Research ArticleCell death

Ca2+-dependent demethylation of phosphatase PP2Ac promotes glucose deprivation–induced cell death independently of inhibiting glycolysis

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Sci. Signal.  09 Jan 2018:
Vol. 11, Issue 512, eaam7893
DOI: 10.1126/scisignal.aam7893
  • Fig. 1 A subset of cancer cell lines is sensitive to glucose deprivation–induced cell death.

    (A) Propidium iodide (PI) exclusion assay using the indicated cell lines cultured in the presence or absence of both 10% serum and 25 mM glucose for 4 hours (U2OS), 6 hours (U251MG), or 8 to 10 hours (remaining cell lines). A representative analysis from three independent experiments is shown. The percentage of PI-positive dead cells is shown. Asterisk (*) indicates cell lines sensitive to glucose deprivation. (B) PI exclusion assay using the indicated cell lines cultured in media containing 10% dialyzed fetal bovine serum (DFBS) with or without 1 mM glucose for 16 hours. Shown is the mean percentage of dead cells ± SD from three independent experiments. (C) Time course of PI exclusion assay in U2OS cells cultured with or without 10% DFBS and 1 mM glucose for the indicated periods. Shown is the mean percentage of dead cells ± SD from three independent experiments. (D) Representative images from phase-contrast microscopy from three independent experiments. Cells were cultured in media containing 10% DFBS with or without 1 mM glucose for 10 hours. Scale bars, 50 μm.

  • Fig. 2 Glucose deprivation–induced cell death is independent of ATP depletion.

    (A) Intracellular adenosine 5′-triphosphate (ATP) amount in U2OS cells treated as indicated for 4 hours. Bars represent the mean (±SD) amount of ATP in each sample relative to the control condition (first bar), quantified from more than three independent experiments. ***P < 0.001, unpaired two-tailed Student’s t test. (B) PI exclusion assay with the indicated cell lines cultured with or without 1 mM 2-deoxy-d-glucose (2-DG) in the absence of glucose for 16 hours. Bars represent the mean percentage of cell death ± SD of three independent experiments. (C) Representative phase-contrast images of U2OS cells from three independent experiments. U2OS cells were treated as indicated for 4 hours. Scale bar, 50 μm.

  • Fig. 3 Glucose deprivation does not induce apoptosis or autophagy.

    (A) Representative Western blotting from three independent experiments of U2OS cells cultured with or without 1 mM glucose for 4 hours. Lysate from ultraviolet (UV)–irradiated U2OS cells serves as a positive control for apoptosis. Z-VAD-FMK was used to inhibit caspase activation. (B) Representative phase-contrast and fluorescent images of three independent experiments. U2OS cells were cultured as indicated for 16 hours and stained with PI before imaging. Z-VAD-FMK was used to inhibit apoptosis. Scale bars, 50 μm. (C) PI exclusion assay with U2OS cells transfected small interfering RNA (siRNA) against the indicated autophagy-associated mRNAs or a control (ctrl) and cultured with or without 1 mM glucose for 4 hours. Shown is the mean percentage of dead cells ± SD from more than three independent experiments. (D) PI exclusion assay with U2OS cells pretreated with or without chloroquine for 12 hours followed by the indicated treatments for 4 hours. Shown is the mean percentage of dead cells ± SD from three independent experiments.

  • Fig. 4 Glucose deprivation induces RIPK1-dependent cell death.

    (A to C) Representative Western blotting of U2OS cells cultured as indicated for 4 hours. Western blotting analysis were performed by regular SDS–polyacrylamide gel electrophoresis (PAGE) (A), Phos-tag SDS-PAGE (B), or regular SDS-PAGE using λ phosphatase–digested lysates (C). All blots are representative of three independent experiments. (D) PI exclusion assay of siRNA-transfected U2OS cells cultured with or without 1 mM glucose for 4 hours. The mean percentage of dead cells ± SD from more than three independent experiments is shown. ***P < 0.001, unpaired two-tailed Student’s t test. (E) Representative phase-contrast and fluorescent images from three independent experiments of U2OS cells cultured as indicated for 16 hours and stained with PI before imaging. Scale bars, 50 μm. (F) Western blotting analysis for RIPK3 in cells cultured with or without 1 mM glucose for 4 hours. Blots are representative of three independent experiments. (G and H) Cells were treated with tumor necrosis factor–α (TNF-α), cycloheximide (CHX), Z-VAD-FMK, and necrostatin-1 (Nec) as indicated for 14 hours. Phase-contrast images are shown (G), and protein lysates were analyzed by Western blotting (H). Data are representative of three independent experiments. Scale bars, 50 μm.

  • Fig. 5 Glucose deprivation induces PP2Ac demethylation.

    (A) Western blotting analysis for PP2Ac in U2OS cells cultured as indicated for 4 hours. NaOH-treated protein extract was used as a positive control for fully demethylated PP2Ac. Blots are representative of three independent experiments. (B and C) Western blotting analysis for PP2Ac in the indicated cell lines treated as indicated for 4 hours. Biological duplicates are shown for each condition. Blots are representative of three independent experiments. (D) Western blotting analysis for PP2Ac in U2OS cells treated as indicated for 4 hours. Blots are representative of three independent experiments. (E) Western blotting analysis for PP2Ac in U2OS cells cultured with various doses of glucose as indicated for 4 hours in the absence of serum. Blots are representative of three independent experiments. (F) Western blotting analysis in U2OS cells cultured with a different dose of glucose as indicated for 4 hours. Blots are representative of three independent experiments.

  • Fig. 6 PP2Ac demethylation is required for glucose deprivation–induced cell death.

    (A) Time course of Western blotting analysis in U2OS cells cultured in the absence of both serum and glucose for the indicated time. Blots are representative of three independent experiments. (B) Western blotting analysis in siRNA-transfected U2OS cells cultured with or without 1 mM glucose for 4 hours. Blots are representative of three independent experiments. (C) Western blotting analysis. U2OS cells were grown with or without cycloheximide in the absence of glucose and serum, for the indicated time. TP53 was used as a positive control for the effect of CHX. Blots are representative of three independent experiments. (D) Western blotting analysis in siRNA-transfected U2OS cells cultured with or without 1 mM glucose for 4 hours. Blots are representative of three independent experiments. (E) Representative phase-contrast images of siRNA-transfected U2OS cells cultured as in (D). Results are representative of three independent experiments. Images were taken by IncuCyte ZOOM System. Scale bar, 50 μm. (F) PI exclusion assay using siRNA-transfected U2OS cells treated as in (D). Shown is the mean percentage of dead cells ± SD from more than three independent experiments. **P < 0.01, *P < 0.05, unpaired two-tailed Student’s t test.

  • Fig. 7 Glucose deprivation induces calcium signaling.

    (A and B) siRNA-transfected U2OS cells were cultured with or without glucose for 4 hours. Protein lysates were analyzed by Western blotting (A). The mean percentage of dead cells ± SD analyzed by PI exclusion assay is shown in (B). **P < 0.01, unpaired two-tailed Student’s t test. Data are from three independent experiments. (C to H) U2OS cells were cultured as indicated for 4 hours and stained with Fluo-4AM for intracellular calcium before imaging. Representative phase-contrast and fluorescent images of cells are shown (C, E, and G; scale bars, 50 μm), and intracellular calcium amounts were quantified (D, F, and H) relative to the control condition (first bar). Data are means ± SD from three independent experiments. ***P < 0.001, unpaired two-tailed Student’s t test. (I to J) Western blotting analysis. U2OS cells were treated as indicated for 4 hours. Blots are representative of three independent experiments. (K) PI exclusion assay of U2OS cells treated as indicated for 4 hours. Bars are mean percentage of dead cells ± SD from three independent experiments.

  • Fig. 8 Glucose deprivation induces plasma membrane depolarization.

    (A) Representative reverse transcription polymerase chain reaction (RT-PCR) for the expression of L-type calcium channel α-1 subunits in U2OS cells from three independent experiments. PCR products were visualized by agarose gel electrophoresis. PCRs without a cDNA were included as negative controls. Expression of TATA box-binding protein (TBP) is shown as a positive control for amplification. (B and C) siRNA-transfected U2OS cells were cultured with or without 1 mM glucose for 4 hours. Cells were stained with Fluo-4AM for intracellular calcium before imaging. Representative phase-contrast and fluorescent images of cells are shown in (B). Scale bars, 50 μm. Protein lysates were analyzed by Western blotting analysis (C). Data are representative of three independent experiments. (D) Representative fluorescent images of DiBAC4-stained U2OS cells for membrane depolarization from three independent experiments. Cells were placed in media as indicated. KCl serves as a positive control. Scale bar, 50 μm. (E to G) Quantification of membrane depolarization from more than three independent experiments. DiBAC4-stained cells were placed in media as indicated, and fluorescent intensity was measured by a plate reader. Bars represent mean (±SD) DiBAC4 fluorescence in each sample relative to the control (first bar). *** P < 0.001, unpaired two-tailed Student’s t test.

  • Fig. 9 Therapeutic intervention targeting calcium signaling and glucose transport.

    (A) Intracellular glucose amounts in cells cultured with or without 25 mM glucose for 4 hours in the absence of serum. Intracellular glucose amounts were normalized against the total protein amount. Shown are means ± SD from three independent replicates. Asterisk (*) indicates cell lines that are sensitive to glucose deprivation. (B) Western blotting analysis for PP2Ac in cells cultured as indicated with 1 mM glucose without serum for 1 day. Blots are representative of three independent experiments. (C) Representative fluorescent images of Fluo-4AM–stained cells for intracellular calcium amounts. Cells were cultured with or without STF-31 in the presence of 1 mM glucose and absence of serum for 1 day. Cells were stained with Fluo-4AM before imaging. Images were representative of three independent experiments. Scale bars, 50 μm. (D) A representative analysis of PI exclusion assay in cells cultured as described in (B). Shown is the percentage of PI-positive dead cells from three independent experiments. (E) Schematic model of glucose deprivation–induced cell death. A, B, and C are PP2A complex subunits; Me, methylation; P, phosphorylation.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/512/eaam7893/DC1

    Fig. S1. Glucose deprivation induces cell death in a subset of cancer cells.

    Fig. S2. 2-DG rescues glucose deprivation–induced cell death that is independent from ROS induction.

    Fig. S3. 2-DG rescues glucose deprivation–induced cell death independently from O-glycosylation.

    Fig. S4. Glucose deprivation–induced cell death is not mediated by apoptosis.

    Fig. S5. Glucose deprivation–induced cell death is independent from autophagy.

    Fig. S6. AMPK is not involved in glucose deprivation–induced cell death.

    Fig. S7. Glucose deprivation induces RIPK1 phosphorylation.

    Fig. S8. Glucose deprivation induces RIPK1-dependent cell death.

    Fig. S9. Glucose deprivation–induced cell death is not mediated by necroptosis.

    Fig. S10. Glucose deprivation induces PP2Ac demethylation.

    Fig. S11. Glucose deprivation induces PP2Ac demethylation and RIPK1 phosphorylation independently from the mTOR signaling pathway.

    Fig. S12. PP2Ac demethylation is required for glucose deprivation–induced cell death.

    Fig. S13. Glucose deprivation induces calcium influx into the cytoplasm.

    Fig. S14. Knockdown efficiency of CACNA1C and CACNA1D.

    Fig. S15. GLUT1 inhibitor increases cytoplasmic calcium concentration in U2OS cells.

    Movie S1. PI staining in the presence of glucose.

    Movie S2. PI staining in the absence of glucose.

    Movie S3. Cell death in the presence of control siRNA and glucose.

    Movie S4. Cell death in the presence of PPME1 siRNA and glucose.

    Movie S5. Cell death in the presence of control siRNA but in the absence of glucose.

    Movie S6. Cell death in the presence of PPME1 siRNA but in the absence of glucose.

    Movie S7. Calcium staining in the presence of glucose.

    Movie S8. Calcium staining in the absence of glucose.

  • Supplementary Materials for:

    Ca2+-dependent demethylation of phosphatase PP2Ac promotes glucose deprivation–induced cell death independently of inhibiting glycolysis

    Ha Yin Lee, Yoko Itahana, Stefan Schuechner, Masahiro Fukuda, H. Shawn Je, Egon Ogris, David M. Virshup, Koji Itahana*

    *Corresponding author. Email: koji.itahana{at}duke-nus.edu.sg

    This PDF file includes:

    • Fig. S1. Glucose deprivation induces cell death in a subset of cancer cells.
    • Fig. S2. 2-DG rescues glucose deprivation–induced cell death that is independent from ROS induction.
    • Fig. S3. 2-DG rescues glucose deprivation–induced cell death independently from O-glycosylation.
    • Fig. S4. Glucose deprivation–induced cell death is not mediated by apoptosis.
    • Fig. S5. Glucose deprivation–induced cell death is independent from autophagy.
    • Fig. S6. AMPK is not involved in glucose deprivation–induced cell death.
    • Fig. S7. Glucose deprivation induces RIPK1 phosphorylation.
    • Fig. S8. Glucose deprivation induces RIPK1-dependent cell death.
    • Fig. S9. Glucose deprivation–induced cell death is not mediated by necroptosis.
    • Fig. S10. Glucose deprivation induces PP2Ac demethylation.
    • Fig. S11. Glucose deprivation induces PP2Ac demethylation and RIPK1 phosphorylation independently from the mTOR signaling pathway.
    • Fig. S12. PP2Ac demethylation is required for glucose deprivation–induced cell death.
    • Fig. S13. Glucose deprivation induces calcium influx into the cytoplasm.
    • Fig. S14. Knockdown efficiency of CACNA1C and CACNA1D.
    • Fig. S15. GLUT1 inhibitor increases cytoplasmic calcium concentration in U2OS cells.
    • Legends for movies S1 to S8

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 2.64 MB

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). PI staining in the presence of glucose.
    • Movie S2 (.mp4 format). PI staining in the absence of glucose.
    • Movie S3 (.mp4 format). Cell death in the presence of control siRNA and glucose.
    • Movie S4 (.mp4 format). Cell death in the presence of PPME1 siRNA and glucose.
    • Movie S5 (.mp4 format). Cell death in the presence of control siRNA but in the absence of glucose.
    • Movie S6 (.mp4 format). Cell death in the presence of PPME1 siRNA but in the absence of glucose.
    • Movie S7 (.mp4 format). Calcium staining in the presence of glucose.
    • Movie S8 (.mp4 format). Calcium staining in the absence of glucose.

    Citation: H. Y. Lee, Y. Itahana, S. Schuechner, M. Fukuda, H. S. Je, E. Ogris, D. M. Virshup, K. Itahana, Ca2+-dependent demethylation of phosphatase PP2Ac promotes glucose deprivation– induced cell death independently of inhibiting glycolysis. Sci. Signal. 11, eaam7893 (2018).

    © 2018 American Association for the Advancement of Science

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