Research ArticleCell Biology

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

See allHide authors and affiliations

Sci. Signal.  03 Mar 2015:
Vol. 8, Issue 366, pp. ra23
DOI: 10.1126/scisignal.2005673

Maintaining mitochondrial calcium uptake

The calcium uniporter complex, which includes the protein MCU, mediates mitochondrial calcium uptake, a process that buffers excess cytosolic calcium and regulates mitochondrial metabolism. Shanmughapriya et al. examined mitochondrial calcium uptake and function in a B lymphocyte cell line deficient in one or more proteins necessary for mediating two types of calcium signals—IICR, calcium released from the endoplasmic reticulum through the calcium-permeable IP3 receptors, and SOCE, calcium influx through store-operated calcium channels. Without IICR or SOCE, the activity of the transcription factor CREB, which bound to the MCU promoter, and the expression and abundance of MCU were reduced, mitochondrial calcium uptake was compromised, and mitochondrial metabolism was altered. Cells deficient in IICR or SOCE lacked an oscillating basal calcium signal. Thus, IICR and SOCE control the capacity of mitochondria to uptake calcium and therefore regulate mitochondrial metabolism.

Abstract

Cytosolic Ca2+ signals, generated through the coordinated translocation of Ca2+ across the plasma membrane (PM) and endoplasmic reticulum (ER) membrane, mediate diverse cellular responses. Mitochondrial Ca2+ is important for mitochondrial function, and when cytosolic Ca2+ concentration becomes too high, mitochondria function as cellular Ca2+ sinks. By measuring mitochondrial Ca2+ currents, we found that mitochondrial Ca2+ uptake was reduced in chicken DT40 B lymphocytes lacking either the ER-localized inositol trisphosphate receptor (IP3R), which releases Ca2+ from the ER, or Orai1 or STIM1, components of the PM-localized Ca2+-permeable channel complex that mediates store-operated calcium entry (SOCE) in response to depletion of ER Ca2+ stores. The abundance of MCU, the pore-forming subunit of the mitochondrial Ca2+ uniporter, was reduced in cells deficient in IP3R, STIM1, or Orai1. Chromatin immunoprecipitation and promoter reporter analyses revealed that the Ca2+-regulated transcription factor CREB (cyclic adenosine monophosphate response element–binding protein) directly bound the MCU promoter and stimulated expression. Lymphocytes deficient in IP3R, STIM1, or Orai1 exhibited altered mitochondrial metabolism, indicating that Ca2+ released from the ER and SOCE-mediated signals modulates mitochondrial function. Thus, our results showed that a transcriptional regulatory circuit involving Ca2+-dependent activation of CREB controls the Ca2+ uptake capability of mitochondria and hence regulates mitochondrial metabolism.

INTRODUCTION

Intracellular calcium ([Ca2+]i) plays an important role in regulating numerous cellular functions. Studies with isolated mitochondria demonstrated that mitochondria can accumulate large amounts of Ca2+ (13). Mitochondrial Ca2+ uptake is mediated by an inner mitochondrial membrane–resident complex that includes the mitochondrial Ca2+ uniporter (MCU) protein. MCU exists as part of a heteromeric complex that consists of MICU1, MICU2, MCUb, MCUR1, EMRE, and SLC25A23 (415). Studies with isolated mitochondria and electrophysiological studies of MCU activity estimated that flux of Ca2+ ions through the MCU exceeds 10,000 Ca2+ ions per second (16). MCU-mediated mitochondrial Ca2+ uptake depends on the mitochondrial membrane potential (Δψm) and is partly inhibited at basal and low concentrations of cytosolic Ca2+ ([Ca2+]c) by the regulator MICU1 (8, 9, 17). Signaling by G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs) or receptor tyrosine kinases that increase [Ca2+]c above 3 μM stimulates MCU current (IMCU), resulting in the accumulation of Ca2+ in the mitochondrial matrix (6, 8). Mitochondria that have accumulated Ca2+ exhibit higher mitochondrial bioenergetics through tricarboxylic acid (TCA) cycle and oxidative phosphorylation (1820). Because regulation of [Ca2+]c is critical for cell survival, one might predict that conditions that produced prolonged increases in [Ca2+]c may stimulate the expression of the genes encoding and the function of mitochondrial Ca2+ transporter proteins.

Ca2+-dependent transcription contributes to the regulation of cellular metabolism, proliferation, differentiation, and cell death (21, 22). Additionally, Ca2+-dependent transcription plays essential roles in controlling immune cell responses (23). Spatiotemporal changes in [Ca2+]c activate various transcription factors, including nuclear factor of activated T cells (NFAT), c-FOS, and cyclic adenosine monophosphate (cAMP) response element–binding protein (CREB) (21, 22). In nonexcitable cells, the major receptors that stimulate cytosolic Ca2+ signals are GPCRs and receptor tyrosine kinases. In both cases, cytosolic Ca2+ signals result from (i) inositol trisphosphate (IP3)–induced Ca2+ release (IICR) through endoplasmic reticulum (ER)–located IP3 receptors (IP3Rs), which are Ca2+-permeable channels, and (ii) store-operated Ca2+ entry (SOCE), which is mediated by STIM-induced activation of Orai channels [also known as Ca2+ release–activated Ca2+ (CRAC) channels], resulting from depletion of the ER Ca2+ store (21, 2330). Mitochondria buffer cytosolic Ca2+ resulting from both IICR and SOCE, but whether expression of genes encoding proteins that mediate mitochondrial Ca2+ uptake are regulated by these Ca2+-responsive transcriptional circuits is unknown.

Here, we examined how Ca2+ signaling cascades affected the activity and abundance of MCU using chicken DT40 lymphocytes deficient in various components that mediate IICR or SOCE. Using STIM, Orai, and IP3R knockout (KO) cells, we found that Ca2+ entry through STIM-activated Orai channels and Ca2+ release through IP3R channels enhanced mitochondrial uptake of Ca2+. Without IICR or SOCE, the cells did not produce spontaneous Ca2+ oscillations, MCU abundance and phosphorylation of CREB were reduced, and cellular metabolism was altered. Chromatin immunoprecipitation (ChIP) and reporter gene assays with HeLa cells indicated that CREB bound the MCU promoter. Reestablishment of cytosolic Ca2+ signals in IP3R or Orai KO cells restored MCU abundance and activity and improved mitochondrial metabolism. These studies established crosstalk between cytoplasmic Ca2+ signaling and mitochondrial Ca2+ buffering mechanisms.

RESULTS

IICR and SOCE alter MCU-mediated mitochondrial Ca2+ uptake rate in permeabilized cells

To determine how mitochondrial Ca2+ uptake was influenced by IICR and SOCE, we used permeabilized DT40 B lymphocytes genetically deficient for IP3R, STIM, or Orai. In addition to testing components of the SOCE complex and IP3Rs, we also examined the effect of KO of the gene encoding phospholipase Cγ2 (PLCγ2), because this enzyme produces IP3 in DT40 cells that is required for IICR and SOCE activation (31). We measured mitochondrial Ca2+ uptake in digitonin-permeabilized IP3R1−/−/2−/−/3−/− triple KO (TKO), PLCγ2−/−, STIM1−/−, STIM2-/, Orai1−/−, Orai2−/−, and Orai1−/−/2−/− double KO (DKO) bathed in intracellular-like medium, containing mitochondrial substrate succinate, thapsigargin, to block uptake of Ca2+ into the ER by sarcoplasmic and ER Ca2+ (SERCA) pump, and Fura-2FF to detect changes in [Ca2+] (6, 32). Mitochondrial Ca2+ uptake was calculated from clearance of bath Ca2+ ([Ca2+]out) (see Materials and Methods). Because Δψm is a driving force for mitochondrial Ca2+ uptake, we confirmed that basal Δψm in the wild-type and KO DT40 cells was similar (fig. S1, A to C).

After the permeabilized cells had reached a steady state mitochondrial membrane potential, we applied six pulses of [Ca2+]out and monitored the reduction in [Ca2+]out after each pulse. We performed the experiments with permeabilized cells that had not been exposed to the Ca2+ ionophore ionomycin (Fig. 1A) and after chronic exposure of the cells to ionomycin before permeabilization (Fig. 1B) (see Materials and Methods). Wild-type, STIM2 KO, and Orai2 KO exhibited similar mitochondrial Ca2+ uptake (Fig. 1, A and C). In contrast, in IP3R TKO, PLCγ2 KO, STIM1 KO, Orai1 KO, and Orai1,2 DKO cells, [Ca2+]out increased after each pulse, indicating compromised mitochondrial uptake (Fig. 1, A and C). Both total accumulated [Ca2+]m and mitochondrial Ca2+ uptake rate in IP3R TKO, PLCγ2 KO, STIM1 KO, Orai1 KO, and Orai1,2 DKO were significantly decreased compared with that in the wild-type cells (Fig. 1, C and D). [Ca2+]out reduced after each pulse (fig. S2A), and the rate of mitochondrial Ca2+ uptake (fig. S2B) was similar in cells exposed to thapsigargin alone or a combination of thapsigargin and acidic intracellular compartment inhibitors (brefeldin A and bafilomycin A). Furthermore, the addition of the mitochondrial uncoupler (7) CCCP, which dissipates Δψm, resulted in the release of similar amounts of Ca2+ in cells exposed to thapsigargin or thapsigargin, brefeldin A, and bafilomycin A (fig. S2C). These controls indicated that other organelles, such as endosomes, did not contribute to the clearance of [Ca2+]out in the permeabilized system.

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).

We examined the extent to which lack of IP3Rs and CRAC channel components affected basal matrix [Ca2+]m. We measured basal [Ca2+]m after dissipation of the Δψm with the CCCP and found that basal [Ca2+]m was significantly decreased in IP3R TKO, PLCγ2 KO, STIM1 KO, Orai1 KO, and Orai1,2 DKO cells (Fig. 1E and fig. S3), suggesting that these cells lacked dynamic changes in [Ca2+]c and the associated signaling.

We hypothesized that the absence of signal-induced Ca2+ transients in IP3R TKO, PLCγ2 KO, STIM1 KO, Orai1 KO, and Orai1,2 DKO lymphocytes results in a slower rate of [Ca2+]m uptake. To investigate this hypothesis, we circumvented IICR and SOCE by chronic ionomycin treatment (2.5 nM; 6 days) to induce a direct increase in [Ca2+]c. Ionophores, such as ionomycin, stimulate lymphocytes by activating proliferation-inducing, Ca2+-dependent kinases (3335). After chronic ionomycin pretreatment, we permeabilized the lymphocytes, added thapsigargin to inhibit ER uptake of Ca2+, and monitored [Ca2+]out in response to pulses of Ca2+. The ionomycin pretreatment rescued mitochondrial Ca2+ uptake rate and accumulation in IP3R TKO, PLCγ2 KO, STIM1 KO, Orai1 KO, and Orai1,2 DKO cells to amounts comparable to those in wild-type cells (Fig. 1, B to D). These results suggested that the Ca2+ signals produced by IICR and SOCE are important for coordinating [Ca2+]m uptake capacity with demand, which may reflect an effect on MCU activity.

IICR and SOCE affect MCU-mediated mitochondrial Ca2+ uptake and IMCU

To provide additional evidence that the results from the permeabilized cells represented altered mitochondrial Ca2+ uptake due to altered cytosolic Ca2+ dynamics, we examined mitochondrial Ca2+ dynamics in cells loaded with Ca2+ indicators that localized to the cytosol or mitochondria. We stimulated intact wild-type lymphocytes with immunoglobulin M (IgM), which produced a robust oscillating increase in [Ca2+]c and a steady increase in [Ca2+]m (fig. S4A). Consistent with IP3R requiring IP3 for activation, the lack of IP3 production in PLCγ2 KO in response to IgM resulted in no changes in [Ca2+]c (Fig. 2A and fig. S4A). Increased fluorescence of the mitochondria-localized fluorophore in response to IgM was substantially reduced in IP3R TKO, PLCγ2 KO, STIM1 KO, Orai1 KO, and Orai1,2 DKO lymphocytes (Fig. 2A and fig. S4A), indicating that without either IICR or SOCE, mitochondrial Ca2+ uptake was reduced. Furthermore, ionomycin treatment restored mitochondrial Ca2+ uptake in STIM1 KO, Orai1 KO, and Orai1,2 DKO lymphocytes (Fig. 2A).

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).

To further verify the defect of mitochondrial Ca2+ uptake in IP3R TKO, STIM1 KO, and Orai1,2 DKO cells, we measured mitochondrial Ca2+ uptake using the genetically encoded mitochondrial Ca2+ reporter, mito-R-GECO1, and recorded the mitochondrial fluorescence signal. Wild-type DT40 cells exposed to thapsigargin exhibited a rapid increase in fluorescence that was similar without or with ionomycin pretreatment (Fig. 2B and fig. S4B). In contrast, mitochondrial Ca2+ uptake was significantly reduced in IP3R TKO, STIM1 KO, and Orai1,2 DKO lymphocytes, whereas pretreatment with ionomycin restored mitochondrial Ca2+ uptake in the KO lymphocytes (Fig. 2B and fig. S4B). Under these conditions, wild-type or the KO DT40 cells treated with thapsigargin exhibited similar cytosolic Ca2+ dynamics (fig. S5, A to D).

We also measured MCU current (IMCU) in isolated mitoplasts from IP3R TKO, STIM1 KO, and Orai1,2 DKO lymphocytes. We compared the currents in cells that had or had not been pretreated with ionomycin for 6 days before mitoplast isolation. Consistent with [Ca2+]m imaging results, mitoplasts from lymphocytes of IP3R TKO, STIM1 KO, and Orai1,2 DKO had markedly reduced IMCU, whereas wild-type mitoplasts displayed classic IMCU (Fig. 2, C and D). As expected, we observed a near-complete restoration of IMCU in IP3R TKO, STIM1 KO, and Orai1,2 DKO mitoplasts after ionomycin pretreatment (Fig. 2, C and D).

Impaired IP3Rs and CRAC-mediated Ca2+ entry reduce CREB activation and reduce the expression and abundance of MCU

One possible mechanism for the impaired mitochondrial Ca2+ uptake in IP3R TKO, PLCγ2 KO, STIM1 KO, Orai1 KO, and Orai1,2 DKO DT40 lymphocytes could be a reduction in the abundance of MCU. Indeed, Western blotting indicated that MCU abundance was low in IP3R TKO, PLCγ2 KO, STIM1 KO, Orai1 KO, and Orai1,2 DKO cells, whereas MCU was more abundant in wild-type, STIM2 KO, and Orai2 KO lymphocytes (Fig. 3, A and B). The abundance of mRNAs for MCU and the MCU paralog MCUb in IP3R TKO and Orai1,2 DKO cells was also decreased compared to wild-type cells (Fig. 3C), suggesting that the decrease in MCU abundance may be due to reduced gene expression. Wild-type and the KO cells had similar amounts of the MCU regulators MICU1 (Fig. 3, D to F) and MCUR1 (fig. S6, A and B). Ionomycin pretreatment restored MCU protein (Fig. 3, A and B) and mRNA abundance (Fig. 3C) in IP3R and CRAC channel component KO cells. The MICU1 paralog MICU2 has nonredundant roles in some cells (12, 36); however, MICU2 was undetectable in DT40 cells possibly due to lack of antibody specificity.

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.

To determine whether the alterations in MCU abundance in the DT40 KO cells occurred in other cells deficient in components of the CRAC channel, we compared the phenotypes of STIM1or STIM2 KO DT40 cells with mouse embryonic fibroblasts (MEFs) with the comparable genetic KOs. In the DT40 lymphocytes, STIM1 KO, but not STIM2 KO, resulted in compromised mitochondrial Ca2+ uptake (Fig. 1) and reduced MCU abundance (Fig. 3), but not MCUR1 abundance (fig. S6). Consistent with these results, the abundance of MCU, but not MCUR1, was reduced in STIM1 KO but not in STIM2 KO MEFs (fig. S7).

To examine the possibility that a reduction in MCU could affect the abundance of IICR and CRAC components, we evaluated the effect of MCU knockdown (KD) on the expression and abundance of IP3R1, STIM1, and Orai1 in HeLa cells. Neither the mRNA nor the protein abundance of IP3R1, STIM1, and Orai1 was altered in MCU KD cells (fig. S8).

Bioinformatic analysis of the human MCU promoter region revealed putative CREB binding sites. CREB is activated by phosphorylation at Ser133 by various signaling cascades, including extracellular signal–regulated kinases (ERKs), which are activated by growth factors, Ca2+ signaling, and stress signaling pathway (3741). Phosphorylated CREB (pCREB) activates transcription of numerous genes through cAMP response elements (CREs). We investigated whether the loss of IP3Rs and CRAC channel components disrupted CREB activity in the DT40 lymphocytes. We measured activation of CREB by monitoring the amount of Ser133-phosphorylated CREB by Western blotting with an antibody specific for pCREB. Abundant pCREB was observed in wild-type, STIM2 KO, and Orai2 KO lymphocytes, whereas pCREB was barely detectable in IP3R TKO, PLCγ2 KO, STIM1 KO, Orai1 KO, and Orai1,2 DKO cells (Fig. 3, G and H). Similar results were observed in MEFs lacking STIM1 but not those lacking STIM2 (fig. S9). Further, increasing [Ca2+]c by bypassing IICR and SOCE with ionomycin increased pCREB in IP3R TKO, PLCγ2 KO, STIM1 KO, Orai1 KO, and Orai1,2 DKO cells (Fig. 3, G and H).

Because CREB phosphorylation occurs in response to increased cAMP, we measured MCU abundance in DT40 lymphocytes stimulated with forskolin to activate adenylate cyclase and increase cAMP. Similar to the effect of ionomycin, forskolin increased pCREB and MCU abundance in the KO cells to amounts indistinguishable from wild-type DT40 cells (fig. S10, A to D). ERK2 is the predominant ERK in DT40 lymphocytes (31, 42), and stimuli that increase ERK phosphorylation and activation also increase CREB activity. Therefore, we examined the ERK2 phosphorylation in the DT40 lymphocytes without or with ionomycin pretreatment. Similar to the abundance of pCREB, the abundance of pERK2 was significantly decreased in IP3R TKO, PLCγ2 KO, STIM1 KO, Orai1 KO, and Orai1,2 DKO cells, and ionomycin pretreatment restored ERK2 phosphorylation to amounts similar to wild-type cells (Fig. 3, I and J). These data suggested that a Ca2+-responsive pathway, possibly involving ERK2, controls the abundance of MCU by regulating the activity of CREB.

Because our data suggested that disruption of basal Ca2+ signaling, resulting in reduced activation of Ca2+-dependent transcriptional regulation, caused the reduced MCU abundance, we measured spontaneous cytosolic Ca2+ transients using Fluo-4 in the DT40 wild-type and KO lymphocytes. We observed Ca2+ oscillations in wild-type, STIM2 KO, and Orai2 KO lymphocytes but not in IP3R TKO, PLCγ2 KO, STIM1 KO, Orai1 KO, and Orai1,2 DKO cells (Fig. 4). Because ionomycin pretreatment restored pCREB and ERK2 abundance and MCU abundance, the data indicated that ERK2 and CREB phosphorylation in DT40 lymphocytes depends on cytosolic Ca2+ dynamics, and this pathway mediates CREB-induced expression of MCU.

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.

To examine the role of extracellular Ca2+ and specifically whether Orai1- and SOCE-mediated Ca2+ influx was necessary for Ca2+-dependent activation of CREB, we cultured wild-type DT40 cells in medium depleted of extracellular Ca2+ with the Ca2+ chelator BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid] over the course of 0, 2, 4, and 6 hours. By 4 hours, the abundance of pCREB was significantly reduced (Fig. 5, A and B). To determine whether SOCE could restore pCREB, MCU abundance, and mitochondrial Ca2+ uptake in the Orai1,2 DKO cells, we expressed Orai1 or the loss-of-function mutant E106A in these cells. We confirmed by Western blot that the DKO cells lacked Orai1 and that both the wild-type Orai1 and E106A mutant were expressed in the cells (Fig. 5C). Expression of wild-type Orai1, but not the E105A mutant, restored pCREB abundance to amounts similar to those in wild-type DT40 cells (Fig. 5, D and E) and also restored MCU abundance (Fig. 5, F and G). Consistent with MCU abundance controlling the mitochondrial Ca2+ uptake in the cells, using a permeabilized cell system, we found that the expression of Orai1 in the DKO cells increased both the amount of Ca2+ accumulated by the mitochondria and the rate of uptake, whereas the E106A mutant did not rescue these mitochondrial phenotypes (Fig. 5, H to J).

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.

CREB binds the MCU promoter and induces gene expression

To examine a link between CREB phosphorylation and MCU expression, we performed ChIP assays. The pCREB antiserum coimmunoprecipitated the MCU promoter sequence from HeLa cells (Fig. 6, A and B). Stimulation of the cells with ionomycin, forskolin, or both increased the amount of MCU bound (Fig. 6, A and B). These data support the model that ionomycin increased MCU abundance and mitochondrial Ca2+ by increasing the pCREB-mediated expression of MCU.

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.

Bioinformatic analysis predicted two putative CRE sites at −620−615 base pairs (bp) (ACGTCA) and +113+117 bp (CGTCA) in the MCU gene. We generated luciferase reporter constructs of the MCU promoter CRE sites and expressed them in HeLa cells (Fig. 6C). One reporter contained only the −620−615 CRE site, one contained only the +113+117 CRE site, one contained the region from −664 to the start site but the −620−615 site was deleted (Δ−620−615), and one contained a scrambled sequence of −620−615 CRE site. Only the −620−615 reporter exhibited expression in response to ionomycin, forskolin, or both (Fig. 6D). Indeed, the −620−615 reporter elicited luciferase activity in unstimulated HeLa cells (Fig. 6D). These data provided additional evidence that CREB stimulates the transcription of MCU.

To examine the effects of Ca2+ dynamics on transcription in a physiological setting, we stimulated wild-type DT40 lymphocytes with IgM (Fig. 6, E and F). Persistent stimulation with IgM triggered both the phosphorylation of CREB, which was increased within 48 hours and continued to increase through 96 hours, and the increased MCU abundance, which appeared after 96 hours. These data suggested that the mitochondrial changes in MCU-dependent Ca2+ uptake may require long-term stimulation.

IICR- and SOCE-mediated Ca2+ signals determine mitochondrial reducing equivalents and cell fate

Mitochondrial Ca2+ accumulation from cytosolic Ca2+ transients regulates a range of mitochondrial enzymes that participate in NADH [reduced form of nicotinamide adenine dinucleotide (NAD+)] generation, the production of metabolic substrates, and the activity of the electron transport chain (20, 43, 44). Therefore, we assessed the effect of IICR- and SOCE-mediated Ca2+ signals on cellular [reduced form of nicotinamide adenine dinucleotide phosphate (NADPH)] and metabolic enzyme activity. We measured the basal steady-state amount of NADPH and basal TCA cycle–dependent NADH production in wild-type and KO cells without and with pretreatment with ionomycin (Fig. 7, A to D). We detected basal steady-state autofluorescence of NADPH. Compared to wild-type DT40 cells, basal NADPH was significantly lower in IP3R TKO, PLCγ2 KO, STIM1 KO, Orai1 KO, and Orai1,2 DKO lymphocytes (Fig. 7, A and C). As predicted from the previous experiments, ionomycin treatment restored the amount of NADPH to that comparable to wild-type cells for the IP3R TKO, STIM1 KO, Orai1 KO, and Orai1,2 DKO cells (Fig. 7, B and C). However, PLCγ2 KO lymphocytes exhibited reduced NADPH even when treated with ionomycin, which may be due to the lack of PLCγ2-derived diacylglycerol-dependent kinase signaling.

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.

To measure the rate of NADPH production, we inhibited the electron transport chain complex I with rotenone, which eliminates NADH consumption, and quantified the change in NADPH between the basal state and the inhibited state. We found that although there was variability in complex I–dependent NADPH consumption among the cells of the various genotypes, these differences were not statistically significant and there was no significant difference between the groups with and without ionomycin pretreatment (Fig. 7, A to D). These results indicated that only NADH production was lower in DT40 KO lymphocytes.

The pyruvate dehydrogenase (PDH) complex generates acetyl-CoA (coenzyme A) for entry into the citric acid cycle (45), which mediates oxidative decarboxylation and produces NADH (19, 46, 47). PDH complex activity is stimulated by Ca2+, because Ca2+ stimulates PDH phosphatase, which dephosphorylates and activates PDH (19). To investigate the role of mitochondrial Ca2+ in this aspect of mitochondrial metabolism, we analyzed PDH activity in mitochondria isolated from wild-type and KO lymphocytes. Consistent with lower mitochondrial Ca2+ uptake in IP3R TKO, PLCγ2 KO, STIM1 KO, Orai1 KO, and Orai1,2 DKO lymphocytes, PDH activity was reduced (Fig. 7E). Cell lysates from lymphocytes treated with ionomycin had significantly increased PDH activity (Fig. 7E). Reduced PDH activity often corresponds with an increased lactate concentration due to increased pyruvate availability for the enzyme lactate dehydrogenase (LDH). Therefore, we measured lactate abundance in both control and ionomycin-treated lymphocytes. Indeed, IP3R TKO, PLCγ2 KO, STIM1 KO, Orai1 KO, and Orai1,2 DKO lymphocytes had increased amounts of lactate, indicating increased concentration of lactate, and ionomycin pretreatment reduced LDH activity, indicating lower concentrations of lactate (Fig. 7F).

The mitochondrial genome (mtDNA) encodes various subunits involved in oxidative phosphorylation and mitochondrial protein synthesis; therefore, we measured mtDNA content relative to that of nuclear DNA in wild-type and the KO lymphocytes. The relative amount of mtDNA was highly variable among the different genotypes and within populations of cells of the same genotype, but the differences were not statistically significant (Fig. 7G), indicating that mtDNA maintenance or replication is independent of IICR and SOCE. Furthermore, the differences between cells pretreated with ionomycin and those not exposed to ionomycin were also not significant. Thus, these data indicated that differences in mtDNA were not responsible for the differences in NADH production and mitochondrial metabolism between wild-type cells and cells lacking IICR and SOCE.

Reduced mitochondrial Ca2+ uptake protects cells from oxidative stress–induced death

Although Ca2+ signals participate in gene transcription and cell proliferation, Ca2+ signals also play a crucial role in necrotic and apoptotic cell death as a consequence of mitochondrial Ca2+ overload (21, 42, 4850). Given that IP3R TKO, PLCγ2 KO, STIM1 KO, Orai1 KO, and Orai1,2 DKO lymphocytes showed reduced mitochondrial Ca2+ uptake, we tested whether eliminating mitochondrial Ca2+ accumulation attenuated oxidant-induced cell death. Wild-type, STIM2 KO, and Orai2 KO lymphocytes exposed to t-butyl hydroperoxide for 6 hours showed a considerable number of cells that stained positive with propidium iodide (PI), indicating that the cells had lost membrane integrity and were susceptible to oxidative stress–induced cell death (Fig. 8, A and B). Strikingly, IP3R TKO, PLCγ2 KO, STIM1 KO, Orai1 KO, and Orai1,2 DKO lymphocytes were resistant to cell death induced by this oxidative stressor (Fig. 8, A and B). These results revealed that the cells with reduced capacity for mitochondrial Ca2+ uptake were protected from oxidative stress–induced cell death.

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.

DISCUSSION

Ca2+ released by IP3Rs is rapidly cleared by plasma membrane (PM)–localized components and mitochondria, which decreases Ca2+ available for SERCA pumps to refill stores (22, 23, 5156). These combined effects lead to extensive ER store depletion and an activation of CRAC channels (57, 58). When depolarized, mitochondria also inhibit CRAC channels in a Ca2+-independent manner (59), and respiring mitochondria enhance the kinetics and extent of slow inactivation of CRAC (53, 60, 61). The influence on slow inactivation of CRAC is mediated by Ca2+ uptake through MCU (4, 5). In addition to influencing the Ca2+-dependent and Ca2+-independent gating of CRAC channels, mitochondria also play a role in STIM1 trafficking because strong mitochondrial depolarization reduces STIM1 puncta at ER-PM junctions without affecting its oligomerization (59). Mitochondrial Ca2+ buffering in the vicinity of the ER contributes to STIM1 oligomerization (62). Our results reveal that IICR- and SOCE-mediated Ca2+ signals also stimulate mitochondrial MCU activity, underscoring the interdependence and connectivity between these Ca2+ transport mechanisms. Here, we revealed that loss of either IP3Rs or the STIM1 and Orai1 channel complex in lymphocytes impaired the rate of mitochondrial Ca2+ uptake and decreased IMCU as a result of the down-regulation of the MCU pore subunit of the uniporter.

Increased [Ca2+]c resulting from either IICR or SOCE activates distinct cellular processes, including secretion, metabolism, proliferation, and cell survival (21, 6366). Additionally, several studies have revealed that diverse Ca2+ signals differentially control gene expression profiles (6769), and aberrant Ca2+-dependent gene regulation has been linked to debilitating human diseases, including cardiac hypertrophy, neurodegenerative disease, and severe combined immunodeficiency. IICR- and SOCE-mediated cytosolic Ca2+ signaling activates several transcription factors, including NFAT and CREB, through either dephosphorylation or phosphorylation, respectively (22). Our results provide evidence that the reduced mitochondrial Ca2+ uptake was due to diminished MCU abundance, and this effect was due to decreased MCU expression mediated by the Ca2+-dependent transcription factor CREB. Cells lacking IICR or SOCE components had barely detectable CREB phosphorylation, decreased MCU expression and MCU abundance, and decreased mitochondrial Ca2+ uptake.

MCUb is a paralog of MCU that can function as a dominant-negative subunit, which reduces MCU complex activity (13). Deletion of MCU decreases MCUb protein abundance without altering MICU1 or MICU2 in human embryonic kidney (HEK) 293T cells (11). Our experiments indicate that loss of IICR- or SOCE-dependent cytosolic Ca2+ signals reduced the mRNAs encoding MCU and MCUb, but not MICU1, suggesting that MCU and MCUb, but not MICU1, expression is regulated by cytosolic Ca2+ transients. Although the abundance of both MCU and MCUb was reduced in the KO lymphocytes, MCUb does not appear to contain a CREB binding site; rather, in silico analysis revealed that NFAT is likely the Ca2+-responsive transcription factor that regulates MCUb. Thus, although both MCU and MCUb are transcriptionally controlled by cytosolic Ca2+ signals, their regulation is likely mediated by different transcription factors.

An interesting question is whether mitochondria contribute to diseases associated with phenotypes predominantly ascribed to deficiencies in STIM or Orai proteins, such as immunodeficiency, autoimmune hemolytic anemia, thrombocytopenia, muscular hypotonia, and disturbed enamel dentition (25, 70). Altered mitochondrial Ca2+ handling may affect STIM and Orai function in disease states. However, expression of genes encoding IICR or SOCE components was unaffected by KD of MCU, suggesting that reduced mitochondrial Ca2+ influx did not affect IICR or SOCE through a transcriptional circuit. This does not preclude transcriptional regulation of genes encoding IP3R, STIM, or Orai expression through other alterations in Ca2+ signaling or that reduced mitochondrial Ca2+ uptake affects IICR or SOCE through nontranscriptional pathways.

This study established a previously unknown link between cytoplasmic and mitochondrial Ca2+ regulatory mechanisms. We revealed that IICR- and SOCE-mediated Ca2+ signals stimulated CREB activation, which influenced Ca2+ uptake through the mitochondrial calcium uniporter through altered expression of MCU. Perturbation of this these cytosolic signals altered cellular metabolism. These results provide a potential mechanism for how the absence of IP3R or STIM and Orai channel components protects cells from mitochondrial Ca2+-induced permeability transition pore opening and cell death (71, 72) by decreasing the abundance of MCU. Thus, it is likely that the down-regulation of MCU yields such protection (73). Moreover, our findings revealed that IICR and SOCE participate in basal calcium signaling that controls mitochondrial metabolism by maintaining the expression of MCU and thus mitochondrial calcium uptake. These results also suggested that cells can adapt their mitochondrial uptake capacity to match the amount of calcium signaling the cell experiences.

MATERIALS AND METHODS

Cell culture

DT40 cells were maintained in suspension culture at 40°C in complete RPMI 1640 [RPMI 1640 medium (Gibco/BLR) supplemented with 10% (v/v) fetal bovine serum (FBS), 1% chicken serum, 2 mM glutamine, 1% antibiotics (penicillin and streptomycin)]. For pretreatment with ionomycin, 2.5 nM was added to the medium for 6 days. MEFs derived from wild-type, STIM1 KO, and STIM2 KO mice were grown in complete Dulbecco’s modified Eagle’s medium (DMEM) [DMEM (Gibco/BLR) supplemented with 10% (v/v) FBS and 1% antibiotics (penicillin and streptomycin)]. HeLa KD cells [negative short hairpin RNA (shRNA) and MCU KD] were grown in complete DMEM supplemented with puromycin (2 μg/ml). All rescue cells (DKO + Orai1, DKO + Orai1 E106A) were grown in complete RPMI 1640 supplemented with G418 (500 μg/ml). To generate Orai1 rescue cells, DT40 Orai1,2 DKO cells were electroporated with either wild-type Orai1 or Orai1 E106A plasmid constructs. After 48 hours, the electroporated cells were selected with G418 (500 μg/ml) for 2 weeks, and the stable clones were maintained in complete RPMI 1640 with G418.

Ca2+ uptake and Δψm measurement in the permeabilized cell system

Cells (1 × 107) were resuspended and permeabilized with digitonin (40 μg/ml) in 1.5 ml of intracellular medium composed of 120 mM KCl, 10 mM NaCl, 1 mM KH2PO4, 20 mM Hepes-tris (pH 7.2), and 2 μM thapsigargin to block the SERCA pump. The experiments were performed in the presence of 5 mM succinate at 37°C with constant stirring. For measuring mitochondrial Ca2+ uptake, the permeabilized cells were suspended in intracellular medium containing 1.0 μM Fura-2FF. Fluorescence was monitored in a multiwavelength excitation, dual-wavelength emission fluorimeter (DeltaRAM, Photon Technology International). Extramitochondrial Ca2+ was recorded as an excitation ratio (340 nm/380 nm) and emission at 510 nm of Fura-2FF fluorescence. Δψm was measured using JC-1 (800 nm) as the ratio of the fluorescence of J-aggregate (570-nm excitation/595-nm emission) and monomer (490-nm excitation/535-nm emission) forms (74, 75). Six 3 μM Ca2+ pulses were added at 50-s interval starting at 600 s, and the changes in Δψm and extramitochondrial Ca2+ fluorescence were monitored. Mitochondrial Ca2+ uptake was derived from the decay of bath [Ca2+] after Ca2+ pulses.

To exclude Ca2+ sequestration by intracellular acidic compartments, mitochondrial Ca2+ uptake was measured in the presence of bafilomycin (1 μM), brefeldin A (1 μM), or a combination of both (76). To verify the mitochondrial Ca2+ uptake, the MCU blocker Ru360 (1 μM) and the mitochondrial uncoupler CCCP (3 μM) were added as indicated.

To assess the resting [Ca2+]m, DT40 lymphocytes were permeabilized with digitonin in intracellular-like medium containing the bath [Ca2+] indicator Fura-2FF (1 μM). After baseline recording, CCCP (3 μM) was added at 150 s to release the mitochondrial Ca2+.

Cytosolic and mitochondrial Ca2+ dynamics

DT40 lymphocytes were transiently transfected with genetically encoded mitochondrial targeted Ca2+ sensor mito-R-GECO1 plasmid by electroporation using the Gene Pulser II electroporation system (Bio-Rad). After 48 hours, the transfected cells were plated on Cell-Tak (BD Biosciences)–coated 25-mm glass coverslips for 1 hour. After 1 min of baseline recording, thapsigargin (2 μM) was added, and the change of mito-R-GECO1 fluorescence was measured with 561-nm excitation on a Carl Zeiss META 510 confocal microscope equipped with a 40× oil objective.

DT40 cells were plated on Cell-Tak–coated 25-mm glass coverslips and loaded with 2 μM Rhod-2 AM (50 min) and 5 μM Fluo-4 AM (30 min) in extracellular medium. After dye loading, cells were washed with dye-free medium and placed on the microscope stage for imaging (68, 53, 72). After 1 min of baseline recording, IgM (1.5 μg/ml) was added and confocal images were recorded every 3 s (510 Meta; Carl Zeiss) with 488- and 561-nm excitation using a 40× oil objective to simultaneously monitor cytoplasmic and mitochondrial Ca2+ dynamics. Images were analyzed and quantified using ZEN 2010 software. To monitor the cytosolic spontaneous Ca2+ oscillations, DT40 cells were loaded with cytosolic Ca2+ indicator Fluo-4 AM as described above. Fluo-4–loaded cells were placed on the microscope stage for monitoring Ca2+ oscillations.

Mitoplast patch-clamp recording

Mitoplast patch-clamp recordings were performed at 30°C as detailed previously (6, 14, 16, 77) with the following modifications. Freshly prepared mitoplasts were stored at 4°C for about 60 to 90 min. Mitoplasts were placed upon the Cell-Tak–coated coverslips and mounted on the microscope. The integrity of the mitoplasts was stable for 15 min at 30°C. Mitoplasts isolated from DT40 B lymphocytes were bathed in a solution containing sodium gluconate (150 mM), KCl (5.4 mM), CaCl2 (5 mM), and Hepes (10 mM) (pH 7.2). The pipette solution contained sodium gluconate (150 mM), NaCl (5 mM), sucrose (135 mM), Hepes (10 mM), and EGTA (1.5 mM) (pH 7.2). After formation of gigohm seals (20 to 35 megohms), the mitoplasts were ruptured with a 200- to 400-mV pulse for 2 to 6 ms. Mitoplast capacitance was measured (2.5 to 3.0 pF). After capacitance compensation, mitoplasts were held at 0 mV and IMCU was elicited with a voltage ramp (from −160 to 80 mV, 120 mV/s).

Samples were discarded if the break-in took longer than 5 s after addition of 5 mM Ca2+. Currents were recorded using an Axon200B patch-clamp amplifier with a Digidata 1320A acquisition board (pCLAMP 10.0 software; Axon Instruments). The pipette solution (5 mM Ca2+) was chosen on the basis of previous measurements (6).

Western blotting analysis

Cell extracts were prepared from ionomycin-treated and untreated DT40 cells, HeLa negative shRNA, HeLa MCU KD (6), wild-type, STIM1 KO, and STIM2 KO MEFs using radioimmunoprecipitation assay buffer [50 mM tris-HCl (pH 7.4), 150 mM NaCl, 0.25% deoxycholic acid, 1 mM EDTA, 1% NP-40, protease inhibitor cocktail (Complete, Roche), and Halt phosphatase inhibitor cocktail (Thermo Scientific)]. Equal amounts of protein (25 μg per lane) were separated on 4 to 12% bis-tris polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and probed with antibodies specific for MCU (custom-made antibody), MICU1 (1:1000, Cell Signaling Technology), MCUR1 (1:1000, Aviva Systems Biology), cyclophilin D (1:1000, Abcam), CREB (1:1000, Cell Signaling Technology), pCREB (1:1000, Cell Signaling Technology), IP3R1 (1:2000, Bethyl Laboratories), STIM1 (1:1000, BD Transduction Laboratories), Orai1 (1:2000, Sigma-Aldrich), ERK (1:2000, Thermo Scientific), and pERK (1:1000, Cell Signaling Technology). The polyclonal rabbit MCU antibody was custom-prepared by YenZym Antibodies, LLC. The following MCU peptide was used for immunization with KLH conjugation: CGVSRHRQQQHHRTVHQR. The polyclonal antibody was affinity-purified, and the specificity was confirmed by enzyme-linked immunosorbent assay.

NADPH measurements

Ionomycin-treated and untreated DT40 cells (10 × 106 cells) were suspended in Hanks’ balanced salt solution (Sigma). Autofluorescence of NADPH was monitored at 350/460 nm (excitation/emission) using a multiwavelength excitation, dual-wavelength emission fluorimeter (DeltaRAM, Photon Technology International) (78). The experiments were performed at 37°C.

Quantitative RT-PCR

Total RNA from DT40 lymphocytes and HeLa cells was isolated using RNeasy Mini Kit from Qiagen and reverse-transcribed using iScript cDNA (complementary DNA) synthesis kit (Bio-Rad). Real-time quantification was performed in a 20-μl reaction, 96-well format [0.2 μl of cDNA; 300 nM of forward and reverse primers; 1× iQ SYBR Green SuperMix (Bio-Rad)] using the StepOnePlus Real-Time PCR Systems (Applied Biosystems). Specific primers that were used for the RT-PCR amplification included the following: chicken MCU, 5′-TTGGCAGAGTGTGAGAGTGG-3′ (forward) and 5′-AATTCCTCGGTCCTCTGCTT-3′ (reverse); chicken MCUb, 5′-AGGCTTCTATGGGTTGGTTTAG-3′ (forward) and 5′-CTCCATGATGTCCCACGAATAG-3′ (reverse); chicken MICU1, 5′-CAGCCCTACAATCCCTAAGAAC-3′ (forward) and 5′-GGAGAAATCGAAGACGGAACA-3′ (reverse); chicken GAPDH, 5′-GGACACTTCAAGGGCACTGT-3′ (forward) and 5′-TCTCCATGGTGGTGAAGACA-3′ (reverse). The relative amount of MCU, MCUb, and MICU1 mRNA normalized to GAPDH was calculated using the StepOnePlus software v2.3.

For quantification of mRNA encoding IP3R1 and CRAC components in HeLa MCU KD cells, TaqMan probes for STIM1 (Life Technologies, assay ID: Hs00162394_m1), ITPR1 (Life Technologies, assay ID: Hs00181881_m1), and ORAI1 (Life Technologies, assay ID: Hs03046013_m1) were used. All data were normalized to human β-actin content (Life Technologies, human ACTB, endogenous control, VIC/MGB probe) and depicted as fold change over negative shRNA cells.

ChIP, quantitative PCR, and luciferase activity

HeLa cells were grown to ~80 to 90% confluency in a 150-mm culture dish containing 20 ml of DMEM. The cells were stimulated with ionomycin (2.5 μM) with or without forskolin (5 μM) for 30 min. ChIP assays were performed using Magna ChIP G kit (Millipore) according to the manufacturer’s instruction. In brief, DNA-protein complexes were crosslinked and immunoprecipitated using ChIP-validated antibodies against RNA polymerase II, pCREB (Cell Signaling Technology), and negative control IgG. Immune complexes were extracted and analyzed by 2% agarose gel after PCR or by quantitative PCR using primers GPH1001674(−)01A (Qiagen) that flank specific regions in the MCU promoter and the SimpleChIP human c-FOS promoter primers (Cell Signaling Technology). Values were normalized by input DNA. Results were depicted as the fold enrichment over basal expression.

HeLa cells (1 × 106) were transfected with luciferase reporter plasmids (4 μg) containing MCU promoter sequence with or without binding elements for CREB using Mirus LT1 transfection reagent. After 48 hours, cells were stimulated with ionomycin (2.5 μM) with or without forskolin (5 μM) for 30 min. The cells were lysed, and luciferase activity was measured (LightSwitch Luciferase Assay Reagent) using a plate reader (Infinite M1000 PRO, Tecan).

PDH activity

Total cell lysate from DT40 lymphocytes were subjected to PDH enzyme activity in a microplate assay (Mitochondria Isolation Kit; #ab11070, Abcam; PDH Microplate Assay Kit, #ab109902, Abcam). The activity is determined by the rate of conversion of NAD+ to NADH, which is coupled with reporter dye, and the absorbance change was recorded at 450 nm (Infinite M1000 PRO, Tecan).

Measurement of lactate

The assay was performed using LDH assay kit (Pierce). Cells (1 × 104) were lysed with 10× lysis buffer for 45 min. The lysate was centrifuged at 1250g for 3 min, and 50 μl of the supernatant was transferred to a 96-well plate with the detection reagents lacking any substrate from the kit. After incubation at room temperature for 30 min, the reaction was terminated, and the formazan was measured by spectrophotometric absorbance at 490 and 680 nm (Infinite M1000 PRO, Tecan). The formation of formazan was proportional to the amount of lactate converted into pyruvate through the reduction of NAD+ to NADH.

Measurement of mtDNA content

DNA was isolated from lymphocytes by SDS–proteinase K digestion, and 2 μg of DNA was digested by Bam HI. Biological triplicate samples for wild-type and KO (with and without ionomycin) were resolved by electrophoresis through a 0.6% agarose gel in 0.5× tris-borate EDTA. Gels were processed and transferred by capillary action. The membrane was ultraviolet crosslinked and probed with a random-primed radiolabeled probe against the nuclear 18S rRNA and signal of the single band quantitated by photostimulated luminescence. The membrane was reprobed with random-primed radiolabeled probe against PCR-amplified mtDNA fragments without stripping and the signal of the mtDNA band quantitated. Each band was discrete. The mtDNA signal was normalized to nuclear DNA and relative mtDNA abundance calculated relative to control (7).

Cell viability measurement

DT40 lymphocytes were exposed to t-butyl hydroperoxide (200 μM) for 6 hours, then the cells were harvested and stained with PI, and cells were sorted with a BD FACSCanto (BD Biosciences) immediately. Dead cells were defined as PI-positive. Relative PI staining was plotted on a logarithmic scale using FlowJo software.

Statistical analysis

Data from multiple experiments were quantified and illustrated as means ± SEM, and differences between groups were analyzed by two-tailed paired t test followed by a post hoc Bonferroni correction. P < 0.00625 was considered statistically significant. To determine the significance within groups, a one-way ANOVA with Tukey correction was performed. P < 0.05 was considered significant. The significance of mRNA and protein abundance was determined as P < 0.05 by Z test. Data were analyzed and plotted with either GraphPad Prism version 5.0 or SigmaPlot 11.0 software.

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.

REFERENCES AND NOTES

Acknowledgments: We thank A. Rao and R. E. Campbell for sharing STIM KO MEFs and mito-R-GECO1 plasmid construct, respectively. We also thank F. Shaikh and M. Cheung for their technical assistance. We thank H. Zhao for help with statistical analysis. Funding: This research was funded by the NIH (R01HL086699, R01HL119306, and R01GM109882 to M.M.). Author contributions: S.S., S.R., N.E.H., and M.M. designed and performed experiments, analyzed data, and wrote the manuscript. X.Z. and J.Y.C. performed electrophysiology experiments and data analysis; J.E.K. and B.A.K. performed and interpreted the mtDNA analysis; K.J.H., S.G., and J.R. performed confocal imaging and luciferase assay; Y.B., Y.Z., D.L.G., and T.K. generated the DT40 KO lymphocytes. N.R.J. and R.C. performed flow cytometry analysis. D.L.G. and T.K. edited the manuscript. M.M. conceived the project and wrote the manuscript. Competing interests: The authors declare that they have no competing interests.
View Abstract

Navigate This Article