Calreticulin Differentially Modulates Calcium Uptake and Release in the Endoplasmic Reticulum and Mitochondria
2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês
10.1074/jbc.m202395200
ISSN1083-351X
AutoresSerge Arnaudeau, Maud Frieden, Kimitoshi Nakamura, Cyril Castelbou, Marek Michalak, Nicolas Demaurex,
Tópico(s)Mitochondrial Function and Pathology
ResumoTo study the role of calreticulin in Ca2+ homeostasis and apoptosis, we generated cells inducible for full-length or truncated calreticulin and measured Ca2+ signals within the cytosol, the endoplasmic reticulum (ER), and mitochondria with “cameleon” indicators. Induction of calreticulin increased the free Ca2+ concentration within the ER lumen, [Ca2+]ER, from 306 ± 31 to 595 ± 53 μm, and doubled the rate of ER refilling. [Ca2+]ER remained elevated in the presence of thapsigargin, an inhibitor of SERCA-type Ca2+ATPases. Under these conditions, store-operated Ca2+ influx appeared inhibited but could be reactivated by decreasing [Ca2+]ER with the low affinity Ca2+ chelatorN,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine. In contrast, [Ca2+]ER decreased much faster during stimulation with carbachol. The larger ER release was associated with a larger cytosolic Ca2+ response and, surprisingly, with a shorter mitochondrial Ca2+ response. The reduced mitochondrial signal was not associated with visible morphological alterations of mitochondria or with disruption of the contacts between mitochondria and the ER but correlated with a reduced mitochondrial membrane potential. Altered ER and mitochondrial Ca2+responses were also observed in cells expressing anN-truncated calreticulin but not in cells overexpressing calnexin, a P-domain containing chaperone, indicating that the effects were mediated by the unique C-domain of calreticulin. In conclusion, calreticulin overexpression increases Ca2+ fluxes across the ER but decreases mitochondrial Ca2+ and membrane potential. The increased Ca2+ turnover between the two organelles might damage mitochondria, accounting for the increased susceptibility of cells expressing high levels of calreticulin to apoptotic stimuli. To study the role of calreticulin in Ca2+ homeostasis and apoptosis, we generated cells inducible for full-length or truncated calreticulin and measured Ca2+ signals within the cytosol, the endoplasmic reticulum (ER), and mitochondria with “cameleon” indicators. Induction of calreticulin increased the free Ca2+ concentration within the ER lumen, [Ca2+]ER, from 306 ± 31 to 595 ± 53 μm, and doubled the rate of ER refilling. [Ca2+]ER remained elevated in the presence of thapsigargin, an inhibitor of SERCA-type Ca2+ATPases. Under these conditions, store-operated Ca2+ influx appeared inhibited but could be reactivated by decreasing [Ca2+]ER with the low affinity Ca2+ chelatorN,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine. In contrast, [Ca2+]ER decreased much faster during stimulation with carbachol. The larger ER release was associated with a larger cytosolic Ca2+ response and, surprisingly, with a shorter mitochondrial Ca2+ response. The reduced mitochondrial signal was not associated with visible morphological alterations of mitochondria or with disruption of the contacts between mitochondria and the ER but correlated with a reduced mitochondrial membrane potential. Altered ER and mitochondrial Ca2+responses were also observed in cells expressing anN-truncated calreticulin but not in cells overexpressing calnexin, a P-domain containing chaperone, indicating that the effects were mediated by the unique C-domain of calreticulin. In conclusion, calreticulin overexpression increases Ca2+ fluxes across the ER but decreases mitochondrial Ca2+ and membrane potential. The increased Ca2+ turnover between the two organelles might damage mitochondria, accounting for the increased susceptibility of cells expressing high levels of calreticulin to apoptotic stimuli. Ca2+ signals control key biological functions, including fertilization, development, cardiac contraction, and secretion of neurotransmitters and hormones (1Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4547) Google Scholar). At the cellular level, Ca2+ can be either a life and death signal, as changes in cytosolic free Ca2+ concentration can control cell growth and proliferation or induce apoptosis, the programmed cell death (2Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1800) Google Scholar). These diverging effects reflect the precise spatial and temporal encoding of Ca2+ signals, which depends largely on the controlled release of Ca2+ from intracellular organelles. The main intracellular Ca2+ store is the endoplasmic reticulum (ER), 1The abbreviations used for: ER, endoplasmic reticulum; Dox, doxycycline; InsP3, inositol 1,4,5-trisphosphate; SOC, store-operated Ca2+ influx; SERCA, sarco(endo)plasmic reticulum Ca2+ transport ATPase, [Ca2+]cyt, [Ca2+]ER, and [Ca2+]mit, cytosolic, ER, and mitochondria-free Ca2+ concentration, respectively; CCh, carbachol; YC, yellow cameleon; DsRed, Red fluorescent protein fromDiscosoma sp.; TPEN, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine; Tg, thapsigargin; Tet, tetracycline; HA, hemagglutinin; TMRM, tetramethylrhodamine methyl ester; CRT, calreticulin; HEDTA, N-(2-hydroxyethyl)ethylenediamine-N,N′,N′-triacetic acid. but mitochondria also take up and release Ca2+ very efficiently and are often strategically located close to Ca2+ sources (see Refs. 3Rizzuto R. Simpson A.W. Brini M. Pozzan T. Nature. 1992; 358: 325-327Crossref PubMed Scopus (798) Google Scholar, 4Rizzuto R. Brini M. Murgia M. Pozzan T. 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Cell Biol. 1997; 137: 633-648Crossref PubMed Scopus (467) Google Scholar), or by providing a local source of Ca2+ for ER refilling (11Arnaudeau S. Kelley W.L. Walsh Jr., J.V. Demaurex N. J. Biol. Chem. 2001; 276: 29430-29439Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). To achieve such precise control over Ca2+ fluxes, the ER and mitochondria are equipped with a variety of Ca2+transport and storage proteins and exert a tight control of the Ca2+ concentration within their lumen. Ca2+fluxes across the ER membrane are stringently dependent on the free Ca2+ concentration within the ER, [Ca2+]ER, as Ca2+ allosterically modulates the activity of the InsP3 receptor, the main Ca2+-release channel of the ER. In addition, changes in [Ca2+]ER regulate the Ca2+permeability of store-operated channels (SOC) at the plasma membrane (12Putney Jr., J.W. McKay R.R. Bioessays. 1999; 21: 38-46Crossref PubMed Scopus (358) Google Scholar). The mechanism of this “capacitative” coupling is still elusive and has been proposed to involve the diffusion of a soluble messenger (13Randriamampita C. Tsien R.Y. Nature. 1993; 364: 809-814Crossref PubMed Scopus (789) Google Scholar), direct interaction between InsP3 receptors and SOC channels (14Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1051) Google Scholar), or a secretion-like docking mechanism (15Patterson R.L. van Rossum D.B. Gill D.L. Cell. 1999; 98: 487-499Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). In addition to these Ca2+ signaling functions, the Ca2+ concentration within the ER lumen and the mitochondrial matrix also affects many functions of these organelles. The activity of several ER resident chaperone proteins is modulated by changes in [Ca2+]ER, which thereby indirectly regulates the processing, sorting, and secretion of cargo proteins (16Corbett E.F. Oikawa K. Francois P. Tessier D.C. Kay C. Bergeron J.J. Thomas D.Y. Krause K.H. Michalak M. J. Biol. Chem. 1999; 274: 6203-6211Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). In mitochondria, Ca2+ directly controls the activity of several dehydrogenases, thereby coupling the cell metabolism to the Ca2+ signal (17Hajnoczky G. Robb-Gaspers L.D. Seitz M.B. Thomas A.P. Cell. 1995; 82: 415-424Abstract Full Text PDF PubMed Scopus (958) Google Scholar, 18Robb-Gaspers L.D. Burnett P. Rutter G.A. Denton R.M. Rizzuto R. Thomas A.P. EMBO J. 1998; 17: 4987-5000Crossref PubMed Scopus (326) Google Scholar). The mitochondrial “decoding” of Ca2+ signals allows cells to quickly respond to an increased energy demand but can be turned into a death signal during concomitant exposure to apoptotic stimuli (reviewed in Ref. 19Duchen M.R. J. Physiol. (Lond.). 1999; 516: 1-17Crossref Scopus (534) Google Scholar). In the presence of ceramide, even physiological Ca2+ responses of mitochondria to InsP3-generating agonists are sufficient to induce apoptosis, possibly via Ca2+-dependent opening of the permeability transition pore (20Szalai G. Krishnamurthy R. Hajnoczky G. EMBO J. 1999; 18: 6349-6361Crossref PubMed Scopus (432) Google Scholar). The Ca2+content of the ER also affects the cell sensitivity to apoptotic stimuli. A decreased [Ca2+]ER was observed in cells overexpressing the antiapoptotic protein Bcl-2 (21Foyouzi-Youssefi R. Arnaudeau S. Borner C. Kelley W.L. Tschopp J. Lew D.P. Demaurex N. Krause K.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5723-5728Crossref PubMed Scopus (388) Google Scholar, 22Pinton P. Ferrari D. Magalhaes P. Schulze-Osthoff K. Di Virgilio F. Pozzan T. Rizzuto R. J. Cell Biol. 2000; 148: 857-862Crossref PubMed Scopus (411) Google Scholar), and a variety of conditions that decreased [Ca2+]ER has been shown to protect cells from ceramide-induced cell death (23Pinton P. Ferrari D. Rapizzi E. Di Virgilio F.D. Pozzan T. Rizzuto R. EMBO J. 2001; 20: 2690-2701Crossref PubMed Scopus (508) Google Scholar). The opposite effect was observed in cells overexpressing the Ca2+-ATPases (SERCA2b) or the ER-resident Ca2+-binding chaperone calreticulin, which increased the Ca2+ content of the ER (23Pinton P. Ferrari D. Rapizzi E. Di Virgilio F.D. Pozzan T. Rizzuto R. EMBO J. 2001; 20: 2690-2701Crossref PubMed Scopus (508) Google Scholar, 24Brini M. Bano D. Manni S. Rizzuto R. Carafoli E. EMBO J. 2000; 19: 4926-4935Crossref PubMed Google Scholar, 25Nakamura K. Bossy-Wetzel E. Burns K. Fadel M.P. Lozyk M. Goping I.S. Opas M. Bleackley R.C. Green D.R. Michalak M. J. Cell Biol. 2000; 150: 731-740Crossref PubMed Scopus (251) Google Scholar). Conversely, cells lacking the calreticulin had a decreased ER Ca2+content and were more resistant to apoptotic stimuli (26Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Muller-Esterl W. Lew D.P. Krause K.H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (240) Google Scholar). Calreticulin-deficient cells, however, had normal [Ca2+]ER levels, suggesting that the ability of calreticulin to modulate the cell sensitivity to apoptotic stimuli might be linked to changes in the total Ca2+ content of the ER rather than to changes in [Ca2+]ER. Calreticulin is a 46-kDa Ca2+-binding chaperone that interacts in a Ca2+-dependent fashion with several ER resident proteins, with unfolded glycoproteins, and with Ca2+ transporters at the ER membrane (27Krause K.H. Michalak M. Cell. 1997; 88: 439-443Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 28Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Biochem. J. 1999; 344: 281-292Crossref PubMed Scopus (697) Google Scholar). Calreticulin is composed of three structural and functional domains as follows: a highly conserved N-terminal domain, involved in chaperone function and in the interactions with other ER chaperones; a proline-rich P-domain, which shares significant amino acid sequence identity with calnexin, calmegin, and CALNUC and is involved in the chaperone function of calreticulin; and a C-terminal domain that binds Ca2+ ions with low affinity and high capacity (29Corbett E.F. Michalak M. Trends Biochem. Sci. 2000; 25: 307-311Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). The Ca2+-binding C-domain has been postulated to be the “Ca2+ sensor” that regulates calreticulin interactions with other proteins (25Nakamura K. Bossy-Wetzel E. Burns K. Fadel M.P. Lozyk M. Goping I.S. Opas M. Bleackley R.C. Green D.R. Michalak M. J. Cell Biol. 2000; 150: 731-740Crossref PubMed Scopus (251) Google Scholar, 29Corbett E.F. Michalak M. Trends Biochem. Sci. 2000; 25: 307-311Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Because of the central role of the ER in Ca2+ signaling, both the chaperoning functions of calreticulin as well as its interactions with ER Ca2+ transporters can interfere with Ca2+ signals. For example, calreticulin inhibits repetitive Ca2+ waves by interacting selectively with distinct isoforms of SERCA2 (30Camacho P. Lechleiter J.D. Cell. 1995; 82: 765-771Abstract Full Text PDF PubMed Scopus (201) Google Scholar, 31John L.M. Lechleiter J.D. Camacho P. J. Cell Biol. 1998; 142: 963-973Crossref PubMed Scopus (182) Google Scholar). On the other hand, conflicting results have been reported regarding the role of calreticulin in the modulation of store-operated Ca2+ influx (SOC). Stable up-regulation of calreticulin in HEK-293 cells inhibits thapsigargin-induced Ca2+ or Mn2+ influx (32Mery L. Mesaeli N. Michalak M. Opas M. Lew D.P. Krause K.H. J. Biol. Chem. 1996; 271: 9332-9339Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar), whereas transient expression in RBL-1 cells only delays the activation of the ICRAC current, to an extent that correlated with the extent of store depletion (33Fasolato C. Pizzo P. Pozzan T. Mol. Biol. Cell. 1998; 9: 1513-1522Crossref PubMed Scopus (63) Google Scholar). Similarly, in Xenopus oocytes overexpressing calreticulin and stimulated with InsP3-generating agonists, SOC inhibition correlated with increased [Ca2+]ER levels as expected from the capacitative mechanism (34Xu W. Longo F.J. Wintermantel M.R. Jiang X. Clark R.A. DeLisle S. J. Biol. Chem. 2000; 275: 36676-36682Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Because of the plethoric effects of the protein and the different expression system used, the role of calreticulin in Ca2+signaling remains controversial. To clarify the role of calreticulin in Ca2+ homeostasis and in apoptosis, we generated cell lines inducible for either the full-length calreticulin, an N-truncated version lacking the chaperoning N-domain, or its chaperone homologue calnexin. The effects of a controlled increase in protein levels on cytosolic, ER, and mitochondrial Ca2+ signals were measured using genetically encoded Ca2+-sensitive “cameleon” indicators. The bright fluorescence and molecular targeting of the probes allowed precise quantification of the changes in free [Ca2+] occurring within the different cell compartments at different times after the induction of protein expression. Dulbecco's modified Eagle's culture medium, fetal calf serum, penicillin, streptomycin, and geneticin were obtained from Invitrogen. Thapsigargin, nigericin, monensin, ATP, and HEPES were purchased from Sigma. Ionomycin was obtained from Calbiochem. Hygromycin B, doxycycline, EGTA, and HEDTA were from Fluka (Buchs, Switzerland). JC-1 and TMRM were from Molecular Probes (Eugene, OR). Transfast transfection reagent was purchased from Promega (Catalys AG, Switzerland). All other chemicals were of analytic grade and were obtained from Fluka or Sigma. The “Ca2+medium” contained 140 mm NaCl, 5 mm KCl, 1 mm MgCl2, 1 mm CaCl2, 10 mm glucose, and 20 mm Hepes, pH 7.4. For the “Ca2+-free medium” CaCl2 was omitted, and 0.5 mm EGTA was included. Drugs were dissolved in dimethyl sulfoxide (Me2SO) or ethanol and diluted in the recording medium on the day of use, at a final solvent concentration <0.1%. Plasmids YC2, YC2.1, and YC4ER were kindly provided by Dr. R. Y. Tsien. Plasmid YC2mit and YC4.1mit were generated as described previously (11Arnaudeau S. Kelley W.L. Walsh Jr., J.V. Demaurex N. J. Biol. Chem. 2001; 276: 29430-29439Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). cDNA encoding full-length or truncated (P + C-domain HA-tagged) rabbit calreticulin and canine calnexin were subcloned into the pTRE plasmid to generate pTRE-CRT, pTRE-P + C and pTRE-CNX expression vectors, respectively. These vectors were used to generate Tet-On inducible cell lines. Plasmid DNAs were purified using a Qiagen column by the Maxi-prep purification protocol recommended by the manufacturer. The Tet-On cell lines were generated by co-transfecting pTRE-CRT, pTRE-P + C, or pTRE-CNX with pTK-Hyg at a ratio 20:1 into HEK-293 cells (HeLa cells) by the Ca2+-phosphate protocol. Transfected cells were selected for growth in the presence of 200 μg of hygromycin B/ml of culture medium. Single colonies of the hygromycin B-resistant cells were tested for doxycycline (Dox)-dependent expression of calreticulin, P + C-domain, and calnexin by Western blotting with anti-calreticulin, anti-HA, and anti-calnexin antibodies. Three cell lines with the highest inducible expression of calreticulin, P + C-domain, and calnexin were selected for this study. HEK-293 or Tet-On cell lines were grown in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum, 2 mml-glutamine, 50 units/ml penicillin, 50 μg/ml streptomycin and were maintained in a humidified incubator at 37 °C in the presence of 5% CO2, 95% air. Cells (∼200,000) were plated on 25-mm glass coverslips. With HEK-293 at 60% of confluency, the cells were transiently transfected with cDNAs encoding the yellow cameleon probes. Cells were imaged 3–5 days after transfection. Stable HEK-293 transfectants were grown in the presence of geneticin (100 μg/ml) for 3 weeks, and ∼20 clones were expanded for each condition and tested for expression of the probes. 2 μg of Dox/ml was added into the culture medium to induce expression of calreticulin, its P + C-domain, or calnexin in Tet-On cell lines. Western blot analysis with the use of goat anti-calreticulin, anti-HA, and rabbit anti-calnexin antibodies was carried out as described (25Nakamura K. Bossy-Wetzel E. Burns K. Fadel M.P. Lozyk M. Goping I.S. Opas M. Bleackley R.C. Green D.R. Michalak M. J. Cell Biol. 2000; 150: 731-740Crossref PubMed Scopus (251) Google Scholar). For indirect immunofluorescence of calreticulin expressing HEK Tet-On cells were plated on coverslips pretreated with polylysine and cultured in the presence or absence of 2 μg of Dox/ml for 72 h. Cells were washed 3 times with PBS, fixed with 3.7% paraformaldehyde for 20 min, and permeabilized with 0.3% Triton X-100 for 20 min. Calreticulin was detected by incubation with a goat anti-calreticulin antibody followed by staining with a rabbit anti-goat antibody conjugated to Texas Red (Jackson ImmunoResearch). Cells plated on 25-mm coverslips were superfused at 37 °C in a thermostatic chamber (Harvard Apparatus, Holliston, MA) equipped with gravity feed inlets and vacuum outlet for solution changes. Dual-emission ratio imaging of [Ca2+] with cameleon probes was performed as described previously (11Arnaudeau S. Kelley W.L. Walsh Jr., J.V. Demaurex N. J. Biol. Chem. 2001; 276: 29430-29439Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Cameleon fluorescence from cells was imaged on a Axiovert S100 TV using a 100×, 1.3 NA oil-immersion objective (Carl Zeiss AG, Feldbach, Switzerland). Cells were excited by the 430 ± 10 nm line from a monochromator (DeltaRam, Photon Technology International Inc., Monmouth Junction, NJ) through a 455DRLP dichroic mirror. Fluorescence emission from the cameleons was imaged using a cooled, 16-bits CCD back-illuminated frame transfer MicroMax camera (Princeton Instruments, Roper Scientific, Trenton, NJ) at two emission wavelengths, using a filter wheel (Ludl Electronic Products, Hawthorn, NY) to alternately change the two emission filters (475DF15 and 535DF25, Omega Optical, Brattleboro, VT). Image acquisition and analysis was performed with the Metamorph/Metafluor 4.1.2 software (Universal Imaging, West Chester, PA). Changes in fluorescence ratio, R = (fluorescence intensity at 535 nm − background intensity at 535 nm)/(fluorescence intensity at 475 nm − background intensity at 475 nm), were calibrated in [Ca2+] using Equation 1, [Ca2+]=KD′(R−Rmin)/(Rmax−R)(1/n)Equation 1 where Rmax and Rmin are the ratios obtained, respectively, in the absence of Ca2+ and at saturating Ca2+. K d′ is the apparent dissociation constant, and n is the Hill coefficient of the Ca2+ calibrations curves obtainedin situ for each cameleon. For better three-dimensional rendering wide field or confocal image stacks were deconvoluted after acquisition on a Silicon Graphics Octane work station using the Huygens 2 software, and shadow projections were constructed using the Imaris software (Bitplane AG, Zurich, Switzerland). To generate cells inducible for calreticulin, we stably transfected HEK-293 cells with a rabbit calreticulin cDNA construct driven by the tetracycline promoter (Tet-ON). The activation of calreticulin gene transcription by doxycycline (Dox), added to the culture medium, was confirmed by immunoblotting with a goat polyclonal CRT antibody (Fig. 1 A). Quantification of the immunoblot indicated that the cellular calreticulin content increased by 2.5-fold within 24 h and remained at this level for up to 5 days in culture. The induction was specific for calreticulin, as addition of Dox had no effect on the expression of other ER luminal chaperones such as ERp57 or Bip (not shown). An immunostaining with a calreticulin-specific antibody confirmed that protein expression was much stronger in Dox-induced cells and still displayed the reticular pattern typical of the ER (Fig.1 B, left). No immunoreactivity was observed in the cytosol or at the plasma membrane, confirming that, after induction, calreticulin remained localized within the ER lumen. The ER structure was not noticeably altered, because Dox induction did not affect the intracellular distribution of the ER-targeted Ca2+indicator YC4ER (Fig. 1 B, right). This indicated that the increase in calreticulin did not interfere with the import, ER retention, or folding efficiency of the GFP-based indicator. Moreover, the Ca2+ affinity of both the ER-targeted probe YC4ER and of the cytosolic probe YC2, measured in situ in cells permeabilized with ionomycin or digitonin, was not affected by the increased expression of calreticulin (Fig.1 C). Thus, Dox induction increased the amount of calreticulin within the ER lumen in a controlled manner, without interfering with the targeting specificity or Ca2+dependence of the cameleon Ca2+ indicators. To assess whether the sustained increase in calreticulin levels interfered with ER Ca2+ homeostasis, we measured the changes in the free Ca2+ concentration within the ER lumen, [Ca2+]ER, using the low affinity ER-targeted ratiometric “cameleon” indicator YC4ER (K D = 290 μm (11Arnaudeau S. Kelley W.L. Walsh Jr., J.V. Demaurex N. J. Biol. Chem. 2001; 276: 29430-29439Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar)). YC4ER measurements revealed that the induction of calreticulin markedly increased the resting [Ca2+]ER levels (Fig.2) with the basal [Ca2+]ER values averaging 306 ± 31 μm in the absence and 595 ± 53 μm72 h after Dox-dependent induction of calreticulin expression. The increase could not be attributed to a specific ER region, as higher [Ca2+]ER levels were observed throughout the ER network in the ratio images (Fig.2 A). Thus, the 2.5-fold increase in calreticulin levels caused, after 3 days of induction, a doubling in the free Ca2+ concentration within the ER lumen. The doubling in resting [Ca2+]ER could reflect either an increased Ca2+ pumping activity, or a decrease in the passive Ca2+ permeability, or “leak” of the ER. To distinguish between these possibilities, we studied the effect of the SERCA inhibitor thapsigargin (Tg) on calreticulin-dependent changes in free ER Ca2+. Tg induced a slow decrease in [Ca2+]ER in both control and calreticulin-induced cells (Fig. 2 B). A linear fit of the initial [Ca2+]ER decay revealed that the kinetics of Ca2+ release were nearly identical (ΔCa2+ = −5.2 ± 0.3 versus−6.0 ± 0.3 μm/s), despite the higher [Ca2+]ER in the calreticulin overexpressers. Consequently, calreticulin-overexpressing cells retained a higher [Ca2+]ER level throughout the course of Tg stimulation. A further decline was observed upon addition of the ionophore ionomycin (Fig. 2, B and D), indicating that the ER Ca2+ store was not fully depleted by Tg. Thus, a block of SERCA ATPases unmasked a nearly identical passive Ca2+ permeability in the ER, regardless of the increase in calreticulin levels. In contrast, upon stimulation with the InsP3-generating agonist carbachol (CCh), [Ca2+]ER decreased much faster in CRT-induced cells, and similar depleted levels were achieved within 100 s of agonist stimulation (Fig. 2, Cand D). The faster kinetics of Ca2+ release (ΔCa2+ = −4.5 ± 0.6 versus −11.4 ± 1.4 μm/min) suggested that calreticulin overexpression increased the InsP3-stimulated Ca2+permeability of the ER. Importantly, re-addition of Ca2+ to the external medium resulted in a rapid increase of the [Ca2+]ER in calreticulin-overexpressing cells (Fig. 2 C). The recovery rates were 1.9-fold higher in calreticulin overexpressers than in control, non-induced cells, at any given [Ca2+]ER (Fig. 2 C, inset). Because this assay measures the net flow of Ca2+ from the external space to the ER, this indicates that both the influx of Ca2+ across the plasma membrane and the ER Ca2+pumping activity were increased in cells expressing high levels of calreticulin. In the absence of agonist stimulation, the increased rates of ER refilling were not balanced by a parallel increase in the endogenous ER Ca2+ permeability, resulting in higher [Ca2+]ER levels at rest. However, induction of calreticulin expression markedly increased the agonist-induced ER Ca2+ permeability, and therefore, upon stimulation, more Ca2+ was released from the ER lumen. To assess how these changes in ER luminal Ca2+ homeostasis influenced Ca2+ signals in the cytosol, we monitored changes in cytoplasmic Ca2+, [Ca2+]cyt, with the cytosolic YC2 probe (K D = 1.24 μm). Ca2+release from ER stores was measured in the absence of external Ca2+, and Ca2+ influx was subsequently measured by re-adding Ca2+ to the external medium. Fig.3 shows that both CCh and Tg elicited a much larger increase in [Ca2+]cyt in calreticulin overexpressing cells, indicating that substantially more Ca2+ was released from the intracellular Ca2+stores. Compared with previous studies using fura-2 (32Mery L. Mesaeli N. Michalak M. Opas M. Lew D.P. Krause K.H. J. Biol. Chem. 1996; 271: 9332-9339Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar), the differences between control and calreticulin overexpresser cells were striking, reflecting the better adequacy of the YC2 probe to quantify [Ca2+]cyt changes in the micromolar range. Subsequent addition of Ca2+ to assess the activity of store-operated Ca2+ channels at the plasma membrane revealed that, as previously reported (32Mery L. Mesaeli N. Michalak M. Opas M. Lew D.P. Krause K.H. J. Biol. Chem. 1996; 271: 9332-9339Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar), Ca2+ influx was severely blunted in Tg-stimulated calreticulin-overexpressing cells (Fig. 3). This decreased influx correlated well with the increased [Ca2+]ER levels measured with YC4ER (Fig. 2) and indicated that, consistent with the capacitative hypothesis, the activity of SOC channels is determined by changes in [Ca2+]ER levels. Accordingly, Ca2+ influx was similar in control and Dox-induced cells stimulated with CCh, which had comparable [Ca2+]ER levels (Figs. 2 and 3). However, in this case the activity of SOC channels could not be readily inferred from the changes in [Ca2+]cyt, because of the concomitant ER Ca2+ pumping activity. Although Ca2+ re-addition produced similar [Ca2+]cyt changes, larger [Ca2+]ER increases were observed in calreticulin-overexpressing cells, indicating that substantially more Ca2+ was taken up by the ER (Fig.2 A). This suggested that the net flux of Ca2+ions across the plasma membrane was, in fact, larger in calreticulin-induced cells but that the Ca2+ entering the cell was rapidly taken up by the ER. Thus, the increased [Ca2+]ER levels observed in the presence of Tg correlated with decreased SOC activity. In contrast, SOC activity was high in calreticulin overexpresser cells stimulated with CCh but did not translate into a larger cytosolic Ca2+ signal because of the high concomitant ER Ca2+ pumping activity. To better assess the effects of high expression of calreticulin on Ca2+ handling, we measured the [Ca2+]ER and [Ca2+]cyt responses at different times following the induction of protein expression. Fig.4 A shows that the resting [Ca2+]ER levels were increased 24 h after Dox-dependent induction of calreticulin expression and remained elevated thereafter. In contrast, Ca2+ influx, taken as the peak [Ca2+]cyt upon Ca2+ re-addition to Tg-treated cells, was inhibited only 3 days after the induction with Dox (Fig. 4 B, circles). The amount of releasable Ca2+ followed a similar delayed time course; the peak of Tg-induced [Ca2+]cytrelease was only marginally increase
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