Research ArticleCell Biology

Ca2+ signals regulate mitochondrial metabolism by stimulating CREB-mediated expression of the mitochondrial Ca2+ uniporter gene MCU

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Sci. Signal.  03 Mar 2015:
Vol. 8, Issue 366, pp. ra23
DOI: 10.1126/scisignal.2005673
  • Fig. 1 Knockout of IP3Rs or CRAC channel components reduces mitochondrial Ca2+ uptake in permeabilized cells, which is restored by ionomycin.

    (A) Mitochondrial Ca2+ uptake detected as reduction in buffer fluorescence of a Ca2+ indicator dye in permeabilized DT40 cells of the indicated genotypes. The experiment was initiated with the application of digitonin (Dg, 40 μg/ml), thapsigargin (Tg, 2 μM), and Fura-2FF (1 μM). After reaching steady state, six pulses of Ca2+ (3 μM) were added and separated by 50 s. Ru360 (1 μM) was applied at 900 s and CCCP (3 μM) at 1050 s. (B) Mitochondrial Ca2+ uptake detected as reduction in buffer fluorescence in permeabilized DT40 cells of the indicated genotypes that had been exposed to ionomycin (2.5 nM) for 6 days before the start of the experiment. Experiments were conducted as in (A). (C) Mitochondrial Ca2+ uptake was quantified as the sum of the successive six areas under the curve after Ca2+ additions for cells of the indicated genotypes either without or with ionomycin pretreatment. (D) The rate of mitochondrial Ca2+ uptake was calculated as 1/τ for the cells of the indicated genotypes either without or with ionomycin pretreatment. (E) Quantification of [Ca2+]m after CCCP addition. In (C) to (E), data are means ± SEM of three to five independent experiments. *P < 0.05, one-way analysis of variance (ANOVA) with Tukey correction (within groups); *P < 0.00625, paired t test with Bonferroni correction (between − and + ionomycin pretreated groups).

  • Fig. 2 Knockout of IP3Rs or CRAC channel components inhibits mitochondrial Ca2+ uptake in intact cells and IMCU.

    (A) Mitochondrial uptake in IgM-stimulated DT40 cells of the indicated genotypes. Cells were pretreated with ionomycin for 6 days where indicated. Mitochondrial Ca2+ was measured in lymphocytes loaded with Rhod-2 AM (2 mM) and Fluo-4 AM. IgM (1.5 μg/ml) was added, and cytosolic fluorescence (Fluo-4 AM) and mitochondrial fluorescence (Rhod-2 AM) were monitored for 600 s. Data plotted are the peak fluorescence after IgM addition and represent means ± SEM of three to four independent experiments. *P < 0.05, one-way ANOVA with Tukey correction. (B) Mitochondrial Ca2+ uptake in DT40 cells of the indicated genotypes expressing mitochondrial genetically encoded Ca2+ sensor, mito-R-GECO1, after thapsigargin (2 μM) addition. Bar graphs represent quantification of mitochondrial Ca2+ uptake for the indicated genotypes either without or with ionomycin treatment. Data are means ± SEM of three independent experiments (17 to 26 cells per condition) (see fig. S4 for representative traces). **P < 0.01, ***P < 0.001, paired t test. f.a.u., fluorescence arbitrary units. (C) IMCU current in mitoplasts derived from IP3R TKO, STIM1 KO, and Orai1,2 DKO DT40 cells without and with ionomycin pretreatment. Traces are a representative single recording of IMCU. (D) IMCU densities (pA/pF) in cells without or with ionomycin pretreatment. Data are means ± SEM of four to seven experiments. *P < 0.05, one-way ANOVA (Tukey test); *P < 0.00625, t test (Bonferroni correction).

  • Fig. 3 Knockout of IP3Rs or CRAC channel components results in reduced MCU protein abundance and MCU expression and CREB phosphorylation.

    (A) MCU abundance in DT40 cells of the indicated genotypes without and with ionomycin pretreatment. Cyclophilin D served as the loading control (lower blots). (B) Quantification of MCU abundance is shown as the mean ± SEM of three independent experiments and is expressed relative to the amount in wild-type (WT) cells under each condition. (C) Relative amount of mRNA for MCU and MCUb in cells of the indicated genotypes with and without ionomycin pretreatment. mRNA abundance was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The relative mRNA abundance was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data are means ± SEM of three to four independent experiments. *P < 0.05, Z test of significance. (D) MICU1 abundance in DT40 cells of the indicated genotypes without or with ionomycin pretreatment. (E) Quantification of MICU1 is shown as the mean ± SEM of three independent experiments calculated as in (B). (F) Relative amount of MICU1 mRNA in cells of the indicated genotypes with and without ionomycin pretreatment. Data are quantified and expressed as in (C). (G) Western blot showing Ser133 phosphorylation of CREB (pCREB) in DT40 cells of the indicated genotypes without and with ionomycin pretreatment. (H) Quantification of pCREB abundance expressed as the percent of pCREB/CREB relative to that in WT cells in the absence of ionomycin and shown as the mean ± SEM of three experiments. *P < 0.05, Z test of significance. (I) Western blot showing phosphorylation of ERK (pERK2) in DT40 cells of the indicated genotypes without and with ionomycin pretreatment. DT40 cells predominantly have ERK2. (J) Quantification of pERK2 abundance expressed as the percent of pERK2/ERK2 relative to that in WT cells in the absence of ionomycin and shown as the mean ± SEM of three experiments. *P < 0.05, Z test of significance.

  • Fig. 4 Knockout of IP3Rs or CRAC channel components eliminates basal Ca2+ dynamics.

    Spontaneous Ca2+ oscillations in DT40 cells of the indicated genotypes under basal condition. The three representative traces depict the spontaneous cytosolic Ca2+ dynamics for each genotype of cells loaded with the cytosolic Ca2+ indicator Fluo-4 AM and imaged by confocal microscopy.

  • Fig. 5 SOCE is required for CREB activation and to maintain MCU abundance.

    (A) Western blot analysis of pCREB abundance in WT DT40 cells deprived of extracellular calcium by exposure to BAPTA (0.5 mM) for the indicated amounts of time. (B) Quantification pCREB expressed as the percent of pCREB/CREB relative to that in the cells at time 0 and shown as the mean ± SEM of three experiments. *P < 0.05, Z test of significance. (C) Reconstitution of Orai1 in Orai1,2 DKO cells. The blot represents the abundance of either WT Orai1 tagged with cyan fluorescent protein (CFP) (Orai1 WT) or the dominant-negative Orai1-E106A-CFP mutant. (D) Western blot analysis of pCREB in WT, Orai1,2 DKO (DKO), and Orai1-reconstituted DT40 cells. (E) Quantification pCREB expressed as percent abundance relative to that in WT cells and shown as the mean ± SEM of three experiments. *P < 0.05, Z test of significance. (F) Abundance of MCU in WT Orai1-reconstituted Orai1,2 DKO cells. CypD, cyclophilin D. (G) Quantification of the abundance of MCU relative to the amount in WT DT40 cells and shown as the mean ± SEM of three experiments. *P < 0.05, Z test of significance. (H) Representative traces depict changes in [Ca2+]out in DT40 cells of indicated genotypes. Experimental procedure as described in Fig. 1A. (I) Mitochondrial Ca2+ uptake was quantified as the sum of the successive six areas under the curve after Ca2+ additions for WT, DKO, and Orai1-reconstituted cells. Data are means ± SEM of three independent experiments. *P < 0.05, one-way ANOVA with Tukey correction. (J) Rate of mitochondrial Ca2+ uptake was calculated as 1/τ from experiments like those shown in (H). Data are means ± SEM of three independent experiments. *P < 0.05, one-way ANOVA with Tukey correction.

  • Fig. 6 CREB binding to the MCU promoter is stimulated by Ca2+ and cAMP signaling.

    (A) ChIP analysis of HeLa cells treated with ionomycin (2.5 μM), forskolin (5 μM), or a combination of both for 30 min using an antibody recognizing pCREB. IgG and an antibody specific for RNA polymerase were used as negative and positive controls, respectively. The PCR products were analyzed qualitatively on agarose gel electrophoresis and compared with controls. Agarose gel images shown are representative of four experiments. Input is 5% of the total DNA used for the immunoprecipitation. (B) The fold enrichment of MCU relative to the matched input control was quantified by qRT-PCR with c-fos as a positive control. Data are means ± SEM of three independent experiments. *P < 0.05, one-way ANOVA (Tukey test). (C) Schematic of the MCU promoter-luciferase constructs. CREB consensus response elements are shown in ovals. (D) HeLa cells were transfected with MCU promoter-luciferase constructs. Cells were stimulated with ionomycin, forskolin, or a combination of both for 12 hours and analyzed for luciferase activity. Data are means ± SEM of three independent experiments. *P < 0.05, one-way ANOVA with Tukey correction. (E and F) Western blot analysis of WT DT40 lymphocyte lysates stimulated with IgM (10 μg/ml) for indicated time points. Blots were probed for antibodies specific for pCREB and MCU, and the relative abundance of pCREB and MCU was quantified. pCREB data are relative to the amount at time 0; MCU data are relative to the amount at time 0. Both are the means ± SEM of three experiments. *P < 0.05, Z test of significance.

  • Fig. 7 KO of IP3Rs or CRAC channel components alters mitochondrial metabolism.

    (A) NADPH fluorescence measurements in DT40 lymphocytes of indicated genotypes before and after rotenone (10 μM) addition. (B) NADPH fluorescence measurements in ionomycin-pretreated DT40 lymphocytes of indicated genotypes. (C) Quantification of basal NADPH fluorescence. (D) Quantification of ΔNADPH fluorescence after rotenone addition. (E) PDH activity in DT40 lymphocytes of the indicated genotypes without and with ionomycin pretreatment. (F) Lactate abundance determined from LDH activity assay with cells of the indicated genotypes without or with ionomycin pretreatment. (G) The relative abundance of mtDNA was normalized to the abundance of nuclear DNA. For all quantified data, data are means ± SEM of three independent experiments. *P < 0.05, one-way ANOVA with Tukey correction for within-group comparisons; *P < 0.00625, paired t test with Bonferroni correction for between-group comparisons.

  • Fig. 8 KO of IP3Rs or CRAC channel components renders cells resistant to oxidative stress.

    (A) DT40 lymphocytes were exposed to t-butyl hydroperoxide (t-BH; 200 μM) for 6 hours. Cell death was assessed by fluorescence-activated cell sorting (FACS) after staining with PI. (B) Quantification of PI-positive cells by FACS. Data are means ± SEM of three independent experiments. *P < 0.05, paired t test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/366/ra23/DC1

    Fig. S1. KO of IP3Rs or CRAC channel components does not alter basal mitochondrial membrane potential (Δψm).

    Fig. S2. Inhibitors of acidic intracellular compartments do not alter mitochondrial Ca2+ uptake in HEK293T cells.

    Fig. S3. KO of IP3Rs or CRAC channel components reduces resting [Ca2+]m.

    Fig. S4. KO of IP3Rs or CRAC channel components reduces mitochondrial Ca2+ uptake after IgM stimulation.

    Fig. S5. Cytosolic Ca2+ dynamics in IP3R TKO, STIM1 KO, and Orai1,2 DKO lymphocytes after thapsigargin stimulation is similar in the presence or absence of ionomycin.

    Fig. S6. KO of IP3Rs and CRAC channel components does not affect MCUR1 abundance.

    Fig. S7. The abundance of MCU depends on STIM1.

    Fig. S8. KD of MCU did not alter the expression or abundance of IP3R and CRAC channel components.

    Fig. S9. pCREB is reduced in STIM1-deficient MEFs.

    Fig. S10. The abundances of MCU and pCREB are similar after exposure of wild-type and KO DT40 cells to forskolin treatment.

  • Supplementary Materials for:

    Ca2+ signals regulate mitochondrial metabolism by stimulating CREB-mediated expression of the mitochondrial Ca2+ uniporter gene MCU

    Santhanam Shanmughapriya, Sudarsan Rajan, Nicholas E. Hoffman, Xueqian Zhang, Shuchi Guo, Jill E. Kolesar, Kevin J. Hines, Jonathan Ragheb, Neelakshi R. Jog, Roberto Caricchio, Yoshihiro Baba, Yandong Zhou, Brett A. Kaufman, Joseph Y. Cheung, Tomohiro Kurosaki, Donald L. Gill,* Muniswamy Madesh*

    *Corresponding author. E-mail: madeshm{at}temple.edu (M.M.); dongill{at}psu.edu (D.L.G.)

    This PDF file includes:

    • Fig. S1. KO of IP3Rs or CRAC channel components does not alter basal mitochondrial membrane potential (ΔΨm).
    • Fig. S2. Inhibitors of acidic intracellular compartments do not alter mitochondrial Ca2+ uptake in HEK293T cells.
    • Fig. S3. KO of IP3Rs or CRAC channel components reduces resting [Ca2+]m.
    • Fig. S4. KO of IP3Rs or CRAC channel components reduces mitochondrial Ca2+ uptake after IgM stimulation.
    • Fig. S5. Cytosolic Ca2+ dynamics in IP3R TKO, STIM1 KO, and Orai1,2 DKO lymphocytes after thapsigargin stimulation is similar in the presence or absence of ionomycin.
    • Fig. S6. KO of IP3Rs and CRAC channel components does not affect MCUR1 abundance.
    • Fig. S7. The abundance of MCU depends on STIM1.
    • Fig. S8. KD of MCU did not alter the expression or abundance of IP3R and CRAC channel components.
    • Fig. S9. pCREB is reduced in STIM1-deficient MEFs.
    • Fig. S10. The abundances of MCU and pCREB are similar after exposure of wild-type and KO DT40 cells to forskolin treatment.

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    Citation: S. Shanmughapriya, S. Rajan, N. E. Hoffman, X. Zhang, S. Guo, J. E. Kolesar, K. J. Hines, J. Ragheb, N. R. Jog, R.Caricchio, Y. Baba, Y. Zhou, B. A. Kaufman, J. Y. Cheung, T. Kurosaki, D. L. Gill, M. Madesh, Ca2+ signals regulate mitochondrial metabolism by stimulating CREB-mediated expression of the mitochondrial Ca2+ uniporter gene MCU. Sci. Signal. 8, ra23 (2015).

    © 2015 American Association for the Advancement of Science

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