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Routes of Ca2+ Shuttling during Ca2+ Oscillations

FOCUS ON THE ROLE OF MITOCHONDRIAL Ca2+ HANDLING AND CYTOSOLIC Ca2+ BUFFERS*
  • László Pecze
    Correspondence
    To whom correspondence should be addressed. Tel.: 41-26-300-85-11; Fax: 41-26-300-97-33
    Affiliations
    Anatomy, Department of Medicine, University of Fribourg, Route Albert-Gockel 1, CH-1700 Fribourg, Switzerland
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  • Walter Blum
    Affiliations
    Anatomy, Department of Medicine, University of Fribourg, Route Albert-Gockel 1, CH-1700 Fribourg, Switzerland
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  • Beat Schwaller
    Affiliations
    Anatomy, Department of Medicine, University of Fribourg, Route Albert-Gockel 1, CH-1700 Fribourg, Switzerland
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  • Author Footnotes
    * This work was partially supported by Swiss National Science Foundation Grants 130680 and 147697 (Sinergia) (to B. S.). The authors declare that they have no conflicts of interest with the contents of this article.
    This article contains a supplemental Excel document showing an example of mathematical simulation.
Open AccessPublished:September 22, 2015DOI:https://doi.org/10.1074/jbc.M115.663179
      In some cell types, Ca2+ oscillations are strictly dependent on Ca2+ influx across the plasma membrane, whereas in others, oscillations also persist in the absence of Ca2+ influx. We observed that, in primary mesothelial cells, the plasmalemmal Ca2+ influx played a pivotal role. However, when the Ca2+ transport across the plasma membrane by the “lanthanum insulation method” was blocked prior to the induction of the serum-induced Ca2+ oscillations, mitochondrial Ca2+ transport was found to be able to substitute for the plasmalemmal Ca2+ exchange function, thus rendering the oscillations independent of extracellular Ca2+. However, in a physiological situation, the Ca2+-buffering capacity of mitochondria was found not to be essential for Ca2+ oscillations. Moreover, brief spontaneous Ca2+ changes were observed in the mitochondrial Ca2+ concentration without apparent changes in the cytosolic Ca2+ concentration, indicating the presence of a mitochondrial autonomous Ca2+ signaling mechanism. In the presence of calretinin, a Ca2+-buffering protein, the amplitude of cytosolic spikes during oscillations was decreased, and the amount of Ca2+ ions taken up by mitochondria was reduced. Thus, the increased calretinin expression observed in mesothelioma cells and in certain colon cancer might be correlated to the increased resistance of these tumor cells to proapoptotic/pronecrotic signals. We identified and characterized (experimentally and by modeling) three Ca2+ shuttling pathways in primary mesothelial cells during Ca2+ oscillations: Ca2+ shuttled between (i) the endoplasmic reticulum (ER) and mitochondria, (ii) the ER and the extracellular space, and (iii) the ER and cytoplasmic Ca2+ buffers.

      Introduction

      The calcium ion (Ca2+) is a universal intracellular messenger that controls a diverse range of cellular processes including cell proliferation, apoptosis, fertilization, neurotransmitter release, and heartbeat among many others (
      • Berridge M.J.
      • Bootman M.D.
      • Roderick H.L.
      Calcium signalling: dynamics, homeostasis and remodelling.
      ). Ca2+ pumps in the plasma membrane (plasma membrane Ca2+-ATPase) and in endoplasmic reticulum (ER)
      The abbreviations used are: ER
      endoplasmic reticulum
      SERCA
      sarco/endoplasmic reticulum Ca2+-ATPase
      ccyt
      cytosolic free Ca2+ concentration
      cnucl
      nuclear free Ca2+ concentration
      cER
      free Ca2+ concentration in the ER lumen
      cmito
      free Ca2+ concentration in the mitochondrial matrix
      MCU
      mitochondrial calcium uniporter
      CCCP
      carbonyl cyanide m-chlorophenylhydrazone
      prMC
      primary mouse mesothelial cells
      EBFP
      enhanced blue fluorescent protein
      InsP3R
      inositol trisphosphate receptor
      InsP3
      inositol trisphosphate
      ΔΨ
      mitochondrial membrane potential
      BAPTA
      1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid
      AM
      acetoxymethyl ester
      CR
      calretinin
      qRT-PCR
      quantitative RT-PCR
      ROI
      region of interest.
      membranes (SERCA) are responsible for the low cytosolic (ccyt) and nuclear free Ca2+ concentrations (cnucl) (50–100 nm) compared with the free Ca2+ concentrations in the extracellular space (1–2 mm) and the ER lumen (cER) (100–500 μm). At rest, the free Ca2+ concentration in the mitochondrial matrix (cmito) is close to the resting ccyt, but it rises to 20–30 μm during stimulation, e.g. in motor nerve terminals in Drosophila melanogaster (
      • Ivannikov M.V.
      • Macleod G.T.
      Mitochondrial free Ca2+ levels and their effects on energy metabolism in Drosophila motor nerve terminals.
      ). Cell activation in a wide range of cell types results in Ca2+ oscillations and in transient waves of increased ccyt (
      • Rooney T.A.
      • Sass E.J.
      • Thomas A.P.
      Characterization of cytosolic calcium oscillations induced by phenylephrine and vasopressin in single fura-2-loaded hepatocytes.
      • Clapham D.E.
      • Lechleiter J.D.
      • Girard S.
      Intracellular waves observed by confocal microscopy from Xenopus oocytes.
      ,
      • Yule D.I.
      • Gallacher D.V.
      Oscillations of cytosolic calcium in single pancreatic acinar cells stimulated by acetylcholine.
      • Martin S.C.
      • Shuttleworth T.J.
      Ca2+ influx drives agonist-activated [Ca2+]i oscillations in an exocrine cell.
      ). These oscillations (or waves) are not restricted to ccyt, but also cnucl (
      • Estrada M.
      • Uhlen P.
      • Ehrlich B.E.
      Ca2+ oscillations induced by testosterone enhance neurite outgrowth.
      ), cER (
      • Pecze L.
      • Schwaller B.
      Characterization and modeling of Ca2+ oscillations in mouse primary mesothelial cells.
      ), and cmito show Ca2+ oscillations (
      • Ishii K.
      • Hirose K.
      • Iino M.
      Ca2+ shuttling between endoplasmic reticulum and mitochondria underlying Ca2+ oscillations.
      ). The spatial extent of the oscillatory Ca2+ signal is also important. (i) In astrocytes, the area of Ca2+ oscillations is sometimes restricted to only one protrusion regulating the release of gliotransmitters; i.e. different oscillatory frequencies can coexist at the same time within the same cell (
      • Wu Y.W.
      • Tang X.
      • Arizono M.
      • Bannai H.
      • Shih P.Y.
      • Dembitskaya Y.
      • Kazantsev V.
      • Tanaka M.
      • Itohara S.
      • Mikoshiba K.
      • Semyanov A.
      Spatiotemporal calcium dynamics in single astrocytes and its modulation by neuronal activity.
      ). (ii) In Xenopus laevis oocytes, regenerative spiral waves of release of free Ca2+ spread through the entire cell (
      • Lechleiter J.
      • Girard S.
      • Peralta E.
      • Clapham D.
      Spiral calcium wave propagation and annihilation in Xenopus laevis oocytes.
      ). (iii) Intercellular Ca2+ waves spreading via gap junctions occur in rat liver epithelial cells upon mechanical stimulation (
      • Frame M.K.
      • de Feijter A.W.
      Propagation of mechanically induced intercellular calcium waves via gap junctions and ATP receptors in rat liver epithelial cells.
      ).
      In cells maintained in vitro, serum starvation followed by readministration leads to intracellular Ca2+ signals, most often in the form of oscillations (
      • Foreman M.A.
      • Smith J.
      • Publicover S.J.
      Characterisation of serum-induced intracellular Ca2+ oscillations in primary bone marrow stromal cells.
      ,
      • Wood A.
      • Wing M.G.
      • Benham C.D.
      • Compston D.A.
      Specific induction of intracellular calcium oscillations by complement membrane attack on oligodendroglia.
      ). The precise mechanism(s) leading to these oscillations is poorly understood because serum contains a large number of known and as yet unidentified growth factors and mitogenic compounds, all potentially participating in this oscillatory activity (
      • Goustin A.S.
      • Leof E.B.
      • Shipley G.D.
      • Moses H.L.
      Growth factors and cancer.
      ). In Swiss 3T3 cells, serum-induced Ca2+ changes are essential but not sufficient to induce NF-κB activation and subsequent DNA synthesis (
      • Sée V.
      • Rajala N.K.
      • Spiller D.G.
      • White M.R.
      Calcium-dependent regulation of the cell cycle via a novel MAPK-NF-κB pathway in Swiss 3T3 cells.
      ). In some cell types, Ca2+ oscillations even persist in the absence of Ca2+ influx across the plasma membrane (
      • Rooney T.A.
      • Sass E.J.
      • Thomas A.P.
      Characterization of cytosolic calcium oscillations induced by phenylephrine and vasopressin in single fura-2-loaded hepatocytes.
      ,
      • Clapham D.E.
      • Lechleiter J.D.
      • Girard S.
      Intracellular waves observed by confocal microscopy from Xenopus oocytes.
      ), whereas in others, Ca2+ oscillations strictly depend on Ca2+ influx (
      • Yule D.I.
      • Gallacher D.V.
      Oscillations of cytosolic calcium in single pancreatic acinar cells stimulated by acetylcholine.
      ,
      • Pecze L.
      • Schwaller B.
      Characterization and modeling of Ca2+ oscillations in mouse primary mesothelial cells.
      ). Mitochondria influence cytosolic Ca2+ oscillations in at least two ways. First, mitochondria produce ATP, which is required for SERCA and plasma membrane Ca2+-ATPase function, that results in Ca2+ extrusion and thus lowering of ccyt. Second, during ccyt oscillations, cmito also manifests oscillations, indicative of a role of mitochondria in shaping and/or modulating ccyt oscillations (
      • Ishii K.
      • Hirose K.
      • Iino M.
      Ca2+ shuttling between endoplasmic reticulum and mitochondria underlying Ca2+ oscillations.
      ). Ca2+ uptake into the mitochondria is determined by both the large negative voltage (−150 to −180 mV) across the inner membrane that results from the proton pumping by the respiratory chain and the Ca2+ concentration gradient between the cytoplasm and matrix (
      • Rizzuto R.
      • Pozzan T.
      Microdomains of intracellular Ca2+: molecular determinants and functional consequences.
      ). The mitochondrial calcium uniporter (MCU) is the key player responsible for the uptake of Ca2+ by mitochondria (
      • De Stefani D.
      • Raffaello A.
      • Teardo E.
      • Szabò I.
      • Rizzuto R.
      A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter.
      ). The MCU has a rather low Ca2+ affinity and operates over a micromolar range of cytosolic Ca2+.
      To address these questions, we performed lanthanum (La3+) insulation experiments where both the Ca2+ influx and efflux across the plasma membrane are blocked (
      • Van Breemen C.
      • Farinas B.R.
      • Gerba P.
      • McNaughton E.D.
      Excitation-contraction coupling in rabbit aorta studied by the lanthanum method for measuring cellular calcium influx.
      ). We hypothesized that under these experimental conditions mitochondria serving as a Ca2+ store/source might substitute for this function normally exerted by the extracellular space. Using a genetically encoded Ca2+ indicator targeted to the mitochondria, we managed to verify this assumption in vitro. In addition, we investigated the effects of the following compounds on ccyt oscillations and mitochondrial Ca2+ handling: the proton uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), the mitochondrial Na+/Ca2+ blocker CGP-37157, the mitochondrial MCU blocker Ru-360, and finally the “Ca2+-buffering” protein calretinin. Based on the experimental findings, we built a mathematical model for Ca2+ oscillations taking into account the various processes implicated in these oscillations.

      Discussion

      Characteristics of mitochondrial Ca2+ transport have not been examined in detail in most cell types. The main reason why we know relatively little about mitochondrial Ca2+ handling is because the molecular identity of the channels involved in mitochondrial transport have only recently been discovered (
      • De Stefani D.
      • Raffaello A.
      • Teardo E.
      • Szabò I.
      • Rizzuto R.
      A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter.
      ,
      • Perocchi F.
      • Gohil V.M.
      • Girgis H.S.
      • Bao X.R.
      • McCombs J.E.
      • Palmer A.E.
      • Mootha V.K.
      MICU1 encodes a mitochondrial EF hand protein required for Ca2+ uptake.
      ,
      • Waldeck-Weiermair M.
      • Jean-Quartier C.
      • Rost R.
      • Khan M.J.
      • Vishnu N.
      • Bondarenko A.I.
      • Imamura H.
      • Malli R.
      • Graier W.F.
      Leucine zipper EF hand-containing transmembrane protein 1 (Letm1) and uncoupling proteins 2 and 3 (UCP2/3) contribute to two distinct mitochondrial Ca2+ uptake pathways.
      ), and specifically targeted, pH- and ΔΨ-insensitive Ca2+ indicators are only currently available (
      • Wu J.
      • Liu L.
      • Matsuda T.
      • Zhao Y.
      • Rebane A.
      • Drobizhev M.
      • Chang Y.F.
      • Araki S.
      • Arai Y.
      • March K.
      • Hughes T.E.
      • Sagou K.
      • Miyata T.
      • Nagai T.
      • Li W.H.
      • Campbell R.E.
      Improved orange and red Ca2+ indicators and photophysical considerations for optogenetic applications.
      ). Nevertheless, there are few models for Ca2+ oscillations where the function of mitochondrial Ca2+ uptake has been taken into account (
      • Marhl M.
      • Schuster S.
      • Brumen M.
      Mitochondria as an important factor in the maintenance of constant amplitudes of cytosolic calcium oscillations.
      ).
      Our experiments affirm previous data that mitochondria, even at the resting state, are able to transport and store Ca2+ ions (
      • Haynes C.L.
      • Buhler L.A.
      • Wightman R.M.
      Vesicular Ca2+-induced secretion promoted by intracellular pH-gradient disruption.
      ). The fast release of the stored Ca2+ from the mitochondria due to the decrease/collapse of the membrane potential indicates that the strongly negative ΔΨ ensures a constant Ca2+ uptake into the mitochondria. This uptake is in a steady-state equilibrium with the constant Ca2+ efflux mediated by the mitochondrial exchangers (
      • De Marchi U.
      • Santo-Domingo J.
      • Castelbou C.
      • Sekler I.
      • Wiederkehr A.
      • Demaurex N.
      NCLX protein, but not LETM1, mediates mitochondrial Ca2+ extrusion, thereby limiting Ca2+-induced NAD(P)H production and modulating matrix redox state.
      ), and the efflux is an electrogenic process (
      • Castaldo P.
      • Cataldi M.
      • Magi S.
      • Lariccia V.
      • Arcangeli S.
      • Amoroso S.
      Role of the mitochondrial sodium/calcium exchanger in neuronal physiology and in the pathogenesis of neurological diseases.
      ). The electrochemical proton gradient across the inner mitochondrial is used to remove the excess Ca2+ ions (
      • Kaasik A.
      • Safiulina D.
      • Zharkovsky A.
      • Veksler V.
      Regulation of mitochondrial matrix volume.
      ). Our recordings show that this basal steady-state mitochondrial Ca2+ concentration can fluctuate, showing “spontaneous” mitochondrial Ca2+ spikes. Most probably this is mediated by an endogenous MCU activator that has not been identified at the molecular level yet. Ca2+ transients in ccyt were previously reported to evoke an increase in cmito, activating both cytoplasmic (
      • Van Breemen C.
      • Farinas B.R.
      • Gerba P.
      • McNaughton E.D.
      Excitation-contraction coupling in rabbit aorta studied by the lanthanum method for measuring cellular calcium influx.
      ) and mitochondrial enzymes (
      • Ivannikov M.V.
      • Macleod G.T.
      Mitochondrial free Ca2+ levels and their effects on energy metabolism in Drosophila motor nerve terminals.
      ). Thus, Ca2+ transients observed selectively in cmito in some prMC (Fig. 2A) might allow for the autonomous activation of mitochondrial enzymes. The Ca2+ ions causing the mitochondrial spike are likely to originate from the cytosolic compartment; however, our results indicate that the amount of Ca2+ ions responsible for the increase in cmito was not sufficient to be detected as a decrease in ccyt. Alternatively, at basal conditions, the equilibrium level of ccyt might be regulated by a rather rapid constant exchange of Ca2+ ions among the cytosol, the extracellular space, and/or the ER compartment.
      The Ca2+ oscillation models usually differ in how they simulate the functions of InsP3R, the channel that transports Ca2+ ions from the ER to the cytosol. The “ccyt/[InsP3]” models (for a review, see Ref.
      • Schuster S.
      • Marhl M.
      • Höfer T.
      Modelling of simple and complex calcium oscillations. From single-cell responses to intercellular signalling.
      ) postulate that the InsP3R has a binding site for InsP3, an activating binding site for Ca2+, and an inhibiting binding site for Ca2+. In these models, all binding sites are localized on the cytoplasmic side, and the function of InsP3 does not depend on cER. Binding of Ca2+ to the activating site and of InsP3 to the InsP3 binding site opens the channel, whereas Ca2+ binding to the inhibiting site closes the InP3R. Moreover, the binding of Ca2+ to the inhibiting site occurs rather slowly and with a lower affinity as compared with the activating site, subsequently resulting in oscillations in ccyt. In these models, the InsP3 concentration uniquely determines the oscillation frequency (
      • De Young G.W.
      • Keizer J.
      A single-pool inositol 1,4,5-trisphosphate-receptor-based model for agonist-stimulated oscillations in Ca2+ concentration.
      ). In the “store loading” models (also called “ccyt/cER” models), the function of InsP3R depends not only on ccyt but also on cER. In these models, the Ca2+ influx across the plasma membrane plays a critical role in determining the oscillation frequency (
      • Pecze L.
      • Schwaller B.
      Characterization and modeling of Ca2+ oscillations in mouse primary mesothelial cells.
      ,
      • Somogyi R.
      • Stucki J.W.
      Hormone-induced calcium oscillations in liver cells can be explained by a simple one pool model.
      ,
      • Dupont G.
      • Goldbeter A.
      One-pool model for Ca2+ oscillations involving Ca2+ and inositol 1,4,5-trisphosphate as co-agonists for Ca2+ release.
      ). At a constant [InsP3], the duration of the interspike period is determined by the velocity of cellular Ca2+ replenishment, which is manifested as a continuous ER loading together with a constant basal ccyt. The experimentally observable sawtooth wave oscillations in cER during the cytoplasmic baseline spiking oscillations are an important argument in favor of the store loading theory (
      • Pecze L.
      • Schwaller B.
      Characterization and modeling of Ca2+ oscillations in mouse primary mesothelial cells.
      ). However, the store loading-based models cannot cope with the fact that in some cells the Ca2+ oscillations do not depend on Ca2+ influx across the plasma membrane. Our experiments and modeling studies revealed that the incorporation of mitochondria as an additional Ca2+ source/store in the store loading-based models considerably augments the quality of the simulations. That is, the modeling predictions are more congruent with the experimental findings, which allows for a better mechanistic understanding. The mitochondrial Ca2+ transport enables the store loading-based models also to display Ca2+ oscillation in the absence of extracellular Ca2+.
      The simulation of the La3+ insulation was previously endeavored by Sneyd et al. (
      • Sneyd J.
      • Tsaneva-Atanasova K.
      • Yule D.I.
      • Thompson J.L.
      • Shuttleworth T.J.
      Control of calcium oscillations by membrane fluxes.
      ). Although their model does not contain mitochondria and moreover ccyt is continuously decreasing during the oscillations, their model reveals important aspects of the Ca2+ oscillations, namely their dependence on the total Ca2+ load of the cell. In their model, the cell has a high resting Ca2+; upon agonist stimulation, the activation of plasma membrane Ca2+-ATPases causes a net loss of Ca2+ from the cells even though the Ca2+ influx is augmented after stimulation (
      • Sneyd J.
      • Tsaneva-Atanasova K.
      • Yule D.I.
      • Thompson J.L.
      • Shuttleworth T.J.
      Control of calcium oscillations by membrane fluxes.
      ). A similar phenomenon is also observed in our model; the total cellular Ca2+ content (ccyt + cER + cmito) determines the response to the La3+ insulation; blocking of the Ca2+ influx and efflux results in an oscillation stop that can either occur after a final Ca2+ spike or directly after La3+ addition, i.e. without a change in ccyt. In contrast to the previous model (
      • Sneyd J.
      • Tsaneva-Atanasova K.
      • Yule D.I.
      • Thompson J.L.
      • Shuttleworth T.J.
      Control of calcium oscillations by membrane fluxes.
      ), basal ccyt levels during the interspike phase of the oscillations remain constant. This is in line with the experiments carried out by us and others (
      • Bird G.S.
      • Putney Jr., J.W.
      Capacitative calcium entry supports calcium oscillations in human embryonic kidney cells.
      ).
      Shuttling of Ca2+ ions between the ER and mitochondria was experimentally demonstrated in the study of Ishii et al. (
      • Ishii K.
      • Hirose K.
      • Iino M.
      Ca2+ shuttling between endoplasmic reticulum and mitochondria underlying Ca2+ oscillations.
      ). They reported that in HeLa cells the cycles of ER/mitochondrion shuttling are repeated until cmito has reached the basal level prior to the stimulation. In our study with prMC, we observed Ca2+ oscillation even (i) when cmito had reached its basal levels or (ii) if cmito had been considerably lowered by CCCP administration. One has to keep in mind that CCCP also results in the collapse of the plasma membrane potential (
      • Lichtshtein D.
      • Kaback H.R.
      • Blume A.J.
      Use of a lipophilic cation for determination of membrane potential in neuroblastoma-glioma hybrid cell suspensions.
      ), which subsequently reduces the plasmalemmal Ca2+ influx (
      • Houamed K.
      • Fu J.
      • Roe M.W.
      • Philipson L.H.
      ). Thus, one reason for the CCCP-evoked stop in oscillations might be a disturbed Ca2+ influx. Moreover, the CCCP-mediated drop in ATP production likely leading to an impairment of the ER Ca2+ transport might also contribute to the oscillation arrest (
      • Walsh C.
      • Barrow S.
      • Voronina S.
      • Chvanov M.
      • Petersen O.H.
      • Tepikin A.
      Modulation of calcium signalling by mitochondria.
      ); i.e. the effects of protonophores are not exclusively attributed to the reduced mitochondrial Ca2+ uptake as was proposed in earlier studies (
      • Ishii K.
      • Hirose K.
      • Iino M.
      Ca2+ shuttling between endoplasmic reticulum and mitochondria underlying Ca2+ oscillations.
      ). When CCCP was administered before serum, it caused a Ca2+ transient due to the mitochondrial release, which was followed by a period of lower resting ccyt. A lower ccyt is a sign of the reduced Ca2+ influx (resting plasmalemmal Ca2+ leakage). There was a similar decrease in resting ccyt when the extracellular free Ca2+ was chelated by EGTA (data not shown).
      The Ca2+ influx across the plasma membrane is important to sustain the Ca2+ oscillations in prMC (
      • Pecze L.
      • Blum W.
      • Schwaller B.
      Mechanism of capsaicin receptor TRPV1-mediated toxicity in pain-sensing neurons focusing on the effects of Na+/Ca2+ fluxes and the Ca2+-binding protein calretinin.
      ) but not in HeLa cells (
      • Ishii K.
      • Hirose K.
      • Iino M.
      Ca2+ shuttling between endoplasmic reticulum and mitochondria underlying Ca2+ oscillations.
      ). The different dependence of these cell types on extracellular Ca2+ for the oscillations might be the result of differences in the contribution/importance of the various Ca2+ shuttling pathways between ER and mitochondria on the one hand and between ER and the extracellular space on the other. Our results indicate that plasmalemmal Ca2+ extrusion systems and mitochondrial Ca2+ uptake channels compete for the Ca2+ ions released from the ER. We hypothesize that in some cells, such as prMC and HEK cells (
      • Bird G.S.
      • Putney Jr., J.W.
      Capacitative calcium entry supports calcium oscillations in human embryonic kidney cells.
      ), the shuttling between the extracellular space and the ER dominates over the shuttling between mitochondria and the ER. However, in HeLa cells and hepatocytes, the ER/mitochondrion shuttling prevails. This might explain why Ca2+ oscillations in some cells are strongly dependent on extracellular Ca2+ ions but not in others.
      Another often neglected aspect about “Ca2+ shuttling” pathways is the contribution of cytosolic Ca2+ buffers present at rather high concentrations in the cytosol of some cell types. They are expected to modulate the Ca2+ shuttling among all compartments, extracellular space, ER, and mitochondria, as well as to transiently affect ccyt (Fig. 7). A strong interdependence between cytoplasmic Ca2+ buffers and mitochondria has been demonstrated before. The expression levels of parvalbumin, a Ca2+-buffering protein with slow binding kinetics, and the mitochondrial volume in fast twitch muscle cells and in parvalbumin-expressing neurons are inversely regulated (Ref.
      • Ducreux S.
      • Gregory P.
      • Schwaller B.
      Inverse regulation of the cytosolic Ca2+ buffer parvalbumin and mitochondrial volume in muscle cells via SIRT1/PGC-1α axis.
      , and for more details, see Ref.
      • Schwaller B.
      The regulation of a cell's Ca2+ signaling toolkit: the Ca2+ homeostasome.
      ). In our study, we observed that overexpression of calretinin modifies Ca2+ signals and associated oscillations. It reduces the amount of Ca2+ ions shuttling both between the ER and mitochondria and between the ER and the cytoplasm. Our model predicts that at calretinin concentrations >1 μm Ca2+ oscillations should be blocked in prMC. This is in apparent contradiction with the experimental results where oscillations still existed in EBFP-calretinin-expressing cells likely expressing levels higher than 1 μm (Fig. 6). However, in our modeling, the Ca2+ microdomain was not considered, and Ca2+ binding characteristics of calretinin (e.g. Kd and kon) might be different in the cytosol of prMC than the parameters determined in vitro (
      • Fridlyand L.E.
      • Tamarina N.
      • Philipson L.H.
      Modeling of Ca2+ flux in pancreatic β-cells: role of the plasma membrane and intracellular stores.
      ). Furthermore, adaptation/compensation mechanisms might be induced in prMC overexpressing calretinin that would still allow for the generation of Ca2+ oscillations.
      Figure thumbnail gr7
      FIGURE 7.Contribution of Ca2+ signaling toolkit components to serum-induced Ca2+ oscillations in prMC. A, in unperturbed (control) prMC in vitro, Ca2+ oscillations are primarily the result of the interplay between Ca2+ from the extracellular space and the ER with some minor contributions of mitochondria. The arrows indicate the shuttling of Ca2+ ions between the different compartments (the thicker the arrow, the more important is this pathway). In prMC, expression of calretinin is virtually absent, excluding an important role of this protein with respect to mobile Ca2+ buffering. B, if cells are subjected to La3+ insulation excluding the exchange of Ca2+ ions via the plasma membrane, the repetitive Ca2+ exchange between the ER and mitochondria allows for the generation of Ca2+ oscillations. C, the addition of the mobile Ca2+ buffer calretinin as observed in reactive mesothelial cells and mesothelioma cells affects Ca2+ oscillations. High expression levels (>1 μm in our model) completely block oscillations; lower levels (≈0.5 μm) reduce the amplitude of ccyt as well as of cmito during Ca2+ oscillations; i.e. calretinin competes with mitochondria, thus reducing the shuttling of Ca2+ ions between the ER and mitochondria.
      Of relevance, calretinin reduced the mitochondrial Ca2+ uptake and Ca2+ accumulation. In human malignant mesothelioma, mostly of the epithelioid type, calretinin is overexpressed (
      • Doglioni C.
      • Dei Tos A.P.
      • Laurino L.
      • Iuzzolino P.
      • Chiarelli C.
      • Celio M.R.
      • Viale G.
      Calretinin: a novel immunocytochemical marker for mesothelioma.
      ). This might cause changes, e.g. a delay or blocking of apoptotic/necrotic processes (
      • Pecze L.
      • Blum W.
      • Schwaller B.
      Mechanism of capsaicin receptor TRPV1-mediated toxicity in pain-sensing neurons focusing on the effects of Na+/Ca2+ fluxes and the Ca2+-binding protein calretinin.
      ,
      • Lukas W.
      • Jones K.A.
      Cortical neurons containing calretinin are selectively resistant to calcium overload and excitotoxicity in vitro.
      ). Thus, the increased calretinin expression in mesothelioma cells and moreover in certain colon cancer (
      • Gotzos V.
      • Wintergerst E.S.
      • Musy J.P.
      • Spichtin H.P.
      • Genton C.Y.
      Selective distribution of calretinin in adenocarcinomas of the human colon and adjacent tissues.
      ) and derived cell lines (
      • Gander J.-C.
      • Bustos-Castillo M.
      • Stüber D.
      • Hunziker W.
      • Celio M.
      • Schwaller B.
      The calcium-binding protein calretinin-22k, an alternative splicing product of the calretinin gene is expressed in several colon adenocarcinoma cell lines.
      ) might be correlated or causally linked to the increased resistance of these tumor cells to the apoptotic/necrotic signals either occurring in healthy physiological conditions or resulting from treatment with chemotherapy drugs such as oxaliplatin or 5-fluorouracil (
      • Boyer J.
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      • Moore S.
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      • Johnston P.G.
      Pharmacogenomic identification of novel determinants of response to chemotherapy in colon cancer.
      ). In support, colon cancer cells resistant to aurora kinase inhibitors are characterized by higher calretinin expression levels (
      • Hrabakova R.
      • Kollareddy M.
      • Tyleckova J.
      • Halada P.
      • Hajduch M.
      • Gadher S.J.
      • Kovarova H.
      Cancer cell resistance to aurora kinase inhibitors: identification of novel targets for cancer therapy.
      ). Moreover, down-regulation of calretinin by lentiviral infection induces apoptosis in mesothelioma cell lines in vitro via an intrinsic mitochondrion-mediated pathway (
      • Blum W.
      • Schwaller B.
      Calretinin is essential for mesothelioma cell growth/survival in vitro: a potential new target for malignant mesothelioma therapy?.
      ). Also down-regulation of calretinin in colon cancer cells is associated with cell growth arrest and increased apoptosis (
      • Gander J.C.
      • Gotzos V.
      • Fellay B.
      • Schwaller B.
      Inhibition of the proliferative cycle and apoptotic events in WiDr cells after down-regulation of the calcium-binding protein calretinin using antisense oligodeoxynucleotides.
      ).

      Author Contributions

      L. P. designed the study, performed the experiments with simulations, and wrote the paper. W. B. provided assistance, contributed to lentivirus production and cloning (CALB2), and performed qRT-PCR. B. S. secured funding, analyzed data, and wrote the paper.

      Acknowledgments

      We are grateful to Simone Eichenberger and Valérie Salicio for excellent technical assistance as well as to Michael Dougoud, Department of Mathematics, University of Fribourg, for verifying the mathematical equations. We thank Dr. D. Trono (École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland) for providing pLVTHM (Addgene plasmid 12247), pMD2G-VSVG (Addgene plasmid 12259), and psPAX2 (Addgene plasmid 12260); Dr. L. Looger for GCaMP3 (Addgene plasmid 22692); and Dr. R. E. Campbell for mito-CAR-GECO1 (Addgene plasmid 46022). pLV-EBF2-nuc was a gift from Dr. P. Tsoulfas (Addgene plasmid 36085).

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