Interplay of the Ca2+-binding Protein DREAM with Presenilin in Neuronal Ca2+ Signaling
2008; Elsevier BV; Volume: 283; Issue: 41 Linguagem: Inglês
10.1074/jbc.m804152200
ISSN1083-351X
AutoresLaura Fedrizzi, Dmitry Lim, Ernesto Carafoli, Marisa Brini,
Tópico(s)Ion channel regulation and function
ResumoThe Ca2+-binding protein DREAM regulates gene transcription and Kv potassium channels in neurons but has also been claimed to interact with presenilins, which are involved in the generation of β-amyloid and in the regulation of the Ca2+ content in the endoplasmic reticulum. The role of DREAM in Ca2+ homeostasis was thus explored in SH-SY5Y cells stably or transiently overexpressing DREAM or a Ca2+-insensitive mutant of it. The overexpression of DREAM had transcriptional and post-transcriptional effects. Endoplasmic reticulum Ca2+ and capacitative Ca2+ influx were reduced in stably expressing cells. The previously shown down-regulation of Na+/Ca2+ exchanger 3 expression was confirmed; it could cause a local increase of subplasma membrane Ca2+ and thus inhibit capacitative Ca2+ influx. DREAM up-regulated the expression of the inositol 1,4,5-trisphosphate receptor and could thus increase the unstimulated release of Ca2+ through it. The transient coexpression of DREAM and presenilin potentiated the decrease of endoplasmic reticulum Ca2+ observed in presenilin-overexpressing cells. This could be due to a direct effect of DREAM on presenilin as the two proteins interacted in a Ca2+-independent fashion. The Ca2+-binding protein DREAM regulates gene transcription and Kv potassium channels in neurons but has also been claimed to interact with presenilins, which are involved in the generation of β-amyloid and in the regulation of the Ca2+ content in the endoplasmic reticulum. The role of DREAM in Ca2+ homeostasis was thus explored in SH-SY5Y cells stably or transiently overexpressing DREAM or a Ca2+-insensitive mutant of it. The overexpression of DREAM had transcriptional and post-transcriptional effects. Endoplasmic reticulum Ca2+ and capacitative Ca2+ influx were reduced in stably expressing cells. The previously shown down-regulation of Na+/Ca2+ exchanger 3 expression was confirmed; it could cause a local increase of subplasma membrane Ca2+ and thus inhibit capacitative Ca2+ influx. DREAM up-regulated the expression of the inositol 1,4,5-trisphosphate receptor and could thus increase the unstimulated release of Ca2+ through it. The transient coexpression of DREAM and presenilin potentiated the decrease of endoplasmic reticulum Ca2+ observed in presenilin-overexpressing cells. This could be due to a direct effect of DREAM on presenilin as the two proteins interacted in a Ca2+-independent fashion. DREAM was originally identified as calsenilin, a Ca2+-binding protein belonging to the family of neuronal calcium sensor proteins (1Buxbaum J.D. Choi E.K. Luo Y. Lilliehook C. Crowley A.C. Merriam D.E. Wasco W. Nat. Med. 1998; 4: 1177-1181Crossref PubMed Scopus (302) Google Scholar). Shortly thereafter, it was found to be identical to the Ca2+-dependent gene silencer DREAM (downstream regulatory element antagonist modulator) (2Carrion A.M. Link W.A. Ledo F. Mellstrom B. Naranjo J.R. Nature. 1999; 398: 80-84Crossref PubMed Scopus (492) Google Scholar) and to one of the interacting proteins (KChIPs) of the voltage-gated Kv channels, KChIP3 (3An W.F. Bowlby M.R. Betty M. Cao J. Ling H.P. Mendoza G. Hinson J.W. Mattsson K.I. Strassle B.W. Trimmer J.S. Rhodes K.J. Nature. 2000; 403: 553-556Crossref PubMed Scopus (840) Google Scholar). The three proteins are the products of a single gene, their function being specified by their cellular location. They contain four EF-hands, of which at least three are operational. As all neuronal calcium sensors, they process the Ca2+ signal by undergoing conformational changes upon Ca2+ binding and upon interacting with target proteins (4Burgoyne R.D. O'Callaghan D.W. Hasdemir B. Haynes L.P. Tepikin A.V. Trends Neurosci. 2004; 27: 203-209Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). The targets of the protein in the cytoplasm have been claimed to be the endoplasmic reticulum (ER) 2The abbreviations used are: ER, endoplasmic reticulum; PS, presenilin; InsP3, inositol 1,4,5-trisphosphate; AEQ, aequorin; PBS, phosphate-buffered saline; erAEQ, endoplasmic reticulum-targeted AEQ; pmAEQ, plasma membrane-targeted AEQ; KRB, Krebs-Ringer buffer; tBuBHQ, 2,5-di-tert-butylhydroquinone; cytAEQ, cytosolic AEQ; BK, bradykinin; CE, 5-(and 6-)carboxyeosin diacetate succinimidyl ester; GST, glutathione S-transferase; RT, reverse transcription; InsP3R, InsP3 receptor; CTF, C-terminal fragment; [Ca2+]er, endoplasmic reticulum [Ca2+]; [Ca2+]c, cytosolic [Ca2+]; WT, wild type. proteins presenilins (PSs) (1Buxbaum J.D. Choi E.K. Luo Y. Lilliehook C. Crowley A.C. Merriam D.E. Wasco W. Nat. Med. 1998; 4: 1177-1181Crossref PubMed Scopus (302) Google Scholar), which are related to familial Alzheimer disease (5De Strooper B. EMBO Rep. 2007; 8: 141-146Crossref PubMed Scopus (281) Google Scholar). Since the three names above have been used to refer to the same protein, hereafter we will only use DREAM. Only few reports have explored the possible roles of DREAM in the regulation of neuronal Ca2+ signals. In the cytoplasm, DREAM has been claimed to counteract the potentially pathogenic effects of mutated PSs (6Leissring M.A. Yamasaki T.R. Wasco W. Buxbaum J.D. Parker I. LaFerla F.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8590-8593Crossref PubMed Scopus (85) Google Scholar), which has been proposed to be due to the potentiation of the inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release from the ER (7Leissring M.A. Parker I. LaFerla F.M. J. Biol. Chem. 1999; 274: 32535-32538Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 8Leissring M.A. Paul B.A. Parker I. Cotman C.W. LaFerla F.M. J. Neurochem. 1999; 72: 1061-1068Crossref PubMed Scopus (163) Google Scholar). DREAM was also claimed to increase the ER Ca2+ content in neuroglioma cells (9Lilliehook C. Chan S. Choi E.K. Zaidi N.F. Wasco W. Mattson M.P. Buxbaum J.D. Mol. Cell. Neurosci. 2002; 19: 552-559Crossref PubMed Scopus (73) Google Scholar); however, the increase had only been inferred from the larger amount of Ca2+ that appeared in the cytoplasm by exposing DREAM-expressing cells to thapsigargin. The effect of DREAM on neuronal Ca2+ could have important implications, given the recent findings of increased DREAM expression in brain samples of Alzheimer disease patients and in neuronal cultures exposed to the amyloid β peptide Aβ42 (10Jo D.G. Lee J.Y. Hong Y.M. Song S. Mook-Jung I. Koh J.Y. Jung Y.K. J. Neurochem. 2004; 88: 604-611Crossref PubMed Scopus (51) Google Scholar). It could be related to previous findings showing that the protein contributed to the production of the Aβ42 peptide and increased neuronal susceptibility to Ca2+-dependent apoptosis (11Jo D.G. Kim M.J. Choi Y.H. Kim I.K. Song Y.H. Woo H.N. Chung C.W. Jung Y.K. FASEB J. 2001; 15: 589-591Crossref PubMed Scopus (67) Google Scholar). However, DREAM has also been associated with the antiapoptotic function of interleukin-3-dependent hematopoietic progenitor cells (12Sanz C. Mellstrom B. Link W.A. Naranjo J.R. Fernandez-Luna J.L. EMBO J. 2001; 20: 2286-2292Crossref PubMed Scopus (86) Google Scholar). In this study, the effect of DREAM on Ca2+ signaling has been investigated in a human neuroblastoma cell line (SH-SY5Y) stably or transiently expressing wild type (WT) DREAM or a Ca2+-insensitive mutated version of it (EFmDREAM), which silences DREAM-sensitive genes permanently. DREAM has been coexpressed together with PSs, and Ca2+ signaling has been compared with that in cells only overexpressing DREAM. Ca2+ was monitored in the cytosolic compartment (13Brini M. Marsault R. Bastianutto C. Alvarez J. Pozzan T. Rizzuto R. J. Biol. Chem. 1995; 270: 9896-9903Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar), in the lumen of the ER (14Montero M. Brini M. Marsault R. Alvarez J. Sitia R. Pozzan T. Rizzuto R. EMBO J. 1995; 14: 5467-5475Crossref PubMed Scopus (265) Google Scholar), and in the space beneath the plasma membrane (15Marsault R. Murgia M. Pozzan T. Rizzuto R. EMBO J. 1997; 16: 1575-1581Crossref PubMed Scopus (162) Google Scholar). The results have revealed a pleiotropic role of DREAM on the homeostasis of Ca2+. As previously found (16Gomez-Villafuertes R. Torres B. Barrio J. Savignac M. Gabellini N. Rizzato F. Pintado B. Gutierrez-Adan A. Mellstrom B. Carafoli E. Naranjo J.R. J. Neurosci. 2005; 25: 10822-10830Crossref PubMed Scopus (76) Google Scholar), DREAM reduced the expression of one of the major neuronal plasma membrane Ca2+ extrusion systems, NCX3 (Na+/Ca2+ exchanger 3), thus elevating Ca2+ in the sub-plasma membrane space and inhibiting capacitative Ca2+ influx. As a consequence, the refilling of the ER stores with Ca2+ was also inhibited. In addition, DREAM up-regulated the InsP3R transcript levels, possibly increasing the Ca2+ leak through the receptor. These effects were transcriptional. However, DREAM also had post-transcriptional effects. It interacted directly with PS and potentiated the PS-promoted efflux of Ca2+ from the ER in transient coexpression experiments. The PS/DREAM interaction was Ca2+-independent. Cell Cultures and Transfection—SH-SY5Y cells were grown in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum, in 75-cm2 flasks; before transfection, they were seeded onto 13-mm glass coverslips and allowed to grow to 80% confluence. Transfection with 0.7 μg of plasmid DNA (or 0.5 μg of each plasmid in cotransfections) was carried out using TransFectin Lipid Reagent (Bio-Rad) according to the manufacturer's instructions. Aequorin (AEQ) measurements were performed 36 h later. Cells plated for Western blotting were collected 24–36 h after transfection. Stable WT DREAM and EFmDREAM clones were generated by transfecting the mammalian expression plasmid pcDNA3 (Invitrogen) containing the WT or EFmDREAM cDNA and were selected in Dulbecco's modified Eagle's medium with 1 mm G418. The same plasmids were used for transient expressions. PS1/pEF6/V5-His-TOPO and PS2/pcDNA3 plasmids were used for PSs transient expression. Immunocytochemistry—For immunofluorescence, SH-SY5Y cells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS; 140 mm NaCl, 2 mm KCl, 1.5 mm KH2PO4, 8 mm Na2HPO4, pH 7.4) for 20 min, washed three times with PBS, and then incubated for 10 min in PBS supplemented with 50 mm NH4Cl. Membranes were permeabilized with a 5-min incubation with 0.1% Triton X-100 in PBS, followed by a 1-h wash with 1% gelatin (type IV, from calf skin) in PBS. The coverslip was processed for the DREAM staining with specific rabbit polyclonal antibody (sc-9142; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a 1:100 dilution in PBS. Staining was carried out with AlexaFluor589 secondary antibody (1:100 dilution in PBS; Invitrogen). Fluorescence was analyzed with a Zeiss Axiovert microscope equipped with a 12-bit digital cooled camera (Micromax-1300Y; Princeton Instruments Inc., Trenton, NJ). Images were acquired using Metamorph software (Universal Imaging Corp., West Chester, PA). Aequorin Measurements—ER Ca2+ content had to be drastically reduced before the reconstitution of functional low affinity recombinant targeted aequorin (erAEQ). To this end, the cells were incubated for 1 h at 4°C in Krebs Ringer modified buffer (KRB; 125 mm NaCl, 5 mm KCl, 1 mm Na3PO4, 1 mm MgSO4, 5.5 mm glucose, 20 mm HEPES, pH 7.4, 37 °C), supplemented with 5 μm coelenterazine n (Invitrogen), the SERCA pump inhibitor 2,5-di-tert-butylhydroquinone (tBuBHQ; 10 μm), and 600 μm EGTA. After this incubation, the cells were washed extensively with KRB supplemented with 2% bovine serum albumin and 1 mm EGTA and transferred to the chamber of a purpose-built luminometer. Transfected cytosolic AEQ (cytAEQ) was reconstituted by incubating the cells for 3 h with 5 μm coelenterazine WT (Invitrogen) in Dulbecco's modified Eagle's medium supplemented with 1% fetal calf serum at 37 °C in a 5% CO2 atmosphere. Low affinity plasma membrane-targeted AEQ (pmAEQ) was reconstituted by incubating the cells for 1–2 h with coelenterazine WT in KRB supplemented with 100 μm EGTA. The procedure was necessary to increase the efficiency of reconstitution of pmAEQ. The additions to the KRB (1 mm CaCl2, 100 nm bradykinin (BK), 100 μm KB-R7943, 20 μm tBuBHQ, and 20 μm 5-(and 6-)carboxyeosin diacetate succinimidyl ester (CE)) were made as specified in the figure legends. The experiments were terminated by lysing the cells with 100 μm digitonin in a hypotonic Ca2+-rich solution (10 mm CaCl2 in H2O) to discharge the remaining AEQ pool. The light signal was collected and calibrated off-line into Ca2+ concentration values, using a computer algorithm based on the Ca2+ response curve of WT and mutant AEQs as previously described (13Brini M. Marsault R. Bastianutto C. Alvarez J. Pozzan T. Rizzuto R. J. Biol. Chem. 1995; 270: 9896-9903Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 17Barrero M.J. Montero M. Alvarez J. J. Biol. Chem. 1997; 272: 27694-27699Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). In brief, a 13-mm round coverslip with the transfected cells was placed in a perfused, thermostated chamber placed in close proximity to a low noise photomultiplier, with a built-in amplifier discriminator. The output of the discriminator was captured by a Thorn-EMI photon counting board and stored for further analyses. Fura-2 Measurements—Fura-2 loading was performed as previously described (18Malgaroli A. Milani D. Meldolesi J. Pozzan T. J. Cell Biol. 1987; 105: 2145-2155Crossref PubMed Scopus (334) Google Scholar); the coverslip was placed on the stage of an inverted fluorescence microscope (Zeiss Axiovert 100TV) connected to a cooled charge-coupled device camera (Micromax 1300Y; Princeton Instruments, Inc.). The sample was illuminated alternately at 340/380 nm, and the emitted light (filtered with an interference filter centered at 510 nm) was collected by the camera. Images were acquired using Metafluor software (Universal Imaging Corp.). The ratio values (1 ratio image/s) were calculated off-line after background subtraction from each single image. DNA Constructs—The plasmids coding for the glutathione S-transferase (GST)/DREAM fusion proteins were constructed including the cDNA of WT DREAM or EFmDREAM in the pGEX4T1 vector (GE Healthcare). The coding regions were amplified by PCR using appropriate pairs of forward 5′-CGG AAT TCC GGC TTG CTC TAG ACA CCA TGG-3′ and reverse 5′-GCC TCG AGC TAG ATG ACA TTC TCA AAC-3′ primers and subsequently cloned into the EcoRI-XhoI restriction site of pGEX4T1. All constructs were completely sequenced. GST Pull-down Assay—GST-WT DREAM and GST-EFmDREAM fusion proteins and GST alone were produced in Escherichia coli (BL21). Protein expression was induced by adding 0.8 mm isopropyl 1-thio-β-d-galactopyranoside to the growing culture (A600 = 0.6), and the cells were incubated at 30 °C for 3 h. Cells were centrifuged at 13,200 × g for 15 min, resuspended in ice-cold lysis buffer (10 mm phosphate buffer, 2.7 mm KCl, 137 mm NaCl, pH 7.4, 0.5 mm phenylmethylsulfonyl fluoride), and disrupted using a sonicator. Cells were then incubated with 1% Triton X-100 for 30 min at 4 °C and centrifuged at 15,700 × g for 30 min. The GST, GST-WT DREAM, and GST-EFmDREAM recombinant proteins were purified by incubating with glutathione-Sepharose 4B at 4 °C for 2 h. The supernatant was removed, and the glutathione-Sepharose 4B pellet was washed three times with ice-cold PBS. SH-SY5Y cells were transfected with PS2/pcDNA3 plasmid. 24–36 h after transfection, a cell extract was prepared by lysing cells in Tris-EDTA buffer (10 mm Tris/HCl, pH 8.0, 1 mm EDTA, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 5 μg/ml pepstatin). Lysis was performed by three cycles of freeze and thaw (–70 °C/37 °C), and lysates were cleared by collecting the supernatants after spinning. Loading of the samples was normalized for the total content of cellular proteins determined by the Bradford assay (Sigma). Different amounts of cell lysate (0.8–2 mg) were added to about 20 μg of GST-beads and mixed by gentle rotation at 4 °C for 2 h. For pull-down in the presence of Ca2+, 2 mm CaCl2 was added. The beads were recovered by centrifugation at 500 × g for 5 min, washed five times with ice-cold PBS, and eluted three times by gentle rotation at 4 °C for 20 min in elution buffer (50 mm Tris-HCl, 10 mm reduced glutathione, pH 8.0) followed by centrifugation at 500 × g for 5 min. Western Blotting Analysis—SH-SY5Y cells were washed twice with PBS and harvested from the culture plates in ice-cold Tris/EDTA buffer. Lysis was performed by three cycles of freeze and thaw (–70 °C/37 °C). Loading of the samples was normalized for the total content of cellular proteins determined by the Bradford assay. Samples were run on a 12% SDS-PAGE Tris/HCl gel and then blotted onto nitrocellulose membrane (GE Healthcare). Western blottings were performed using the polyclonal antibody anti-DREAM (sc-9142; Santa Cruz Biotechnology), the mouse monoclonal antibody anti-GST (sc-138; Santa Cruz Biotechnology), the polyclonal antibody PC235 (Oncogene) that recognizes the PS2 full-length protein of 54 kDa and the C-terminal fragment (CTF) of 20 kDa, the polyclonal antibody PC267 (Oncogene) that recognizes the PS1 CTF of 18 kDa, the mouse monoclonal anti-β-tubulin (D-10, Santa Cruz Biotechnology), and in the mouse monoclonal anti-β-actin (Sigma). Detection was carried out by incubation with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology) for 1 h and 30 min. The proteins were visualized by the chemiluminescent reagent Immun-Star horseradish peroxidase (Bio-Rad). Densitometric analyses were performed by using the Kodak1D image analysis software (Kodak Scientific Imaging Systems, New Haven, CT). Means of densitometric measurements of independent experiments, normalized by the endogenous β-actin or β-tubulin values, were compared by Student's t test. The results shown in the figures are representative of at least three separate experiments. RT-PCR and Quantitative RT-PCR Analysis—Total RNA from neuroblastoma cell culture was prepared using TRIzol reagent (Invitrogen). PCR assays were performed using specific primers designed using Primer3 software. RT-PCR cycling parameters were as follows: 95 °C for 5 min, 35 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 15 s. The reaction was performed with PlatinumTaq DNA polymerase (Invitrogen) in the presence of 5% dimethyl sulfoxide. For NCX3, the primers used for amplification were as follows: forward, 5′-GAC AGT AGA AGG GAC AGC CA-3′; reverse, 5′-CTA GTT TGG GGT GTT CAC CC-3′. The results were normalized as shown by parallel amplification of the glyceraldehyde-3-phosphate dehydrogenase cDNA. Glyceraldehyde-3-phosphate dehydrogenase primers used were as follows: forward, 5′-CAA GGT CAT CCA TGA CAA CTT TG-3′; reverse, 5′-GGG CCA TCC ACA GTC TTC TG-3′. Quantitative RT-PCR was performed on a Rotor-Gene 3000 platform (Corbet Research, Sydney, Australia). The PCR cycling parameters were as follows: 94 °C for 7 min, 45 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 15 s. An amount of cDNA corresponding to 1–10 ng of total RNA was amplified in 25 μl of a mixture containing 12.5 μl of Platinum SYBR-Green qPCR SuperMix-UGD (Invitrogen) and a 2-μl primer mixture (2.5 μm each) for each sample. The primers used were as follows: for DREAM, 5′-CAC CTA TGC ACA CTT CCT CTT CA-3′ (forward) and 5′-ACC ACA AAG TCC TCA AAG TGG A-3′ (reverse); for InsP3R2, 5′-GGA CAT CGT GTC CCT GTA CG-3′ (forward) and 5′-TGA ACT TCT TGG GAG GGT TG-3′ (reverse); for InsP3R3, 5′-GGA CAT CGT CTC CCT GTA CG-3′ (forward) and 5′-CAC CAC ACA GCG GTC ATC-3′ (reverse); for SERCA2b, 5′-CCT CTA TGT CGA ACC CTT GC-3′ (forward) and 5′-GCA GGC TGC ACA CAC TCT T-3′ (reverse); for calreticulin, 5′-TCA AGG AGC AGT TTC TGG AC-3′ (forward) and 5′-GTG CAT CCT GGC TTG TCT G-3′ (reverse); for calnexin, 5′-GCT AAG AGG CCA GAT GCA GA-3′ (forward) and 5′-TCA TGT CAT TGA GCA GAT TTC C-3′ (reverse); for Grp78/BiP, 5′-TGT CCC CTT ACA CTT GGT ATT G-3′ (forward) and 5′-CAA ATG TAC CCA GAA GAT GAT TG-3′ (reverse). The relative amount of amplified DNA was calculated as described (19Pfaffl M.W. Nucleic Acids Res. 2001; 29: 2002-2007Crossref Scopus (25592) Google Scholar) using hypoxanthine-guanine phosphoribosyl-transferase cDNA as endogenous control. The hypoxanthine-guanine phosphoribosyltransferase primers used were as follows: forward, 5′-TTG GAT ACA GGC CAG ACT TTG TT-3′; reverse, 5′-CTG AAG TAC TCA TTA TAG TCA AGG GCA TA-3′. Statistical Analysis—Data are reported as means ± S.D. Statistical differences were evaluated by Student's two-tailed t test for impaired samples, with p value 0.01 being considered statistically significant. Generation of Stable Clones of SH-SY5Y Cells Expressing WT and EFmDREAM—Expression vectors for WT DREAM and EFmDREAM were transfected into SH-SY5Y cells. A number of stable clones were obtained following G418 selection. The expression level of DREAM was verified in all selected clones by Western blotting on total cell lysates. Fig. 1A shows blots of representative DREAM-expressing clones. The specific DREAM antibody recognized a doublet of ∼50 kDa, corresponding to a dimer of the DREAM protein. Untransfected HeLa and untransfected SH-SH5Y cell lysates were used as negative and positive controls, respectively (endogenous DREAM is present in neuroblastoma cells but not in HeLa cells). A quantitative estimate by densitometric analysis of the doublet showed that the overexpressed DREAM was 2–4-fold higher than the endogenous DREAM. C12#4 (EFm) and D5#1 (WT) clones were selected for Ca2+ measurements, but similar results were obtained on two other independent clones for each cell type. Quantitative RT-PCR was also carried out. Fig. 1B shows the quantification, indicating that the DREAM mRNA rose about 6–7-fold above the endogenous content. Fig. 1C shows the immunolocalization of overexpressed WT and mutated DREAM; in both cases, a clear cytosolic and reticular distribution pattern was evident. The overexpression of a Ca2+-insensitive EFmDREAM mutant in the cerebellum of transgenic mice had been previously found to significantly reduce NCX3 mRNA and protein levels, increasing the basal concentration of Ca2+ in cultured cerebellar granules (16Gomez-Villafuertes R. Torres B. Barrio J. Savignac M. Gabellini N. Rizzato F. Pintado B. Gutierrez-Adan A. Mellstrom B. Carafoli E. Naranjo J.R. J. Neurosci. 2005; 25: 10822-10830Crossref PubMed Scopus (76) Google Scholar). It was thus decided to investigate whether DREAM influenced the transcription of NCX3 also in SH-SY5Y cells we used. RT-PCR analysis indicated a reduction of about 25% in the transcript of NCX3 in the EFmDREAM cell clone, but no changes were detected in the WT clone (Fig. 1D). Values were normalized by the content of glyceraldehyde-3-phosphate dehydrogenase mRNA. EFmDREAM acts as a constitutive repressor of transcription (i.e. as a dominant active mutant), since its inability to bind Ca2+ does not permit its detachment from the promoter region of the gene. For this reason, its effects on gene transcription are expected to be more marked and evident than those of the WT DREAM. The Overexpression of WT DREAM and EFmDREAM Decreases the Resting ER Ca2+ Content but Not the Agonist-stimulated Ca2+ Release—Since no work has so far directly analyzed ER free Ca2+ in neuroblastoma cells overexpressing DREAM, it was decided to directly monitor it with erAEQ (14Montero M. Brini M. Marsault R. Alvarez J. Sitia R. Pozzan T. Rizzuto R. EMBO J. 1995; 14: 5467-5475Crossref PubMed Scopus (265) Google Scholar) under resting conditions and following cell stimulation with an agonist coupled to the generation of InsP3. Fig. 2A shows that unstimulated cells overexpressing the two DREAM variants had significantly lower resting ER Ca2+ than control cells. The reduction was about 25% in both clones: 317 ± 32 μm (n = 30) in control cells, 241 ± 19 μm (n = 26) in the EFmDREAM cell clone, and 232 ± 32 μm (n = 9) in WT DREAM clone (p < 0.001). After BK stimulation, the release of Ca2+ set the ER Ca2+ content at about 100 μm in the clones expressing the two DREAM variants and to about 150 μm in control cells (144 ± 16 μm (n = 9) in control cells, 98 ± 26 μm (n = 11) in EFmDREAM clone, and 98 ± 3 μm (n = 4) in WT DREAM clone; p < 0.001). Thus, considering the different starting levels of ER Ca2+, the net amount of Ca2+ released from the ER store by InsP3 remained essentially constant in the three cell types. It was considered important to clarify the mechanism by which the overexpression of WT and EFmDREAM reduced the basal amount of ER Ca2+. The release of Ca2+ through passive leak channels (i.e. in the absence of agonists that would open the InsP3 receptor (InsP3R)) was evaluated after blocking Ca2+ uptake by the sarcoendoplasmic Ca2+ ATPase (SERCA pump). Cytosolic Ca2+ elevations were monitored in KRB supplemented with 1 mm CaCl2 using cytAEQ (13Brini M. Marsault R. Bastianutto C. Alvarez J. Pozzan T. Rizzuto R. J. Biol. Chem. 1995; 270: 9896-9903Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar), after adding the SERCA pump inhibitor tBuBHQ (20Dettbarn C. Palade P. J. Pharmacol. Exp. Ther. 1998; 285: 739-745PubMed Google Scholar). The increase of cytosolic Ca2+ due to the enhanced Ca2+ leak from the ER was the same in control cells, in the WT DREAM, and in EFmDREAM clones (Fig. 2B). To investigate whether the changes in ER Ca2+ content were related to the activity of DREAM as transcriptional repressor, quantitative analysis of the transcripts of ER proteins, such as the SERCA pump, the InsP3R, and the Ca2+-dependent chaperones calnexin and calreticulin, was performed. In addition to buffering Ca2+ in the ER lumen (21Krause K.H. Michalak M. Cell. 1997; 88: 439-443Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar), the last two proteins also regulate InsP3Rs activity (22Arnaudeau S. Frieden M. Nakamura K. Castelbou C. Michalak M. Demaurex N. J. Biol. Chem. 2002; 277: 46696-46705Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Another Ca2+ storage-related protein, Grp78/BiP (23Lievremont J.P. Rizzuto R. Hendershot L. Meldolesi J. J. Biol. Chem. 1997; 272: 30873-30879Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar), which is involved in the Ca2+-activated ER stress response (24Ellgaard L. Helenius A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 181-191Crossref PubMed Scopus (1676) Google Scholar), was also investigated. Quantitative RT-PCR on total RNA extracted from the three different batches of cells using the primers indicated under "Experimental Procedures" only showed differences for the transcript of the InsP3R2, which was increased by about 25% in the cells expressing WT and EFmDREAM (Fig. 2C). This finding was not surprising, since in some cases, DREAM has been described to activate transcription, rather than inhibit it, by acting on the promoters of certain genes (25Scsucova S. Palacios D. Savignac M. Mellstrom B. Naranjo J.R. Aranda A. Nucleic Acids Res. 2005; 33: 2269-2279Crossref PubMed Scopus (34) Google Scholar). The cytosolic transients generated by the release of Ca2+ through the opening of the InsP3R were then analyzed using cytAEQ or the fluorescent Ca2+ indicator fura-2. The two probes yielded similar results. In agreement with the findings that the amount of Ca2+ released by InsP3R was the same in the controls and in the two DREAM-expressing clones, the heights of the BK-induced cytosolic Ca2+ transients were about the same in the three cell types. The peaks of the transients were 2.95 ± 0.32 μm (n = 8) in control cells, 2.85 ± 0.12 μm (n = 10) in the EFmDREAM clone, and 2.78 ± 0.24 μm (n = 8) in the WT DREAM clone (Fig. 3A). Previous work on cerebellar granules from EFmDREAM transgenic mice, which expressed reduced amounts of NCX3, had shown slower kinetics of the post-transient decline of the Ca2+ traces (16Gomez-Villafuertes R. Torres B. Barrio J. Savignac M. Gabellini N. Rizzato F. Pintado B. Gutierrez-Adan A. Mellstrom B. Carafoli E. Naranjo J.R. J. Neurosci. 2005; 25: 10822-10830Crossref PubMed Scopus (76) Google Scholar). A similar effect was found in the SH-SY5Y clones overexpressing EFmDREAM (i.e. a slower return of the postpeak Ca2+ trace to basal level); the t/2 decay of the peak was 13.9 ± 1.8s (n = 6) in control cells and 17.6 ± 1.6s (n = 9) in the EFmDREAM clone (p < 0.001). However, the WT DREAM clone behaved like control cells (14.5 ± 1.8 s, n = 8), possibly because the reduction of NCX3 in the plasma membrane was below detection level in the WT DREAM clone (see Fig. 1D). In principle, the overexpressed DREAM could have buffered cytosolic Ca2+, decreasing the amount available to the SERCA pump and thus the ER Ca2+ content. Fura-2 was used to evaluate the resting cytosolic Ca2+, since AEQ is inadequate to monitor Ca2+ at the low nanomolar level. Fura-2 signals (ratio of fluorescence emitted by illuminating cells at 340 and 380 nm) detected in DREAM clones were similar to those in control cells, suggesting that the Ca2+ levels were similar in all three cell batches (Fig. 3B) (i.e. the differences in the ER Ca2+ content were not due to the Ca2+ buffering effect of overexpressed DREAM). The effects of DREAM were analyzed in more detail by monitoring cytosolic Ca2+ in the presence of specific inhibitors of the three different Ca2+ transporter proteins that have roles in the reestablishment of the post-transient cytosolic Ca2+ conditions: tBuBHQ as a SERCA inhibitor, CE as a plasma membrane Ca2+-ATPase inhibitor (26Gatto C. Hale C.C. Xu W. Milanick M.A. Biochemistry. 1995; 34: 965-972Crossref PubMed Scopus (72) Google Scholar), and KB-R7943 as an NCX inhibitor (27Iwamoto T. Watano T. Shigekawa M. J. Biol. Chem. 1996; 271: 22391-22397Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). Fig. 4A indi
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