Antiapoptotic Activity of Akt Is Down-regulated by Ca2+ in Myocardiac H9c2 Cells
2004; Elsevier BV; Volume: 279; Issue: 49 Linguagem: Inglês
10.1074/jbc.m407225200
ISSN1083-351X
AutoresChie Yasuoka, Yoshito Ihara, Satoshi Ikeda, Yoshiyuki Miyahara, Takahito Kondo, Shigeru Kohno,
Tópico(s)Cell death mechanisms and regulation
ResumoCell survival signaling of the Akt/protein kinase B pathway was influenced by a change in the cytoplasmic free calcium concentration ([Ca2+]i) for over 2 h via the regulation of a Ser/Thr phosphatase, protein phosphatase 2Ac (PP2Ac), in rat myocardiac H9c2 cells. Akt was down-regulated when [Ca2+]i was elevated by thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, but was up-regulated when it was suppressed by 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA-AM), a cell permeable Ca2+ chelator. The inactivation of Akt was well correlated with the susceptibility to oxidant-induced apoptosis in H9c2 cells. To investigate the mechanism of the Ca2+-dependent regulation of Akt via the regulation of PP2A, we examined the transcriptional regulation of PP2Acα in H9c2 cells with Ca2+ modulators. Transcription of the PP2Acα gene was increased by thapsigargin but decreased by BAPTA-AM. The promoter activity was examined and the cAMP response element (CRE) was found responsible for the Ca2+-dependent regulation of PP2Acα. Furthermore, phosphorylation of CRE-binding protein increased with thapsigargin but decreased with BAPTA-AM. A long term change of [Ca2+]i regulates PP2Acα gene transcription via CRE, resulting in a change in the activation status of Akt leading to an altered susceptibility to apoptosis. Cell survival signaling of the Akt/protein kinase B pathway was influenced by a change in the cytoplasmic free calcium concentration ([Ca2+]i) for over 2 h via the regulation of a Ser/Thr phosphatase, protein phosphatase 2Ac (PP2Ac), in rat myocardiac H9c2 cells. Akt was down-regulated when [Ca2+]i was elevated by thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, but was up-regulated when it was suppressed by 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA-AM), a cell permeable Ca2+ chelator. The inactivation of Akt was well correlated with the susceptibility to oxidant-induced apoptosis in H9c2 cells. To investigate the mechanism of the Ca2+-dependent regulation of Akt via the regulation of PP2A, we examined the transcriptional regulation of PP2Acα in H9c2 cells with Ca2+ modulators. Transcription of the PP2Acα gene was increased by thapsigargin but decreased by BAPTA-AM. The promoter activity was examined and the cAMP response element (CRE) was found responsible for the Ca2+-dependent regulation of PP2Acα. Furthermore, phosphorylation of CRE-binding protein increased with thapsigargin but decreased with BAPTA-AM. A long term change of [Ca2+]i regulates PP2Acα gene transcription via CRE, resulting in a change in the activation status of Akt leading to an altered susceptibility to apoptosis. Calcium (Ca2+) plays a signaling role in many important cellular functions, such as fertilization, embryonic pattern formation, differentiation, proliferation, contraction, secretion, and metabolism (1Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. 2000; 1: 11-21Crossref Scopus (4493) Google Scholar). The versatility of the Ca2+-signaling mechanism in terms of speed, amplitude and spatio-temporal patterning enables elevations of Ca2+ to regulate many processes of cell activity. Ca2+ exhibits cross-talk between a variety of signaling pathways (1Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. 2000; 1: 11-21Crossref Scopus (4493) Google Scholar). Ca2+ affects the protein kinase A pathway by regulating the metabolism of cAMP. It also activates nitric-oxide (NO) synthase to generate NO, which in turn activates the cGMP pathway through the activation of guanylyl cyclase. The Ras/mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; BAPTA-AM, 1,2-bis (o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxymethyl) ester; CRE, cAMP response element; CREB, CRE-binding protein; ER, endoplasmic reticulum; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; PI3K, phosphatidylinositol 3-kinase; LDH, lactate dehydrogenase; CaMK, calmodulin kinase; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; EMSA, electrophoretic mobility shift assay. and Ca2+/calmodulin/calmodulin kinase (CaMK) pathways are also controlled by Ca2+ (1Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. 2000; 1: 11-21Crossref Scopus (4493) Google Scholar, 2Agell N. Bachs O. Rocamora N. Villalonga P. Cell. Signal. 2002; 14: 649-654Crossref PubMed Scopus (353) Google Scholar). On the other hand, a cellular Ca2+ overload or the perturbation of intracellular Ca2+ compartmentalization can cause cytotoxicity and trigger apoptosis or necrosis (3Orrenius S. Zhivotovsky B. Nicotera P. Nat. Rev. 2003; 5: 552-565Crossref Scopus (2447) Google Scholar, 4Rizzuto R. Pinton P. Ferrari D. Chami M. Szabadkai G. Magalhaes P.J. Virgilio F. Pozzan T. Oncogene. 2003; 22: 8619-8627Crossref PubMed Scopus (394) Google Scholar). Under such circumstances, various Ca2+-dependent signaling cascades with kinases and phosphatases directly or indirectly influence cellular signaling. Protein kinase C family has been proposed to play an important role in the Ca2+-mediated signaling of apoptosis (5Murriel C.L. Mochly-Rosen D. Arch. Biochem. Biophys. 2003; 420: 246-254Crossref PubMed Scopus (116) Google Scholar). Calcineurin/PP2B, a Ca2+-dependent Ser/Thr phosphatase (6Crabtree G.R. J. Biol. Chem. 2001; 276: 2313-2316Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar), also appears to be involved in apoptosis (7Klumpp S. Krieglstein J. Curr. Opin. Pharmacol. 2002; 2: 458-462Crossref PubMed Scopus (130) Google Scholar). Together, these findings show that Ca2+ has a pivotal role in the regulatory mechanism of signaling pathways in cell survival and death, although the precise mechanism of Ca2+-dependent cross-talk has not been fully clarified. Akt/protein kinase B is a pleckstrin homology domain-containing Ser/Thr kinase (8Brazil D.P. Hemmings B.A. Trends Biochem. Sci. 2001; 26: 657-664Abstract Full Text Full Text PDF PubMed Scopus (1043) Google Scholar, 9Scheid M.P. Woodgett J.R. FEBS Lett. 2003; 546: 108-112Crossref PubMed Scopus (346) Google Scholar, 10Franke T.F. Hornik C.P. Segev L. Shostak G.A. Sugimoto C. Oncogene. 2003; 22: 8983-8998Crossref PubMed Scopus (1001) Google Scholar). Akt is presently recognized as a cell survival or an antiapoptotic cellular signaling mediator. Akt is activated through a growth factor receptor-mediated activation of the phosphatidylinositol 3-kinase (PI3K) pathway (10Franke T.F. Hornik C.P. Segev L. Shostak G.A. Sugimoto C. Oncogene. 2003; 22: 8983-8998Crossref PubMed Scopus (1001) Google Scholar). With growth factor signals, Akt is recruited to the plasma membrane and is activated through phosphorylation at Ser-473 and Thr-308 by phosphatidylinositol 3-phosphate-dependent protein kinase-1 (PDK1) or integrin-linked kinase (9Scheid M.P. Woodgett J.R. FEBS Lett. 2003; 546: 108-112Crossref PubMed Scopus (346) Google Scholar, 10Franke T.F. Hornik C.P. Segev L. Shostak G.A. Sugimoto C. Oncogene. 2003; 22: 8983-8998Crossref PubMed Scopus (1001) Google Scholar). Akt can phosphorylate Bad, caspase-9, and forkhead-related transcription factors, leading to their inactivation and to enhanced cell survival (8Brazil D.P. Hemmings B.A. Trends Biochem. Sci. 2001; 26: 657-664Abstract Full Text Full Text PDF PubMed Scopus (1043) Google Scholar, 9Scheid M.P. Woodgett J.R. FEBS Lett. 2003; 546: 108-112Crossref PubMed Scopus (346) Google Scholar, 10Franke T.F. Hornik C.P. Segev L. Shostak G.A. Sugimoto C. Oncogene. 2003; 22: 8983-8998Crossref PubMed Scopus (1001) Google Scholar). Inhibitor of nuclear factor κB (IκB) kinase is also phosphorylated by Akt leading to an up-regulation of its activity and resulting in a promotion of the nuclear factor κB (NFκB)-mediated inhibition of apoptosis (8Brazil D.P. Hemmings B.A. Trends Biochem. Sci. 2001; 26: 657-664Abstract Full Text Full Text PDF PubMed Scopus (1043) Google Scholar, 11Ozes O.N. Mayo L.D. Gustin J.A. Pfefer S.R. Pfeffer L.M. Donner D.B. Nature. 1999; 401: 82-85Crossref PubMed Scopus (1904) Google Scholar, 12Romashkova J.A. Makarov S.S. Nature. 1999; 401: 86-90Crossref PubMed Scopus (1670) Google Scholar) Akt has been found to be involved in cell death following the withdrawal of extracellular signaling factors, oxidative and osmotic stress, irradiation, treatment with drugs and ischemic stress (10Franke T.F. Hornik C.P. Segev L. Shostak G.A. Sugimoto C. Oncogene. 2003; 22: 8983-8998Crossref PubMed Scopus (1001) Google Scholar). However, in spite that a variety of cellular stressors influence cells through Ca2+ signaling, the number of studies on Akt signaling and Ca2+ is limited. As for Akt, Ca2+-dependent activation was reported in several studies (13Yano S. Tokumitsu H. Soderling T.R. Nature. 1998; 396: 584-587Crossref PubMed Scopus (536) Google Scholar, 14Huber M. Hughes M.R. Krystal G. J. Immunol. 2000; 165: 124-133Crossref PubMed Scopus (53) Google Scholar). On the other hand, there was a report that the activation of Akt is independent of Ca2+ (15Conus N.M. Hemmings B.A. Pearson R.B. J. Biol. Chem. 1998; 273: 4776-4782Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In contrast, we found that Akt was suppressed by an elevation of [Ca2+]i in myocardiac H9c2 cells overexpressing the calreticulin gene (16Kageyama K. Ihara Y. Goto S. Urata Y. Toda G. Yano K. Kondo T. J. Biol. Chem. 2002; 277: 19255-19264Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). In the cells overexpressing calreticulin, protein phosphatase 2A (PP2A) was up-regulated by Ca2+ to decrease the phosphorylation level of Akt, and the inactivated status of Akt was well correlated with the susceptibility to apoptosis in H9c2 cells under conditions for differentiation induced by retinoic acid. Collectively, these results suggest that the Ca2+-dependent regulatory mechanism of Akt signaling may be important to a variety of apoptotic signaling mechanisms, although how has not been fully clarified. In the present study, to investigate the mechanism of the Ca2+-dependent regulation of Akt signaling, we examined the influence of a change of [Ca2+]i on susceptibility to oxidative stress-induced cell injury and on the Akt signaling pathway in myocardiac H9c2 cells. We show that the Ca2+-dependent regulation of PP2Acα gene transcription is controlled through the cAMP responsive element (CRE), resulting in a change in the activation status of Akt leading to an altered susceptibility to apoptosis. Antibodies and Reagents—Antibodies against Akt, phospho-Akt (Ser-473), and phospho-Akt (Thr-308), CRE-binding protein (CREB), and phospho-CREB (Ser-133) were purchased from Cell Signaling Technology (Beverly, MA). The antibody against Sp1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The reagents used in the study were all of high grade and from Sigma or Wako Pure Chemicals (Osaka, Japan). Cell Culture—H9c2 cells from embryonic rat heart (16Kageyama K. Ihara Y. Goto S. Urata Y. Toda G. Yano K. Kondo T. J. Biol. Chem. 2002; 277: 19255-19264Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 17Hescheler J. Meyer R. Plant S. Krautwurst D. Rosenthal W. Schultz G. Circ. Res. 1991; 69: 1476-1486Crossref PubMed Scopus (414) Google Scholar) were obtained from American Type Culture Collection (CRL-1446). H9c2 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. Before reaching confluence, the cells were split, and plated at low density in culture medium containing 10% fetal bovine serum. Measurement of Cytoplasmic Free Ca2+—The cytoplasmic free Ca2+ concentration, [Ca2+]i, was measured using Fura-2-AM essentially as described previously (16Kageyama K. Ihara Y. Goto S. Urata Y. Toda G. Yano K. Kondo T. J. Biol. Chem. 2002; 277: 19255-19264Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Briefly, cultured cells on glass coverslips were loaded with 5 μm Fura-2-AM (Dojindo, Kumamoto, Japan) for 20 min in Earle's balanced salt solution (EBSS) in the presence of 0.01% pluronic acid F-127. After four washes with EBSS, the cover glass was positioned in a quartz cuvette containing 3.5 ml of fresh EBSS at a 45° angle to both excitation and emission light paths. The fura-2 fluorescence was determined at 37 °C using a spectrofluorophotometer operating at an emission wavelength of 505 nm with an excitation wavelength of 340 and 380 nm. The maximal signal (Rmax) was obtained by adding ionomycin at a final concentration of 4 μm. Then the minimal signal (Rmin) was obtained by adding EGTA at a final concentration of 7.5 mm, followed by Tris-free base to a final concentration of 30 mm, to increase the pH to 8.3. R is the ratio (F1/F2) of the fluorescence of Ex 340 nm, Em 505 nm (F1) to that of Ex 380 nm, Em 505 nm (F2). The actual Ca2+ concentration was calculated as Kd X (R - Rmin)/(Rmax - R) with the Kd equal to 224 nm (18Grynkiewcz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Google Scholar). Lactate Dehydrogenase (LDH) Release Assay—After 4 h of treatment with 5 μm thapsigargin or 10 μm BAPTA-AM or not, cells were incubated with 75 μm hydrogen peroxide (H2O2) for 0–120 min. The LDH activity was assayed by using a MTX LDH kit (Kyokuto Seiyaku, Tokyo, Japan) according to the manufacturer's instructions. Briefly, 50 μl of supernatant was transferred to a 96-well plate, then 50 μl of coloring reagent was added and incubated for 45 min at room temperature. After 100 μl of stop solution was added, absorbance was measured at 560 nm with a microplate reader. The LDH release was shown as a rate of LDH released in the medium to total cellular LDH. TUNEL Assay—Apoptosis was detected flow cytometrically by the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) method (19Gavrieli Y. Sherman Y. Benn-Sasson S.A. J. Cell Biol. 1992; 119: 493-501Crossref PubMed Scopus (9167) Google Scholar) using an ApopTag Plus Fluorescein in situ Apoptosis Detection kit (Serologicals, Norcross, GA). Briefly, cells were harvested and fixed in 70% ethanol, treated with terminal deoxynucleotidyl transferase for 1 h, and then with fluorescein isothiocyanate (FITC)-conjugated antidigoxigenin for 1 h at room temperature, and washed with phosphate-buffered saline (pH 7.0) (PBS) containing 0.1% Triton X-100. The fluorescence intensity was measured at 530 nm using a flow cytometer (BD Biosciences, San Jose, CA). Northern Blot Analysis—The full-length rat PP1α catalytic subunit and PP2A catalytic α cDNAs were generously provided by Dr. Kunimi Kikuchi (Hokkaido University, Japan) (20Kitamura K. Mizuno Y. Sasaki A. Yasui A. Tsuiki S. Kikuchi K. J. Biochem. 1991; 109: 307-310PubMed Google Scholar, 21Kitagawa Y. Tahira T. Ikeda I. Kikuchi K. Tsuiki S. Sugimura T. Nagao M. Biochim. Biophys. Acta. 1988; 951: 123-129Crossref PubMed Scopus (46) Google Scholar). A PstI-SmaI fragment of 600 bp and EcoRI-PvuII fragment of 680 bp were prepared from the cDNAs of PP1αc and PP2Acα, respectively, and used as cDNA probes. The probes were labeled with 32P using a Random Primer Labeling kit (Takara Biomedicals, Shiga, Japan). The isolation of cytoplasmic RNA and Northern blotting were essentially performed as described before (16Kageyama K. Ihara Y. Goto S. Urata Y. Toda G. Yano K. Kondo T. J. Biol. Chem. 2002; 277: 19255-19264Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Isolated RNAs (10 μg) were electrophoresed on a 1% agarose gel containing 0.6 m formaldehyde, transferred to a nylon membrane, and then hybridized with 32P-labeled probes. Autoradiographed membranes were analyzed using a BAS5000 bioimage analyzer (Fuji Photo Film). Immunoblot Analysis—Cultured cells were harvested and lysed for 20 min at 4 °C in lysis buffer (20 mm Tris-HCl, pH 7.5, 130 mm NaCl, 1% Nonidet P-40, and 10% glycerol including protease inhibitors (20 μm amidinophenylmethanesulfonyl fluoride, 50 μm pepstatin, and 50 μm leupeptin)). The supernatants obtained on centrifugation at 8,000 × g for 10 min were used in subsequent experiments. Protein samples were subjected to 10% SDS-PAGE under reducing conditions and then transferred to nitrocellulose membrane as described (22Ihara Y. Sakamoto Y. Mihara M. Shimizu K. Taniguchi N. J. Biol. Chem. 1997; 272: 9629-9634Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The membrane was blocked with 5% skim milk or 3% bovine serum albumin in Tris-buffered saline (pH 7.5) containing 0.05% Tween 20. The blots were coupled with the peroxidase-conjugated secondary antibodies, washed, and then developed using the ECL chemiluminescence delection kit (Amersham Biosciences) according to the manufacturer's instructions. Akt Activity Assay—Akt acitivity was assayed using an Akt assay kit (Cell Signaling Technology) according to the manufacturer's protocol. Briefly, Akt was immunoprecipitated from cell lysates using the anti-Akt antibody, and then the immunoprecipitates were incubated at 30 °C for 30 min in an assay mixture containing an Akt substrate, GSK-3α/β fusion protein. Phosphorylated proteins were separated by 12.5% SDS-PAGE and then transferred to nitrocellulose membrane to detect phosphorylated GSK-3α/β using an anti-phosphorylated GSK-3α/β (Ser-21/9) antibody. Protein Phosphatase Assay—Protein Ser/Thr phosphatase activity was assayed photometrically using Ser/Thr phosphatase assay kit 1 (Upstate Biotechnology, Lake Placid, NY), according to the manufacturer's directions. The activity was assayed in the presence or absence of 10 nm okadaic acid, and the okadaic acid-sensitive activity was estimated as PP2A-specific activity. The phosphopeptide (RK(pT)IRR) was used as a phosphatase substrate. Protein concentrations were determined using a BCA assay kit (Pierce). Generation of Luciferase Reporter Constructs—A ∼1.6-kb fragment of rat PP2Acα gene promoter (-1350 to +258) (23Kitagawa Y. Shima H. Sasaki K. Nagao M. Biochim. Biophys. Acta. 1991; 1089: 339-344Crossref PubMed Scopus (18) Google Scholar) was amplified with rat genome by PCR using Pfu turbo DNA polymerase (Stratagene). The primers used are as follows; a forward primer (5′-GATCTCAGGTACTTTCTTCCGGAACACTAG-3′) and a reverse primer (5′-GTCCAGCTCCTTGGTGAACAACTTC-3′). The PCR product was subcloned into pUC18 to obtain pUC18-pro-PP2Ac. The nucleotide sequence was confirmed by sequencing with an ALFexpress II system (Amersham Biosciences). pUC18-pro-PP2Ac was digested with HindIII, and the resulting fragment containing the promoter region from -1209 to +258 was inserted into the HindIII site of the reporter vector pGL3-Basic (Stratagene) to give pGL3-pro-PP2Ac. To generate deleted mutants of the luciferase reporter construct, pGL3-pro-PP2Ac was digested with SacI and XhoI, and deletion mutants were made using a deletion kit for kilo sequence (Takara Biomedicals). Site-directed Mutagenesis for Luciferase Vectors—In vitro mutagenesis was performed with pGL3-pro-PP2Ac-del (-279 to +258) and del (-145 to +258) as templates by using a QuikChange site-directed mutagenesis kit (Stratagene). Oligonucleotides used are as follows: GC box (-155), 5′-CCCTCCCCGCGGGAGGACCACAACCCAAAAGCGAAGCCACTTCC-3′; CRE (-26), 5′-CCTGACGCCGGCGTGTGGTCACCACGCCGGGCGGCCGCCATTAC-3′. The nucleotide sequences were confirmed by sequencing with an ALFexpress II system (Amersham Biosciences). Luciferase Activity Assay—Each vector was transfected into H9c2 cells by using Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. After 24 h of transfection, cells were treated with thapsigargin (5 μm) or BAPTA-AM (10 μm), or left untreated for the periods indicated in the text. Then luciferase activity was assayed with cellular extracts by using a dual-luciferase reporter assay system (Promega). Electrophoretic Mobility Shift Assay—The electrophoretic mobility shift assay (EMSA) for the GC box and CRE was performed as described previously (24Muroya T. Ihara Y. Ikeda S. Yasuoka C. Miyahara Y. Urata Y. Kondo T. Kohno S. Biochem. Biophys. Res. Commun. 2003; 309: 905-910Crossref Scopus (15) Google Scholar). Briefly, oligonucleotides were labeled with [γ-32P]ATP using T4 polynucleotide kinase and then annealed to double-strand oligonucleotides. Specific oligonucleotides for the GC box and CRE were prepared according to the nucleotide sequences of the rat PP2Acα promoter region. Oligonucleotides used are as follows: GC box (-155), 5′-CGGGAGGACCACGCCCCAAAAGCGAAGC-3′; GC box (-155)-Mt, 5′-CGGGAGGACCACAACCCAAAAGCGAAGC-3′; CRE (-26), 5′-GACGCCGGCCTGACGTCACCACGCC-3′; CRE (-26)-Mt, 5′-GACGCCGGCCTGTGGTCACCACGCC -3′. Binding reactions were carried out in 15 μl of reaction mixture (25 mm Tris, pH 7.0, 6.25 mm MgCl2, 0.5 mm EDTA, 0.5 mm dithiothreitol, 50 mm KCl, and 10% glycerol) containing 10 μg of nuclear extract, and 25 ng of labeled oligonucleotide. For the supershift assay, specific antibodies were added to the reaction mixture during the 30-min binding reaction. Indirect Immunofluorescence Microscopy—After treatment with 5 μm thapsigargin or 10 μm BAPTA-AM for 2 h, cells incubated on Lab-Tek chamber slides (Nunc) were fixed with 3% paraformaldehyde for 20 min at room temperature and washed three times with PBS. Cells were permeabilized in 1% Triton X-100 in PBS for 10 min and washed three times with PBS. They were blocked with 1% bovine serum albumin in PBS for 30 min at room temperature, washed three times with PBS, and then incubated with antiphospho-CREB (Ser-133) overnight at 4 °C. Cells were washed with PBS four times and incubated with FITC-labeled anti-rabbit IgG antibody for 30 min in a dark room. The immunoreactive signals were visualized by indirect immunofluorescence microscopy. Elevation of [Ca2+]i Gradually Accelerates Cell Damage and Apoptosis in H9c2 Cells—To modify the level of [Ca2+]i in H9c2 cells, the cells were treated with 5 μm thapsigargin (25Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci., U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (3010) Google Scholar), an inhibitor for sarcoplasmic/endoplasmic reticulum Ca2+ ATPase to increase [Ca2+]i, or with 10 μm BAPTA-AM (26Pan Z. Bhat M.B. Nieminen A.-L. Ma J. J. Biol. Chem. 2001; 276: 32257-32263Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), a cell permeable Ca2+ chelator to decrease [Ca2+]i, for 0–4 h. As shown in Fig. 1A, with thapsigargin, [Ca2+]i shows a transient increase within the first 10 min. It then decreases to the basal level till 30 min, but again increases and retains elevated until 120 min, before gradually decreasing to the initial level. In contrast, with BAPTA-AM, [Ca2+]i shows a continuous lowering during the treatment. To investigate the influence of the long term change of [Ca2+]i on susceptibility to oxidative stress-induced cell injury, cells were treated with Ca2+ modulators (i.e. thapsigargin and BAPTA-AM) for 4 h then exposed to 75 μm hydrogen peroxide (H2O2) for 0–120 min, and cell damage was examined at predetermined times using the LDH release assay as described in the methods. In Fig. 1B, the release of LDH by H2O2 was observed only at 120 min in the medium of untreated cells. However, the release by H2O2 was initially observed at 60 min and increased at 120 min in the cells treated with thapsigargin. In contrast, BAPTA-AM treatment completely suppressed the release of LDH by H2O2 throughout the 120 min. Next, to investigate whether apoptosis is involved in the mechanism of thapsigargin-induced cell damage, cells were treated with Ca2+ modulators for 4 h then exposed to H2O2 (75 μm) for 2 h, and apoptosis was examined. Fig. 1C shows that TUNEL-positive fluorescence intensity was increased slightly by H2O2 (upper), but was significantly enhanced after the pretreatment with thapsigargin (middle). In contrast, no change was observed in the fluorescent intensity of the BAPTA-AM-treated cells with or without H2O2 (lower). Collectively, these results indicate that the susceptibility to apoptosis was enhanced with a long term elevation of [Ca2+]i, but was suppressed with the lowering of [Ca2+]i in the cells under oxidative stress with H2O2, suggesting that the continuous change of [Ca2+]i influences the susceptibility to apoptosis. The Long Term Change of [Ca2+]i Influences Akt Signaling in H9c2 Cells—To know the role of [Ca2+]i in cell survival signaling, we focused on the effect of Ca2+ modulators on Akt signaling. Previously, we found that the Akt signaling pathway is responsible for the cytoprotective mechanism in H9c2 cells under conditions of stress such as serum starvation with retinoic acid (16Kageyama K. Ihara Y. Goto S. Urata Y. Toda G. Yano K. Kondo T. J. Biol. Chem. 2002; 277: 19255-19264Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), and oxidative stress with hydrogen peroxide (27Murata H. Ihara Y. Nakamura H. Yodoi J. Sumikawa K. Kondo T. J. Biol. Chem. 2003; 278: 50226-50233Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Although elevations in Ca2+ act as a signal, a prolonged increase in the concentration of Ca2+ can be lethal (1Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. 2000; 1: 11-21Crossref Scopus (4493) Google Scholar). Moreover, cell signaling molecules including transcription factors are activated differentially by the amplitude and duration of the response to Ca2+ (28Dolmetsch R.E. Lewis R.S. Goodnow C.C. Healy J.I. Nature. 1997; 386: 855-858Crossref PubMed Scopus (1564) Google Scholar). In the present study, H9c2 cells were treated with Ca2+ modulators such as thapsigargin (5 μm) and BAPTA-AM (10 μm) for over 2 h to induce a long term change of [Ca2+]i. After the treatments, the phosphorylation levels of Ser-473 and Thr-308 of Akt were examined. The phosphorylation of Thr-308 located in the kinase catalytic domain of Akt is necessary for the activation, and the phosphorylation of Ser-473 located in the regulatory domain of Akt supports the activation. As shown in Fig. 2, A (right) and B, the treatment with BAPTA-AM increased the phosphorylation of Akt both at Ser-473 and Thr-308, and the phosphorylation level increased to a maximum at 2 h, and was sustained thereafter till 4 h. In contrast, the phosphorylation of Akt decreased in a time-dependent manner with thapsigargin (Fig. 2, A (left) and B). Next we examined whether Ca2+ modulators also have an effect on the kinase activity of Akt. Fig. 2C shows that Akt activity is suppressed after the treatment with thapsigargin, but increased with BAPTA-AM. These results were consistent with the change in the phosphorylation status of Akt on treatment with Ca2+ modulators such as thapsigargin and BAPTA-AM. Together, Akt signaling was suppressed by the long term elevation of [Ca2+]i with thapsigargin, but was enhanced by the long term lowering of [Ca2+]i with BAPTA-AM. This suggests a Ca2+-dependent regulation of Akt signaling in H9c2 cells. Furthermore, the Ca2+-induced suppression of Akt signaling was compatible with the enhanced susceptibility to apoptosis in H9c2 cells treated with H2O2 (Fig. 1, B and C). The Expression of PP2Acα Is Transcriptionally Regulated by Ca2+Modulators—3-Phosphoinositide-dependent protein kinase (PDK1) is known to be responsible for phosphorylating Akt at Thr-308, and is activated by both phosphatidylinositol (3,4,5)-trisphosphate and phosphatidylinositol (3,4,)-bisphosphate, products of PI3K (9Scheid M.P. Woodgett J.R. FEBS Lett. 2003; 546: 108-112Crossref PubMed Scopus (346) Google Scholar, 10Franke T.F. Hornik C.P. Segev L. Shostak G.A. Sugimoto C. Oncogene. 2003; 22: 8983-8998Crossref PubMed Scopus (1001) Google Scholar). To investigate whether the upstream kinases are involved in the regulation of Akt by the change of [Ca2+]i, the activities for PI3K and PDK1 were measured in the cells treated with Ca2+ modulators. However, neither activities showed any significant change even if the cells were treated with thapsigargin or BAPTA-AM for 0–4 h (data not shown). Therefore, we focused on Ser/Thr protein phosphatases that could dephosphorylate and inactivate Akt to regulate the Akt signaling pathway (7Klumpp S. Krieglstein J. Curr. Opin. Pharmacol. 2002; 2: 458-462Crossref PubMed Scopus (130) Google Scholar). To investigate whether [Ca2+]i levels affect the expression of protein Ser/Thr phosphatases, the cells were treated with 5 μm thapsigargin or 10 μm BAPTA-AM for 0–4 h, and transcriptional levels were estimated by Northern blot analysis for protein phosphatase 2A catalytic subunit α (PP2Acα) and protein phosphatase 1α catalytic subunit (PP1αc). In Fig. 3A, the level of PP2Acα mRNA was increased by thapsigargin but decreased by BAPTA-AM. In contrast, the mRNA level of PP1αc was not significantly changed by the Ca2+ modulators. In the immunoblot analysis, the protein level of PP2Acα was increased by thapsigargin but decreased by BAPTA-AM (Fig. 3B). However, the protein level of PP1αc was not influenced by thapsigargin or BAPTA-AM. The protein level of calcineurin/PP2B was not influenced by thapsigargin or BAPTA-AM either (data not shown). These results were consistent with results of the change of transcriptional levels for the phosphatases in the cells treated with each Ca2+ modulator. The enzymatic activity of PP2A was also assayed in the cells treated with thapsigargin or BAPTA-AM for 0–4 h. As shown in Fig. 3C, the activity of PP2A increased with thapsigargin by ∼2-fold compared with that of untreated cells. In contrast, the activity was slightly suppressed after 2 h treatment with BAPTA-AM. Collectively, these results indicate that PP2Acα expression is transcriptionally regulated by the long term change of [Ca2+]i to control the phosphorylation status of target molecules including Akt. Inhibition of PP2A Activity by Okadaic Acid Enhanc
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