratio and propidium iodide (PI) in Ire1α−/− MEFs treated with tunicamycin for the indicated times. White dashed lines indicate the shape of the cell experiencing ER membrane permeabilization. (I) The MERO-GFP ratio of each cell shown in (H) at the indicated times. (J) Fates of Ire1α−/− MEFs treated with tunicamycin. n = 30 cells imaged over three independent experiments. (K) Mitochondrial calcium monitored by Rhod-2 and MERO-GFP ratio in WT MEFs treated with thapsigargin. Statistical significance was calculated by Student’s t test. (L) ROS and MERO-GFP ratios in WT MEFs treated with thapsigargin. Statistical significance was calculated by Student’s t test. (M) ROS generation and MERO-GFP ratios in Perk knockout (Perk−/−) MEFs treated with thapsigargin. Statistical significance was calculated by Student’s t test. (N) Induction of mCherry fluorescence driven by the human Chop promoter and MERO-GFP ratios in HEK293 cells treated with thapsigargin. Statistical significance was calculated by Student’s t test. n = at least three biological replicates. Representative blots and images are shown. *P < 0.05; **P < 0.01. Error bars show SD. Scale bars, 20 μm.

  • Fig. 6 ER membrane permeabilization in pathophysiological conditions.

    (A) Immunoblot analysis of calreticulin, GRP94, VAPB, and GAPDH in cytosolic fractions of cerebrums from four mice with TMCAO. # shows the pair of control cerebrum and infarcted cerebrum from the same sample. (B) Left: Immunoblot analysis of GRP78, VAPB, COXIV, and GAPDH in cytosolic and membrane fractions of cardiac tissue with ischemia/reperfusion (I/R) or sham operation. Right: Ratios of cytosolic GRP78 (cyto)/membrane GRP78 of three independent pairs of mice with ischemia/reperfusion or sham operation. Statistical significance was calculated by unequal variance t test. n = 3 mice per each condition. (C) Immunoblot analysis of GRP94, VAPB, and GAPDH in cytosolic and membrane fractions of cerebrums from floxed-WFS1 mice crossed with Nestin-Cre transgenic mice. Each lane represents a separate animal. n = 3 mice per each condition. Representative blots are shown.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/382/ra62/DC1

    Fig. S1. Leakage of ER contents to the cytosol precedes the apoptotic cascade.

    Fig. S2. Phosphorylation of Bnip3.

    Fig. S3. Role of Bnip3 in the leakage of ER calcium to the cytosol.

    Fig. S4. Subcellular localization of Bnip3.

    Fig. S5. Gel filtration of GRP94.

    Fig. S6. Confirmation of attenuation of IRE1 signaling by small-molecule inhibitors.

    Fig. S7. Leakage of ER contents to the cytosol in Xbp1+/+ and Xbp1−/− cells.

    Fig. S8. Time-course analysis of the phosphorylation of JNK and leakage of ER contents.

    Fig. S9. Degradation mechanism of Bnip3.

    Fig. S10. Abundance of Bnip3 and phosphorylation of JNK and LC3-II in wild-type cells and Bax/Bak DKO cells.

    Fig. S11. Live-cell imaging of MERO-GFP–expressing IRE1 knockout cells under ER stress conditions.

    Fig. S12. Time-lapse ratio imaging of ER membrane permeabilization in IRE1 knockout cells.

    Fig. S13. Time-lapse ratio imaging of HEK293 cells expressing cytosolic roGFP under ER stress conditions.

    Fig. S14. Fluorescent imaging of HEK293 cells expressing the mCherry-Chop reporter under ER stress conditions.

    Fig. S15. Scheme of ER membrane permeabilization by ER stress.

  • Supplementary Materials for:

    IRE1 prevents endoplasmic reticulum membrane permeabilization and cell death under pathological conditions

    Kohsuke Kanekura, Xiucui Ma, John T. Murphy, Lihua J. Zhu, Abhinav Diwan, Fumihiko Urano*

    *Corresponding author. E-mail: urano{at}dom.wustl.edu

    This PDF file includes:

    • Fig. S1. Leakage of ER contents to the cytosol precedes the apoptotic cascade.
    • Fig. S2. Phosphorylation of Bnip3.
    • Fig. S3. Role of Bnip3 in the leakage of ER calcium to the cytosol.
    • Fig. S4. Subcellular localization of Bnip3.
    • Fig. S5. Gel filtration of GRP94.
    • Fig. S6. Confirmation of attenuation of IRE1 signaling by small-molecule inhibitors.
    • Fig. S7. Leakage of ER contents to the cytosol in Xbp1+/+ and Xbp1−/− cells.
    • Fig. S8. Time-course analysis of the phosphorylation of JNK and leakage of ER contents.
    • Fig. S9. Degradation mechanism of Bnip3.
    • Fig. S10. Abundance of Bnip3 and phosphorylation of JNK and LC3-II in wild-type cells and Bax/Bak DKO cells.
    • Fig. S11. Live-cell imaging of MERO-GFP–expressing IRE1 knockout cells under ER stress conditions.
    • Fig. S12. Time-lapse ratio imaging of ER membrane permeabilization in IRE1 knockout cells.
    • Fig. S13. Time-lapse ratio imaging of HEK293 cells expressing cytosolic roGFP under ER stress conditions.
    • Fig. S14. Fluorescent imaging of HEK293 cells expressing the mCherry-Chop reporter under ER stress conditions.
    • Fig. S15. Scheme of ER membrane permeabilization by ER stress.

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    Citation: K. Kanekura, X. Ma, J. T. Murphy, L. J. Zhu, A. Diwan, F. Urano, IRE1 prevents endoplasmic reticulum membrane permeabilization and cell death under pathological conditions. Sci. Signal. 8, ra62 (2015).

    © 2015 American Association for the Advancement of Science

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