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• W2146245819 abstract "Inositol 1,4,5-trisphosphate receptor-deficient (IP3RKO) B-lymphocytes were used to investigate the functional relevance of type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) and its cleavage by caspase-3 in apoptosis. We showed that inositol 1,4,5-trisphosphate receptor-deficient cells were largely resistant to apoptosis induced by both staurosporine (STS) and B-cell receptor (BCR) stimulation. Expression of either the wild-type IP3R1 or an N-terminal deletion mutant (Δ1-225) that lacks inositol 1,4,5-trisphosphate-induced Ca2+ release activity restored sensitivity to apoptosis and the consequent rise in free cytosolic Ca2+ concentration ([Ca2+]i). Expression of caspase-3-non-cleavable mutant receptor, however, dramatically slowed down the rate of apoptosis and prevented both Ca2+ overload and secondary necrosis. Conversely, expression of the “channel-only” domain of IP3R1, a fragment of the receptor generated by caspase-3 cleavage, strongly increased the propensity of the cells to undergo apoptosis. In agreement with these observations, caspase inhibitors impeded apoptosis and the associated rise in [Ca2+]i. Both the staurosporine- and B-cell receptor-induced apoptosis and increase in [Ca2+]i could be induced in nominally Ca2+-free and serum-free culture media, suggesting that the apoptosis-related rise in [Ca2+]i was primarily because of the release from internal stores rather than of influx through the plasma membrane. Altogether, our results suggest that IP3R1 plays a pivotal role in apoptosis and that the increase in [Ca2+]i during apoptosis is mainly the consequence of IP3R1 cleavage by caspase-3. These observations also indicate that expression of a functional IP3R1 per se is not enough to generate the significant levels of cytosolic Ca2+ needed for the rapid execution of apoptosis, but a prior activation of caspase-3 and the resulting truncation of the IP3R1 are required. Inositol 1,4,5-trisphosphate receptor-deficient (IP3RKO) B-lymphocytes were used to investigate the functional relevance of type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) and its cleavage by caspase-3 in apoptosis. We showed that inositol 1,4,5-trisphosphate receptor-deficient cells were largely resistant to apoptosis induced by both staurosporine (STS) and B-cell receptor (BCR) stimulation. Expression of either the wild-type IP3R1 or an N-terminal deletion mutant (Δ1-225) that lacks inositol 1,4,5-trisphosphate-induced Ca2+ release activity restored sensitivity to apoptosis and the consequent rise in free cytosolic Ca2+ concentration ([Ca2+]i). Expression of caspase-3-non-cleavable mutant receptor, however, dramatically slowed down the rate of apoptosis and prevented both Ca2+ overload and secondary necrosis. Conversely, expression of the “channel-only” domain of IP3R1, a fragment of the receptor generated by caspase-3 cleavage, strongly increased the propensity of the cells to undergo apoptosis. In agreement with these observations, caspase inhibitors impeded apoptosis and the associated rise in [Ca2+]i. Both the staurosporine- and B-cell receptor-induced apoptosis and increase in [Ca2+]i could be induced in nominally Ca2+-free and serum-free culture media, suggesting that the apoptosis-related rise in [Ca2+]i was primarily because of the release from internal stores rather than of influx through the plasma membrane. Altogether, our results suggest that IP3R1 plays a pivotal role in apoptosis and that the increase in [Ca2+]i during apoptosis is mainly the consequence of IP3R1 cleavage by caspase-3. These observations also indicate that expression of a functional IP3R1 per se is not enough to generate the significant levels of cytosolic Ca2+ needed for the rapid execution of apoptosis, but a prior activation of caspase-3 and the resulting truncation of the IP3R1 are required. Apoptosis is a highly regulated and evolutionarily conserved form of cell death that plays an important role in normal embryonic development and maintenance of adult tissue homeostasis (1Arends M.J. Wyllie A.H. Int. Rev. Exp. Pathol. 1991; 32: 223-254Crossref PubMed Scopus (1394) Google Scholar). Apoptotic cell death involves a characteristic sequence of morphological and biochemical features (2Wyllie A.H. Kerr J.F. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-306Crossref PubMed Scopus (6728) Google Scholar, 3Crompton M. Biochem. J. 1999; 341: 233-249Crossref PubMed Scopus (2119) Google Scholar). Most, but not all, forms of apoptotic cell death processes are characterized by the activation of a family of aspartate-specific cysteine proteases called caspases that cleave a wide range of cellular proteins leading to the manifestation of the major phenotypes in apoptosis (4Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2450) Google Scholar). Early studies of glucocorticoid-induced cell death suggested that an increase in the cytosolic free Ca2+ concentration ([Ca2+]i) was a key component of the apoptotic process (5Kaiser N. Edelman I.S. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 638-642Crossref PubMed Scopus (168) Google Scholar). Various reports have since then established that a prolonged and up-regulated intracellular Ca2+ signal is a general feature of apoptosis (6McConkey D.J. Hartzell P. Amador-Perez J.F. Orrenius S. Jondal M. J. Immunol. 1989; 143: 1801-1806PubMed Google Scholar, 7Nicotera P. Orrenius S. Cell Calcium. 1998; 23: 173-180Crossref PubMed Scopus (403) Google Scholar, 8Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1780) Google Scholar, 9Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell Biol. 2000; 1: 11-21Crossref PubMed Scopus (4491) Google Scholar). Apoptosis-related cleavage of a range of regulatory proteins and ion channels seems to be common to many apoptotic paradigms. During apoptosis, caspase-3, the main effector caspase, cleaves a wide array of cellular proteins including many that play significant roles in intracellular Ca2+ regulation such as the Ca2+/calmodulin-dependent protein kinase IV (10McGinnis K.M. Whitton M.M. Gnegy M.E. Wang K.K. J. Biol. Chem. 1998; 273: 19993-20000Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), plasma membrane Ca2+ ATPase (11Paszty K. Verma A.K. Padanyi R. Filoteo A.G. Penniston J.T. Enyedi A. J. Biol. Chem. 2002; 277: 6822-6829Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 12Schwab B.L. Guerini D. Didszun C. Bano D. Ferrando-May E. Fava E. Tam J. Xu D. Xanthoudakis S. Nicholson D.W. Carafoli E. Nicotera P. Cell Death Differ. 2002; 9: 818-831Crossref PubMed Scopus (232) Google Scholar), Na+/Ca2+ exchanger (13Orrenius S. Zhivotovsky B. Nicotera P. Nat. Rev. Mol. Cell Biol. 2003; 4: 552-565Crossref PubMed Scopus (2445) Google Scholar), and the β-subunit of the Na+/K+-ATPase (14Dussmann H. Rehm M. Kogel D. Prehn J.H. J. Cell Sci. 2003; 116: 525-536Crossref PubMed Scopus (94) Google Scholar). Caspase cleavage could bring about a gain or loss of function on the target proteins leading to aberrant intracellular Ca2+ regulation that can directly influence the commitment of cells to apoptosis. Inositol 1,4,5-trisphosphate (IP3) 1The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; BCR, B-cell antigen receptor; IICR, IP3-induced Ca2+ release; IP3R-KO cells, DT40 cells lacking all three IP3R isoforms; STS, staurosporine; WT-, wild-type; F, farad; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; FITC, fluorescein isothiocyanate; PI, propidium iodide; z-, benzyloxycarbonyl; fmk, fluoromethyl ketone. receptors (IP3Rs) are ubiquitous intracellular Ca2+ release channels, and their involvement in apoptosis has been demonstrated in different cell types. It was initially reported that the mRNA and protein levels of IP3R3 increase during apoptosis in lymphocytes, with no change in the IP3R1 level (15Khan A.A. Soloski M.J. Sharp A.H. Schilling G. Sabatini D.M. Li S.H. Ross C.A. Snyder S.H. Science. 1996; 273: 503-507Crossref PubMed Scopus (245) Google Scholar). Also, expression of an antisense cDNA construct of IP3R3 blocked the dexamethasone-induced apoptosis and increase in [Ca2+]i, whereas that of IP3R1 had no effect. Subsequently, however, it was reported that Jurkat cells deficient in IP3R1 were resistant to apoptosis induced by Fas, dexamethasone, and γ-irradiation despite the presence of IP3R3 (16Jayaraman T. Marks A.R. Mol. Cell. Biol. 1997; 17: 3005-3012Crossref PubMed Scopus (235) Google Scholar). IP3Rs are functionally redundant in chicken B-lymphocytes as apoptosis induced by B-cell receptor (BCR) stimulation was significantly inhibited only in cells deficient of all three receptors (17Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (375) Google Scholar). The reason for these discrepancies is a matter of speculation, but together the reports indicate that Ca2+ flux through IP3Rs plays a fundamental role in apoptotic cell death induced by various stimuli. A more direct involvement of IP3Rs in apoptosis was demonstrated by reports that identified IP3R1 as a substrate of caspase-3 during apoptosis (18Hirota J. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; 274: 34433-34437Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 19Haug L.S. Walaas S.I. Ostvold A.C. J. Neurochem. 2000; 75: 1852-1861Crossref PubMed Scopus (29) Google Scholar). IP3R1, but not IP3R2 or IP3R3, contains a single DEVD-specific cleavage site for caspase-3 at amino acids 1888-1891 (mouse sequence), and this sequence is conserved in rat and human proteins. Caspase-3-mediated degradation may actually explain a previous observation that the IP3R1 level shows a dramatic decrease following dexamethasone treatment of thymocytes and S49 cells (15Khan A.A. Soloski M.J. Sharp A.H. Schilling G. Sabatini D.M. Li S.H. Ross C.A. Snyder S.H. Science. 1996; 273: 503-507Crossref PubMed Scopus (245) Google Scholar). Cleavage by caspase-3 removes the cytoplasmic segment of IP3R1 comprising the IP3-binding domain and most of the regulatory domain. This cleavage obviously abolishes the IP3-induced Ca2+ release (IICR) activity of the receptor and produces a “channel-only” domain that apparently remains constitutively open in transiently transfected COS-1 and HeLa cells (20Nakayama T. Hattori M. Uchida K. Nakamura T. Tateishi Y. Bannai H. Iwai M. Michikawa T. Inoue T. Mikoshiba K. Biochem. J. 2004; 377: 299-307Crossref PubMed Scopus (78) Google Scholar). The significance of this cleavage either to the process of cell death or to the apoptosis-related increase in [Ca2+]i is not yet clear. Because IP3R1 is the most ubiquitous isoform of the IP3R family, its direct involvement in apoptosis as a caspase-3 substrate could have a far reaching physiological significance. Therefore, we set out to investigate the functional relevance of IP3R1 and its cleavage by caspase-3 to apoptosis induced by staurosporine (STS) and BCR stimulation using IP3R-deficient chicken B-lymphocytes (IP3R-KO) that stably express various mutants of the IP3R1. We report here that expression of either the wild-type IP3R1 or a mutant receptor that is no longer activated by IP3 could render the IP3R-KO cells susceptible to apoptosis and support the consequent rise in [Ca2+]i. Pretreatment of the cells with caspase inhibitors blocked the cleavage of IP3R1 and the rise in [Ca2+]i as well as apoptosis. A functional receptor that was mutated at the caspase-3 cleavage site significantly slowed down the kinetics of apoptosis and prevented both Ca2+ overload and secondary necrosis. In contrast, stable expression of the channel-only domain, corresponding to the C-terminal fragment generated by caspase-3, predisposed the cells to undergo apoptosis. Both STS- and BCR-induced apoptotic cell death and the associated rise in [Ca2+]i could also be induced in nominally Ca2+-free culture medium, suggesting Ca2+ release from internal stores as the primary cause of the [Ca2+]i rise rather than a major influx from the extracellular medium. Our data also indicate that IP3R1 plays a pivotal role in apoptosis not necessarily through its IICR activity but mainly as a substrate of caspase-3. The perturbance of intracellular Ca2+ homeostasis during the execution phase of apoptosis seems to be related to the cleavage of IP3R1 by caspase-3. Accordingly, the amplification of apoptotic signals and the rapid execution of apoptosis necessitate a prior activation of caspase-3 and the resulting truncation of the IP3R1. In addition, our results suggest that the specific pattern of changes in [Ca2+]i during apoptosis in different cell types may be related to the relative distribution of IP3R1 among the different tissues. Reagents—The polyclonal antibody (Rbt04) raised against amino acids 2735-2749 of mouse IP3R1 has been described previously (21Parys J.B. De Smedt H. Missiaen L. Bootman M.D. Sienaert I. Casteels R. Cell Calcium. 1995; 17: 239-249Crossref PubMed Scopus (119) Google Scholar). STS was purchased from Sigma. Mouse anti-chicken IgM (clone M-4) was from Southern Biotech (Birmingham, AL). Caspase inhibitors and colorimetric caspase-3 substrate were obtained from Bachem (Bubendorf, Switzerland). Cells and Culture Conditions—The DT40 chicken B-lymphocytes lacking all three IP3Rs (IP3R-KO) were a kind gift from Dr. T. Kurosaki (Tokyo, Japan). IP3R-KO cells were maintained in RPMI 1640 medium containing 10% fetal calf serum, 1% chicken serum, 50 μm 2-mercaptoethanol, 85 units/ml penicillin, 85 μg/ml streptomycin, and 3.5 mml-glutamine in a humidified incubator at 5% CO2 and 37 °C. These culture media and additives and Dulbecco's modified Eagle's medium with no added calcium (catalog no. 21068) were purchased from Invitrogen. DNA Constructs and Transfection—Mouse cerebellum IP3R1 cDNA (a kind gift from Dr. K. Mikoshiba, Tokyo, Japan) in pcDNA-3.1(+) vector was used as a template to generate different mutants of the receptor. Mutagenesis was carried out using the QuikChange XL site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. To construct the caspase-3-non-cleavable mutant of IP3R1 (IP3R1Δcasp), a fragment containing the region 2129-6819 of the full-size IP3R1 cDNA, flanked by two BamHI restriction sites, was first subcloned into the pBlueScript II SK(+) vector. This construct was then used as template for the insertion of the mutations using 5′-GGGAAACAAAAAGAAAGATATCGAAGTGGCCAGGGATGCCCCGTC-3′ as a forward primer and 5′-GAGGGGGCATCCCTGGCCACTTCGATATCTTTCTTTTTGTTTCCC-3′ as a reverse primer (underlined are the inserted mutations). These oligonucleotides replace the known caspase-3 cleavage site DEVD, encoded between nucleotides 2584 and 2594 with IEVA, thereby mutating the aspartic acids at positions 1888 and- 1891 to isoleucine and alanine, respectively. The mutated IP3R1 region in the recombinant vector was then recovered with BamHI and religated into a BamHI-digested pcDNA3.1(+)/IP3R1. The N-terminal deletion mutant of the IP3R1 lacking the first 225 amino acids ((Δ1-225)- IP3R1) was constructed as described earlier (22Bultynck G. Szlufcik K. Nadif Kasri N. Assefa Z. Callewaert G. Missiaen L. Parys J.B. De Smedt H. Biochem. J. 2004; 381: 87-96Crossref PubMed Scopus (98) Google Scholar), and the deletion mutant (Δ1-1891)IP3R1 encoding the 95-kDa caspase-3-generated C-terminal region of the receptor was constructed by PCR amplification of the relevant region and subsequent substitution in the pcDNA3.1(+)/IP3R1 plasmid. The sequences of the different constructs were confirmed by the automated fluorescent sequencing system (Amersham Biosciences). All constructs, including that of the wild-type receptor (WT-IP3R1), were transfected into IP3R-KO cells by electroporation using a Gene Pulser apparatus (Bio-Rad). Briefly, about 107 cells in 0.5 ml of serum-free medium were transferred to a 4-mm electroporation cuvette (Eurogentec, Seraing, Belgium) and pulsed at 550 V and 25 μF in the presence of 100 μg of plasmids. The electroporated cells were incubated in 30 ml of normal culture medium for 24 h before starting the selection with 1.5 mg of G418/ml to generate stable cell lines. IP3R1 Subcellular Localization—The cells (5 × 105) were attached to poly-l-lysine-coated, 2-well chambered slides (Nalge Nunc, Naperville, IL) for 3 h before fixation in 3% paraformaldehyde for 15 min at room temperature. The fixed cells were then permeabilized with 0.5% Triton X-100 in PBS for 5 min, and the nonspecific binding sites were blocked with 20% goat serum in PBS for 1 h at room temperature. The cells were then incubated for 1 h with the Rbt04 primary antibody in PBS containing 1.5% goat serum. Subsequently, the slides were washed three times with PBS and incubated with the Alexa Fluor 488 goat anti-rabbit secondary antibody (Molecular Probes) in PBS containing 5% goat serum. BODIPY-thapsigargin and MitoTracker dyes (Molecular Probes) were used to visualize endoplasmic reticulum (ER) and mitochondria, respectively. BODIPY-thapsigargin was added together with the secondary antibody. Mitochondria were visualized by incubating the cells with MitoTracker for 30 min at 37 °C prior to fixation. Images were acquired using an LSM510 confocal laser-scanning microscope (Carl Zeiss, Germany) with a Plan-Neofluar ×100 numerical aperture 1.3 oil immersion objective. Excitation wavelengths of 488 nm for Alexa Fluor 488 and 543 nm for BODIPY-thapsigargin and MitoTracker were used. Emission fluorescence was monitored by a photomultiplier fitted with a BP filter of 505-530 nm for Alexa Fluor 488 and with an LP filter of 585 nm for BODIPY-thapsigargin and MitoTracker. Preparation of Cell Lysates and Microsomes—Following treatments, cells were harvested and washed in ice-cold PBS before preparation of lysates as described previously (23Assefa Z. Vantieghem A. Garmyn M. Declercq W. Vandenabeele P. Vandenheede J.R. Bouillon R. Merlevede W. Agostinis P. J. Biol. Chem. 2000; 275: 21416-21421Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Total microsomes were prepared as described previously with minor modifications (21Parys J.B. De Smedt H. Missiaen L. Bootman M.D. Sienaert I. Casteels R. Cell Calcium. 1995; 17: 239-249Crossref PubMed Scopus (119) Google Scholar). Briefly, cells were harvested by centrifugation for 5 min at 400 × g and washed twice with ice-cold PBS without Ca2+ and Mg2+. Cell pellets were then resuspended in homogenization buffer (10 mm Tris/HCl, pH 7.4, 1 mm EGTA, 0.8 mm benzamidine, 0.2 mm phenylmethylsulfonyl fluoride, 0.5 μg/ml leupeptin, 0.5 μg/ml aprotinin, 0.5 μg/ml pepstatin A) and homogenized on ice with a probe sonicator (MSE Ltd., UK). Total microsomes were obtained by centrifugation for 25 min at 125,000 × g. The membranous pellet was then resuspended in end medium (20 mm Tris/HCl, pH 7.4, 300 mm sucrose, 0.8 mm benzamidine, and 0.2 mm phenylmethylsulfonyl fluoride). Cell lysates and microsomal preparations were frozen in liquid nitrogen and stored at -80 °C. Protein concentrations were determined using either the BCA protocol (Pierce) or the Lowry method (24Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.F. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) with bovine serum albumin as a standard. Cleavage of IP3R1 by Recombinant Caspase-3—For in vitro assay of IP3R1 cleavage by caspase-3, microsomes (200 μg) were incubated with purified active recombinant human caspase-3 (Pharmingen, BD Biosciences) at 37 °C for 1 h. The samples were then analyzed by Western blot using the Rbt04 antibody against the C-terminal region of IP3R1. Briefly, the samples were subjected to electrophoresis on 3-8% Trisacetate SDS-polyacrylamide gradient gels (Invitrogen), transferred to a polyvinylidene difluoride membrane, and subsequently incubated with the primary antibody (1:3000 dilution) and alkaline phosphatase-conjugated secondary antibody (1:8000 dilution). The immunoreactive bands were developed using the enhanced chemifluorescence reagent from Amersham Biosciences and then detected by the Storm840 FluorImager equipped with the ImageQuant software (Amersham Biosciences). Induction and Analysis of Apoptosis—Cells were seeded at a density of 0.5 × 106 cells/ml (1.5 ml of medium) in 12-well plates before treatment with either STS or anti-chicken IgM and then incubated for a specified period of time before harvesting. In some experiments, the anti-chicken IgM antibody was supplemented with anti-mouse IgM to further cross-link the surface IgM. For analysis of apoptosis, the protocol included in the annexin V-FITC apoptosis detection kit from Pharmingen was used as provided. Cell death detection was performed on a Coulter Epics flow cytometer (Beckman-Coulter Inc., Miami, FL) using the standard emission filters for green (FL1) and red (FL3) fluorescence photomultipliers. The Expo32 MultiCOMP software from Coulter Corporation was used to analyze the data. Cells having a reduced overall volume and staining with annexin V-FITC while retaining the plasma membrane integrity (propidium iodide (PI)-negative) were regarded as apoptotic. Primary necrosis was identified as the loss of plasma membrane integrity without a clear reduction in cell volume and no annexin staining. Primary necrotic cells stain with PI only. Cells that stain with both annexin V-FITC and PI were considered as those undergoing secondary necrosis, subsequent to apoptotic cell death. Caspase-3 Assay—Cells treated with STS or anti-chicken IgM antibody and incubated for the indicated time were harvested in ice-cold buffer containing 50 mm Tris/HCl, pH 7.6, 150 mm NaCl, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 10 μg of each/ml leupeptin, aprotinin, and pepstatin A, 1 mm dithiothreitol, and 1% Triton X-100. Caspase-3 assay was then performed by the colorimetric CaspACE assay system (Promega) exactly as recommended by the manufacturer, using 50-100 μg of protein/assay. After incubation for 3 h at 37 °C, A405 readings were taken using a 96-well plate reader. Analysis of [Ca2+]iby Flow Cytometry—Measurement of [Ca2+]i was performed essentially as described previously (25Vermes I. Haanen C. Reutelingsperger C. J. Immunol. Methods. 2000; 243: 167-190Crossref PubMed Scopus (639) Google Scholar, 26Scoltock A.B. Bortner C.D. St. J. Bird G. Putney Jr., J.W. Cidlowski J.A. J. Biol. Chem. 2000; 275: 30586-30596Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Briefly, 106 cells were first treated with STS or anti-chicken IgM for a specific period of time and then loaded with either 3 μm Fluo-3/AM or 6 μm Fura red/AM (Molecular Probes) in 200 μl of culture medium for 30 and 90 min, respectively, at 37 °C. At the end of the incubation period, extra medium was added to make the final cell density up to about 106 cells/ml. 15,000 cells/sample were analyzed for the Ca2+-dependent increase in Fluo-3 fluorescence and the Ca2+-dependent decrease in the Fura red fluorescence emission by a Coulter Epics flow cytometer by exciting the cells at 488 nm. Expression of Different IP3R1 Constructs in IP3R-deficient Cells—The basic structure of IP3R1 comprises the N-terminal IP3-binding domain, the regulatory domain, and the C-terminal channel domain as shown schematically in Fig. 1A. Previous studies have shown that caspase-3 mediates the cleavage of mouse IP3R1 at Asp-1891 in cells undergoing apoptosis (18Hirota J. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; 274: 34433-34437Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 19Haug L.S. Walaas S.I. Ostvold A.C. J. Neurochem. 2000; 75: 1852-1861Crossref PubMed Scopus (29) Google Scholar). In this study, we aimed to investigate the exact role of the IP3R1 channel activity and the significance of this cleavage in apoptotic cell death. For this purpose, we first generated a caspase-non-cleavable mutant (IP3R1Δcasp) by introducing the mutations D1888I and D1891A (Fig. 1B) using mouse IP3R1 as a template. IP3R1Δcasp possesses the same level of IICR activity as that of the WT-IP3R1 in stably expressing cell lines (data not shown). We also generated a deletion mutant lacking the first 225 amino acids ((Δ1-225)IP3R1). This segment immediately precedes the ligand-binding core (Fig. 1C) and has been designated as a suppressor domain because of the observation that its deletion significantly enhances the affinity of the receptor for IP3 binding (27Yoshikawa F. Morita M. Monkawa T. Michikawa T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1996; 271: 18277-18284Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 28Yoshikawa F. Uchiyama T. Iwasaki H. Tomomori-Satoh C. Tanaka T. Furuichi T. Mikoshiba K. Biochem. Biophys. Res. Commun. 1999; 257: 792-797Crossref PubMed Scopus (42) Google Scholar). However, we (22Bultynck G. Szlufcik K. Nadif Kasri N. Assefa Z. Callewaert G. Missiaen L. Parys J.B. De Smedt H. Biochem. J. 2004; 381: 87-96Crossref PubMed Scopus (98) Google Scholar) and others (29Uchida K. Miyauchi H. Furuichi T. Michikawa T. Mikoshiba K. J. Biol. Chem. 2003; 278: 16551-16560Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) have observed that despite the higher level of IP3 binding, (Δ1-225)IP3R1 lacks any detectable IICR activity. The use of this mutant will help clarify whether the role of IP3R1 in apoptosis requires its IICR activity. Finally, a mutant receptor that lacks amino acids 1-1891 ((Δ1-1891)IP3R1) and mainly comprising the channel domain (as generated by caspase-3 cleavage of IP3R1) was created by PCR amplification of the specific region (Fig. 1D). All constructs including the WT-IP3R1 were transfected by electroporation into chicken embryonic B-lymphocytes (DT40 cells) lacking all three IP3R isoforms (IP3R-KO cells), and permanent cell lines were then established by selection with G418. The level of expression of all constructs in cell lines was comparable, as determined by Western blot analysis of membrane preparations (data not shown). In addition, the subcellular localization of the constructs was determined using confocal laser-scanning microscopy by co-staining of the IP3R1 (Rbt04 antibody and Alexa Fluor 488-conjugated secondary antibody) and the ER (BODIPY-thapsigargin) or mitochondria (MitoTracker). Representative images from the cells expressing the (Δ1-1891)IP3R1 construct are shown in Fig. 1, E and F. The truncated IP3R1 was expressed exclusively in the perinuclear region and was strongly associated with the distribution of the ER. It was excluded from mitochondria and the plasma membrane. These results are in agreement with the subcellular localization of a similar construct in other cell types (20Nakayama T. Hattori M. Uchida K. Nakamura T. Tateishi Y. Bannai H. Iwai M. Michikawa T. Inoue T. Mikoshiba K. Biochem. J. 2004; 377: 299-307Crossref PubMed Scopus (78) Google Scholar). Cleavage of IP3R1 in Vitro and in Cells Undergoing Apoptosis—The first data demonstrating a possible involvement of IP3R1 in cell death were published a few years ago showing that cells deficient in IP3R1 were resistant to apoptosis (16Jayaraman T. Marks A.R. Mol. Cell. Biol. 1997; 17: 3005-3012Crossref PubMed Scopus (235) Google Scholar). This study, together with reports that caspase-3 mediates the cleavage of IP3R1 (18Hirota J. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; 274: 34433-34437Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 19Haug L.S. Walaas S.I. Ostvold A.C. J. Neurochem. 2000; 75: 1852-1861Crossref PubMed Scopus (29) Google Scholar), prompted us to examine the relevance of IP3R1 cleavage to the cell death processes and its effect on intracellular Ca2+ distribution. First, to examine whether the cleavage process could occur in vitro as well as in stable cell lines, crude microsomal preparations from WT-IP3R1 and IP3R1Δcasp cells were incubated with recombinant human caspase-3. In addition, we also studied IP3R1 degradation in cells treated with 50 nm STS to induce apoptosis. The extent of receptor degradation was then analyzed by Western blotting. As shown in Fig. 2A, recombinant caspase-3 cleaved IP3R1 in vitro in a dose-dependent manner generating exactly the same 95-kDa fragment as in cells undergoing STS-induced apoptosis. No such cleavage product could be detected in microsomal preparations from IP3R1Δcasp cells. Pretreatment of WT-IP3R1 cells with 100 μm z-VAD-fmk (a pan-caspase inhibitor) or z-DEVD-fmk (a specific inhibitor of caspase-3) completely inhibited the degradation of IP3R1 during STS-induced apoptosis (data not shown). These results confirm that caspase-3 was responsible for the cleavage of IP3R1 in cells undergoing apoptosis. The same 95-kDa fragment was also generated in (Δ1-225)IP3R1 cells induced to undergo apoptosis by either 50 nm STS or BCR cross-linking using 15 μg/ml anti-chicken IgM (Fig. 2B). Again, no cleavage product was detected in IP3R1Δcasp cells. These results suggest that the process of IP3R1 cleavage by caspase-3 is probably common to a wide range of apoptosis-inducing agents. Importantly, the results from the (Δ1-225)IP3R1 cells indicate that the initial caspase-3 activation during both STS- and anti-chicken IgM-induced apoptosis does not require IICR activity. Role of Caspase-3-mediated Cleavage of IP3R1 in the Process of Apoptotic Cell Death—Previous studies have shown that the degradation of IP3R1 by caspase-3 inhibits IICR activity in microsomal preparations from cerebellum (18Hirota J. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; 274: 34433-34437Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) and in digitonin-permeabilized A7r5 cells. 2Z. Assefa, G. Bultynck, K. Szlufcik, N. Nadif Kasri, E. Vermassen, J. Goris, L. Missiaen, G. Callewaert, J. B. Parys, and H. De Smedt, unpublished results. However, the point at which Ca2+ is involved in apoptosis and the possible contribution of the IP3R1" @default.
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