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• W2014294890 abstract "BI-1 (Bax inhibitor-1) is an evolutionarily conserved multitransmembrane protein that resides in the endoplasmic reticulum (ER) and that has documented cytoprotective functions in both animals and plants. Recent studies indicate that BI-1 shares in common with Bcl-2/Bax family proteins the ability to regulate the amounts of Ca2+ that can be released from the ER by agents, such as the ER-Ca2+-ATPase (SERCA) inhibitor thapsigargin (TG). Using an ER-targeted, Ca2+ indicator (cameleon), with characteristics optimized for measuring ER Ca2+ ([Ca2+]er), we studied the effects of BI-1 on [Ca2+]er in resting and TG-treated cells. Similar to cells overexpressing antiapoptotic Bcl-2 or Bcl-XL, overexpression of BI-1 resulted in lower resting [Ca2+]er, with concomitantly less Ca2+ released into the cytosol upon stimulation by TG and with a higher rate of Ca2+ leakage from the ER. Co-expression of SERCA restored levels of [Ca2+]er to normal, showing opposing actions of the ER-Ca2+ATPase and BI-1 on ER Ca2+ homeostasis. Conversely, cells with deficient BI-1 have increased [Ca2+]er, and release more Ca2+ into the cytosol when challenged with TG. In BI-1-deficient cells, Bcl-XL fails to reduce [Ca2+]er, indicating that BI-1 functions downstream of Bcl-XL. In bax-/-bak-/- double knock-out cells, both BI-1 and Bcl-XL retained their ability to reduce [Ca2+]er, suggesting that BI-1 and Bcl-XL operate downstream of or parallel to Bax/Bak. The findings reveal a hierarchy of functional interactions of BI-1 with Bcl-2/Bax family proteins in regulating ER Ca2+ homeostasis. BI-1 (Bax inhibitor-1) is an evolutionarily conserved multitransmembrane protein that resides in the endoplasmic reticulum (ER) and that has documented cytoprotective functions in both animals and plants. Recent studies indicate that BI-1 shares in common with Bcl-2/Bax family proteins the ability to regulate the amounts of Ca2+ that can be released from the ER by agents, such as the ER-Ca2+-ATPase (SERCA) inhibitor thapsigargin (TG). Using an ER-targeted, Ca2+ indicator (cameleon), with characteristics optimized for measuring ER Ca2+ ([Ca2+]er), we studied the effects of BI-1 on [Ca2+]er in resting and TG-treated cells. Similar to cells overexpressing antiapoptotic Bcl-2 or Bcl-XL, overexpression of BI-1 resulted in lower resting [Ca2+]er, with concomitantly less Ca2+ released into the cytosol upon stimulation by TG and with a higher rate of Ca2+ leakage from the ER. Co-expression of SERCA restored levels of [Ca2+]er to normal, showing opposing actions of the ER-Ca2+ATPase and BI-1 on ER Ca2+ homeostasis. Conversely, cells with deficient BI-1 have increased [Ca2+]er, and release more Ca2+ into the cytosol when challenged with TG. In BI-1-deficient cells, Bcl-XL fails to reduce [Ca2+]er, indicating that BI-1 functions downstream of Bcl-XL. In bax-/-bak-/- double knock-out cells, both BI-1 and Bcl-XL retained their ability to reduce [Ca2+]er, suggesting that BI-1 and Bcl-XL operate downstream of or parallel to Bax/Bak. The findings reveal a hierarchy of functional interactions of BI-1 with Bcl-2/Bax family proteins in regulating ER Ca2+ homeostasis. Cell death is essential for development of most multicellular organisms, and its dysregulation contributes to many diseases characterized by inappropriate cell loss or cell accumulation. Bcl-2/Bax family proteins are central regulators of cell death in animals. Many Bcl-2/Bax family members reside in the membranes of mitochondria, where they play well defined roles in controlling release of apoptosis-inducing proteins from these organelles and regulating other aspects of mitochondrial function (1Reed J.C. Jürgensmeier J.M. Matsuyama S. Biochim. Biophys. Acta. 1998; 1366: 127-137Crossref PubMed Scopus (351) Google Scholar). Besides mitochondria, some members of the Bcl-2/Bax family also localize to membranes of the ER, where their functions are less clear at present. Overexpression of antiapoptotic Bcl-2 or Bcl-XL or ablation of proapoptotic Bax and Bak has been variably reported to result in lower basal concentrations of free Ca2+ in the ER ([Ca2+]er) and to reduce the amount of Ca2+ released from this organelle in response to SERCA inhibitors or agonists of inositol triphosphate-regulated Ca2+ channels (IP3Rs) 4The abbreviations used are: IP3R, inositol triphosphate-regulated Ca2+ channel; TG, thapsigargin; MEF, mouse embryo fibroblast; CFP, cyan-emitting fluorescent protein; YFP, yellow-emitting fluorescent protein; HBSS, Hanks' balanced salt solution; RT, reverse transcription; ER, endoplasmic reticulum; shRNA, short hairpin RNA; HA, hemagglutinin; [Ca2+]er, endoplasmic reticulum Ca2+; MPT, mitochondrial permeability transition. 4The abbreviations used are: IP3R, inositol triphosphate-regulated Ca2+ channel; TG, thapsigargin; MEF, mouse embryo fibroblast; CFP, cyan-emitting fluorescent protein; YFP, yellow-emitting fluorescent protein; HBSS, Hanks' balanced salt solution; RT, reverse transcription; ER, endoplasmic reticulum; shRNA, short hairpin RNA; HA, hemagglutinin; [Ca2+]er, endoplasmic reticulum Ca2+; MPT, mitochondrial permeability transition. (2Foyouzi-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 (383) Google Scholar, 3Lam M. Dubyak G. Chen L. Nuñez G. Miesfeld R.L. Distelhorst C.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6569-6573Crossref PubMed Scopus (611) Google Scholar, 4Pinton P. Ferrari D. Rapizzi E. Di Virgilio F. Pozzan T. Rizzuto R. EMBO J. 2001; 20: 2690-2701Crossref PubMed Scopus (500) Google Scholar, 5Pinton P. Ferrari D. Magalhaes P. Schulze-Osthoff K. Di Virgilio F. Pozzan T. Rizzuto R. J. Cell Biol. 2000; 148: 857-862Crossref PubMed Scopus (408) Google Scholar, 6Scorrano L. Oakes S.A. Opferman J.T. Cheng E.H. Sorcinelli M.D. Pozzan T. Korsmeyer S.J. Science. 2003; 300: 135-139Crossref PubMed Scopus (1230) Google Scholar, 7Zong W.X. Li C. Hatzivassiliou G. Lindsten T. Yu Q.C. Yuan J. Thompson C.B. J. Cell Biol. 2003; 162: 59-69Crossref PubMed Scopus (506) Google Scholar, 8Palmer A.E. 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A. 2005; 102: 105-110Crossref PubMed Scopus (387) Google Scholar, 17Jones R.G. Bui T. White C. Madesh M. Krawczyk C.M. Lindsten T. Hawkins B.J. Kubek S. Frauwirth K.A. Wang Y.L. Conway S.J. Roderick H.L. Bootman M.D. Shen H. Foskett J.K. Thompson C.B. Immunity. 2007; 27: 268-280Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 18Li C. Wang X. Vais H. Thompson C.B. Foskett J.K. White C. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 12565-12570Crossref PubMed Scopus (134) Google Scholar, 19White C. Li C. Yang J. Petrenko N.B. Madesh M. Thompson C.B. Foskett J.K. Nat. Cell Biol. 2005; 7: 1021-1028Crossref PubMed Scopus (357) Google Scholar, 20Chen R. Valencia I. Zhong F. McColl K.S. Roderick H.L. Bootman M.D. Berridge M.J. Conway S.J. Holmes A.B. Mignery G.A. Velez P. Distelhorst C.W. J. Cell Biol. 2004; 166: 193-203Crossref PubMed Scopus (347) Google Scholar). BI-1 (Bax inhibitor 1), also known as TEGT (21Walter L. Dirks B. Rothermel E. Heyens M. Szpirer C. Levan G. Gunther E. Mamm. Genome. 1994; 5: 216-221Crossref PubMed Scopus (29) Google Scholar), was identified as a suppressor of Bax-induced cell death in a yeast-based screen for mammalian cytoprotective proteins (22Xu Q. Reed J.C. Mol. Cell. 1998; 1: 337-346Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). BI-1 is an evolutionarily conserved, multitransmembrane protein that resides in the ER in both animal and plant cells (22Xu Q. Reed J.C. Mol. Cell. 1998; 1: 337-346Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 23Bolduc N. Ouellet M. Pitre F. Brisson L.F. Planta. 2003; 216: 377-386Crossref PubMed Scopus (70) Google Scholar, 24Kawai-Yamada M. Jin L. Yoshinaga K. Hirata A. Uchimiya H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12295-12300Crossref PubMed Scopus (197) Google Scholar, 25Ihara-Ohori Y. Nagano M. Muto S. Uchimiya H. Kawai-Yamada M. Plant Physiol. 2007; 143: 650-660Crossref PubMed Scopus (90) Google Scholar) and that displays cytoprotective activity when overexpressed in animal or plant cells (23Bolduc N. Ouellet M. Pitre F. Brisson L.F. Planta. 2003; 216: 377-386Crossref PubMed Scopus (70) Google Scholar, 26Bolduc N. Brisson L.F. FEBS Lett. 2002; 532: 111-114Crossref PubMed Scopus (43) Google Scholar, 27Kawai M. Pan L. Reed J.C. Uchimiya H. FEBS Lett. 1999; 464: 143-147Crossref PubMed Scopus (131) Google Scholar, 28Kawai-Yamada M. Ohori Y. Uchimiya H. Plant Cell. 2004; 16: 21-32Crossref PubMed Scopus (207) Google Scholar, 29Matsumura H. Nirasawa S. Kiba A. Urasaki N. Saitoh H. Ito M. Kawai-Yamada M. Uchimiya H. Terauchi R. Plant J. 2003; 33: 425-434Crossref PubMed Scopus (155) Google Scholar, 30Sanchez P. de Torres M. Grant M. Plant J. 2000; 21: 393-399Crossref PubMed Google Scholar, 31Chae H.-J. Ke N. Chen S. Kim H.-R. Godzik A. Dickman M. Reed J.C. Gene (Amst.). 2003; 323: 101-113Crossref PubMed Scopus (160) Google Scholar). Antisense-mediated knockdown of BI-1 expression increases sensitivity of animal and plant cells to certain stress-induced forms of cell death (22Xu Q. Reed J.C. Mol. Cell. 1998; 1: 337-346Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 26Bolduc N. Brisson L.F. FEBS Lett. 2002; 532: 111-114Crossref PubMed Scopus (43) Google Scholar). Targeted ablation of the bi-1 gene in mice results in increased sensitivity to cell death induced by ischemia-reperfusion injury and pharmacological inducers of ER stress (32Chae H.J. Kim H.R. Bailly-Maitre B. Zhu X. Ke N. Krajewska M. Krajewski S. Cui J. Digicaylioglu M. Thomas M. Kress C.L. Babendure J. Tsien R.Y. Lipton S.A. Reed J.C. Mol. Cell. 2004; 15: 355-366Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 33Bailly-Maitre B. Fondevila C. Kaldas F. Droin N. Luciano F. Ricci J.E. Croxton R. Krajewska M. Zapata J.M. Kupiec-Weglinski J.W. Farmer D. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2809-2814Crossref PubMed Scopus (145) Google Scholar), whereas ablation of the gene encoding BI-1 in plants increases sensitivity to several biotic and abiotic stimulators of cell death (34Watanabe N. Lam E. Plant J. 2006; 45: 884-894Crossref PubMed Scopus (160) Google Scholar). Similar to Bcl-2/Bax family proteins, a role for BI-1 in regulating the amounts of Ca2+ released from ER by SERCA inhibitors, such as TG, was recently demonstrated using BI-1-overexpressing and bi-1-/- cells (32Chae H.J. Kim H.R. Bailly-Maitre B. Zhu X. Ke N. Krajewska M. Krajewski S. Cui J. Digicaylioglu M. Thomas M. Kress C.L. Babendure J. Tsien R.Y. Lipton S.A. Reed J.C. Mol. Cell. 2004; 15: 355-366Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 35Westphalen B.C. Wessig J. Leypoldt F. Arnold S. Methner A. Cell Death Differ. 2005; 12: 304-306Crossref PubMed Scopus (46) Google Scholar) and subsequently confirmed in plant cells (25Ihara-Ohori Y. Nagano M. Muto S. Uchimiya H. Kawai-Yamada M. Plant Physiol. 2007; 143: 650-660Crossref PubMed Scopus (90) Google Scholar). Moreover, BI-1 has been reported to associate with Bcl-2 and Bcl-XL, based on co-immunoprecipitation experiments (22Xu Q. Reed J.C. Mol. Cell. 1998; 1: 337-346Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar), suggesting the possibility of collaborations among these proteins. Using genetically engineered cell lines, we show that BI-1 functions downstream of Bcl-2 family proteins in a hierarchical pathway regulating ER Ca2+ homeostasis. Plasmids—The plasmids, pcDNA3 BI-1 HA, pcDNA3 myc-Bcl-XL, pcDNA3 SERCA2b, ER-cameleon, and pMito-YC2, have been described (8Palmer A.E. Jin C. Reed J.C. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17404-17409Crossref PubMed Scopus (521) Google Scholar, 32Chae H.J. Kim H.R. Bailly-Maitre B. Zhu X. Ke N. Krajewska M. Krajewski S. Cui J. Digicaylioglu M. Thomas M. Kress C.L. Babendure J. Tsien R.Y. Lipton S.A. Reed J.C. Mol. Cell. 2004; 15: 355-366Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 36Zhang H. Xu Q. Krajewski S. Krajewska M. Xie Z. Fuess S. Kitada S. Pawloski K. Godzik A. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2597-2602Crossref PubMed Scopus (161) Google Scholar, 37Lytton J. Westlin M. Hanley M.R. J. Biol. Chem. 1991; 266: 17067-17071Abstract Full Text PDF PubMed Google Scholar, 38Filippin L. Abad M.C. Gastaldello S. Magalhaes P.J. Sandona D. Pozzan T. Cell Calcium. 2005; 37: 129-136Crossref PubMed Scopus (94) Google Scholar). To generate retroviral expression construct pCLXSN/BI-1-HA, pcDNA3-BI-1-HA was digested with XhoI to release the BI-1-HA fragment, which was then filled in with Klenow, digested with EcoRI, and inserted into the pCLXSN vector (Imgenex, 10041P), using standard molecular cloning methods. Cell Culture—Early passage HeLa cells (P5-P15) were obtained from ATCC (Manassas, VA) and cultured in Dulbecco's modified Eagle's medium low glucose (1 g/liter; Invitrogen) supplemented with 10% fetal bovine serum, 1 mm l-glutamine, and antibiotics. HeLa cells were transfected by Fugene 6 (Roche Applied Science). Mouse embryo fibroblast (MEF) cells were cultured in Dulbecco's modified Eagle's medium high glucose (4.5 g/liter) (Irvine Scientific) supplemented with 10% fetal bovine serum, 1 mm l-glutamine, and antibiotics. Immortalized bax-/-bak-/- double knock-out MEFs were provided by S. J. Korsmeyer (6Scorrano L. Oakes S.A. Opferman J.T. Cheng E.H. Sorcinelli M.D. Pozzan T. Korsmeyer S.J. Science. 2003; 300: 135-139Crossref PubMed Scopus (1230) Google Scholar). Immortalized bi-1-/- and bi-1+/+ MEFs (32Chae H.J. Kim H.R. Bailly-Maitre B. Zhu X. Ke N. Krajewska M. Krajewski S. Cui J. Digicaylioglu M. Thomas M. Kress C.L. Babendure J. Tsien R.Y. Lipton S.A. Reed J.C. Mol. Cell. 2004; 15: 355-366Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar) were generated by infecting cells with SV40 T antigen-producing retrovirus in the presence of 8 μg/ml Polybrene for 3 h and then diluting the Polybrene to 2 μg/ml and culturing for an additional 3 days. The infected cells were then split at 1:20 and selected for single colonies in medium containing 1 mg/ml G418 for 10 days. The MEF cells were then transfected by Fugene 6 (Roche Applied Science) or infected with protein-encoding retroviruses (see below) for imaging studies. Stable HeLa cell lines with BI-1 knockdown (by short hairpin RNA (shRNA)) were generated using retrovirus infection. 293T cells were first transfected (using Lipofectamine 2000) with 2 μg of VSVG, 4 μg of cytomegalovirus-gag polymerase, and 5 μg of either BI-1 shRNA (Open Biosystems, V2SH_153887) or a control shRNA to produce viruses that can express the corresponding shRNA. Virus-containing medium was collected, filtered, and used to infect early passage HeLa cells. The infected cells were then diluted and selected for single colonies in medium containing 1 μg/ml puromycin for 15 days. BI-1 knockdown was confirmed by reverse transcription (RT)-PCR. Retroviruses were generated by the same method as for infecting MEFs. Ca2+ Imaging—HeLa cells (4 × 105) were plated on 35-mm glass-bottom dishes (MatTek, Ashland, MA) and transfected the next day using Fugene 6 (Roche Applied Science) with 0.08 μg of ER-cameleon-encoding plasmid and either 1 μg of pcDNA3 control (“neo”), 1 μg of pcDNA3-BI-1-HA, or 0.5 μg of pcDNA3-BI-1-HA and 0.5 μg of pcDNA3-SERCA2b. To obtain mitochondrial Ca2+, HeLa cells were transfected with 0.3 μg of Mito-YC2-encoding plasmid and 1.7 μg of pcDNA3 control (“vector”) or 1.7 μg of pcDNA3-BI-1-HA. MEF cells (8 × 104 to 1.6 × 105) were plated and transfected with 0.4 μg of ER-cameleon-encoding plasmid and 1.6 μg of pcDNA3 control (“vector”), 1.6 μg of pcDNA3-BI-1-HA or pcDNA3-myc-Bcl-XL. Cells were imaged 2-3 days after transfection using either a Zeiss Axiovert microscope with a cooled CCD camera, controlled by MetaFluor version 6.1 software (Universal Imaging, Downington, PA), or an inverted Olympus IX81 fluorescence microscope equipped with a thermo-controlled stage (Warner Instruments Inc., Hamden, CT) with oil immersion objective UAPO ×40/340, numerical aperture 1.35-0.65, fitted with a cooled Cascade 512B camera (Photometrics, Inc., Tucson, AZ) and employing MetaFluor software, version 7.1.4 (Molecular Devices, Downingtown, PA). The cells were imaged either in Ca2+-containing or Ca2+-free Hanks' balanced salt solution (HBSS) (Invitrogen), containing 20 mm HEPES (pH 7.4), 1-2 g/liter d-glucose (osmolarity ∼300). Dual emission ratio imaging of the cameleons was accomplished with the Zeiss system using a 436DF20 excitation filter, a 450DRLP dichroic mirror, and two emission filters (475DF30 for ECFP, 535DF25 for EYFP) controlled by a Lambda 10-2 filter wheel (Sutter Instrument Co., Novato, CA). With the Olympus system, we used ultra-high speed wavelength switcher DG4 (Sutter Instrument Co.) with the D436/20× excitation filter, with 455 long pass dichroic mirror, and D480/40m and D535/30m emission filters for cyan-emitting fluorescent protein (CFP) and yellow-emitting fluorescent protein (YFP), respectively, installed in a Sutter Lambda 10B filter wheel. All filters were obtained from Chroma Technology Co. (Rockingham, VT). For cameleon imaging, the yellow and cyan intensities were measured 5-7 times every 15 s, and the average ratios were calculated after background subtraction for each individual cell. Mito-YC2 cells were imaged using the same parameters as the ER-cameleon cells. The ER-cameleon calibration was performed as described (8Palmer A.E. Jin C. Reed J.C. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17404-17409Crossref PubMed Scopus (521) Google Scholar). To deduce [Ca2+]er, Rmin was obtained by treating the cells with 3 mm EGTA and 2 μm ionomycin, and Rmax was determined by treating the cells with 25 μm digitonin, followed by 5-10 mm Ca2+, 1 mm ATP, and 1 mm Mg2+. The Rmin and Rmax values were then used to convert cell data to [Ca2+]er as described previously (8Palmer A.E. Jin C. Reed J.C. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17404-17409Crossref PubMed Scopus (521) Google Scholar). Fura-2-based imaging of cytosolic Ca2+ was performed as described previously (8Palmer A.E. Jin C. Reed J.C. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17404-17409Crossref PubMed Scopus (521) Google Scholar). In brief, the cells were incubated in HBSS containing 4 μm Fura-2/AM (Molecular Probes, Inc., Eugene, OR) with 0.04% Pluronic F-127 for 30 min at room temperature in the dark. Cells were then washed with HBSS and incubated for 15 min before changing to fresh HBSS for imaging. Excitation ratio imaging for Fura-2 was accomplished by using 350- and 380-nm excitation filters (both 10-nm bandwidths), and (for the Zeiss system) a 450-nm dichroic mirror with a 535/45 emission filter or (for the Olympus system) a 400 long pass dichroic mirror with a D340X filter. To calibrate Fura-2, Rmin was obtained by treating the cells with 8 μm ionomycin and 10 mm EGTA in Ca2+-deficient HBSS, and Rmax was obtained by treating the cells with 2 μm ionomycin and 20 mm Ca2+. The 350/380 nm ratio was then converted to [Ca2+]i, as described (39Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Immunoblotting—Total protein was isolated from HeLa cells homogenized in detergent-containing buffer, normalized for protein content (50 μg/sample), and analyzed by SDS-PAGE (8-16% gels) and immunoblotting using the following primary antibodies: anti-HA (3F10; Roche Applied Science), anti-Bax (40Krajewski S. Krajewska M. Shabaik A. Miyashita T. Wang H.-G. Reed J.C. Am. J. Pathol. 1994; 145: 1323-1333PubMed Google Scholar), anti-Bcl-2, anti-Bcl-XL (41Krajewski S. Krajewska M. Shabaik A. Wang H.-G. Irie S. Fong L. Reed J.C. Cancer Res. 1994; 54: 5501-5507PubMed Google Scholar), anti-Grp94 (SPA-851; Stressgen Biotechnologies), anti-tubulin (catalog number 317; Santa Cruz Biotechnology), anti-calreticulin (GTX20004; GeneTex), anti-SERCA2 (GTX30342; GeneTex), and anti-IP3R1, -2, and -3 (sc-6093/7277/7278; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Antibody detection was accomplished by using horseradish peroxidase-conjugated secondary antibodies (anti-mouse IgG or anti-rabbit IgG; Amersham Biosciences) and an enhanced chemiluminescence method (Amersham Biosciences). RT-PCR—Total RNAs were isolated from cells using the RNeasy kit (74104; Qiagen), reverse-transcribed using the Superscript RT-PCR kit (10928-034; Invitrogen), following the manufacturer's instructions. cDNAs were then used as template for PCR amplification using a BI-1-specific primer pair as well as a CPH-specific primer pair as control. Cell Death Assay—HeLa and MEF cell lines were treated with 2 μm TG or 1.0 μg/ml TRAIL in Dulbecco's modified Eagle's medium low glucose (1 g/liter; Invitrogen) supplemented with 10% fetal bovine serum, 1 mm l-glutamine, and antibiotics for 12 h to induce apoptosis. Both floating and attached cells were then collected and labeled using the annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit (catalog number K101-100; BioVision), following a protocol provided by the manufacturer. 104 cells from each condition were analyzed by fluorescence-activated cell sorting to identify cells into four groups: annexin-negative/PI-negative, annexin-negative/PI-positive, annexin-positive/PI-negative, and annexin-positive/PI-positive. Aliquots of the collected cells were also suspended at 106 cells/ml in PBS with 2 μg/ml Hoechst dye, and the percentage of cells with apoptotic nuclear morphology was determined by UV microscopy. BI-1 Alters Steady-state ER Ca2+ Levels—Our previous investigations showed that mammalian BI-1 regulates a cell death pathway linked to ER stress and modulates the levels of releasable Ca2+ from ER, as measured by a Ca2+-sensitive ratiometric dye (Fura-2) that reports cytosolic free Ca2+ concentrations, in experiments where cells were treated with SERCA inhibitor, TG, to release Ca2+ into cytosol (32Chae H.J. Kim H.R. Bailly-Maitre B. Zhu X. Ke N. Krajewska M. Krajewski S. Cui J. Digicaylioglu M. Thomas M. Kress C.L. Babendure J. Tsien R.Y. Lipton S.A. Reed J.C. Mol. Cell. 2004; 15: 355-366Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Westphalen et al. (35Westphalen B.C. Wessig J. Leypoldt F. Arnold S. Methner A. Cell Death Differ. 2005; 12: 304-306Crossref PubMed Scopus (46) Google Scholar) also measured ER [Ca2+] in BI-1-overexpressing cells using first generation ER-targeted Ca2+-sensitive fluorescent proteins that report [Ca2+] via fluorescence resonance energy transfer. To lay a foundation for our studies on the relation of BI-1 to ER [Ca2+], we compared the effects of BI-1 overexpression and BI-1 knockdown, achieved with shRNAs on steady-state Ca2+ concentrations in the ER ([Ca2+]er). To directly measure [Ca2+]er, we utilized an improved ER-targeted, Ca2+-sensitive fluorescent protein, termed D1ER-cameleon, that consists of tandem fusions of CFP and YFP, separated by Ca2+-responsive elements (mutant calmodulin and a complementary mutant version of the calmodulin-binding peptide M13) (8Palmer A.E. Jin C. Reed J.C. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17404-17409Crossref PubMed Scopus (521) Google Scholar). Binding of Ca2+ to the calmodulin moiety induces binding to M13, bringing CFP and YFP into a new orientation that enhances fluorescence resonance energy transfer. The characteristics of the ER-targeted cameleon used here have been reported in detail (8Palmer A.E. Jin C. Reed J.C. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17404-17409Crossref PubMed Scopus (521) Google Scholar). The cameleon was targeted to ER by the addition of an N-terminal calreticulin signal sequence and C-terminal ER retention sequence (KDEL), and its localization to ER was confirmed by microscopy (supplemental Fig. S1). This improved ER-targeted cameleon has been used to study the effects of Bcl-2 and Bcl-XL on ER Ca2+ dynamics (8Palmer A.E. Jin C. Reed J.C. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17404-17409Crossref PubMed Scopus (521) Google Scholar), confirming and extending prior studies. We first tested the effects of BI-1 overexpression on [Ca2+]er using a gene transfer approach, where HeLa cells were co-transfected with plasmids encoding ER-targeted cameleon and either control vector or BI-1-encoding plasmid, measuring fluorescence resonance energy transfer and [Ca2+]er in individual cells by previously established ratiometric methods that normalize for differences in levels of protein production (8Palmer A.E. Jin C. Reed J.C. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17404-17409Crossref PubMed Scopus (521) Google Scholar, 42Miyawaki A. Griesbeck O. Heim R. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2135-2140Crossref PubMed Scopus (727) Google Scholar, 43Miyawaki A. Llopis J. Heim R. McCaffery J.M. Adams J.A. Ikura M. Tsien R.Y. Nature. 1997; 388: 882-887Crossref PubMed Scopus (2614) Google Scholar). Immunoblotting confirmed expression of the epitope-tagged BI-1 protein in cells transfected with BI-1-HA but not in control-transfected cells (Fig. 1A). Although individual cells varied, on average, BI-1-overexpressing cells contained significantly lower resting [Ca2+]er, as represented by the YFP/CFP ratio (Fig. 1B) (BI-1-HA = 1.85 ± 0.02, n = 24; vector = 2.05 ± 0.02, n = 24, p < 0.01 by Student's t test). For some experiments, we also converted the YFP/CFP ratio to an estimated [Ca2+]er by a calibration method, as described previously (8Palmer A.E. Jin C. Reed J.C. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17404-17409Crossref PubMed Scopus (521) Google Scholar). Recognizing that accurate calibrations of the maximum YFP/CFP ratio (Rmax) are difficult to obtain due to the toxic effects of permeabilizing cells in the presence of high extracellular Ca2+ (reviewed in Ref. 44Palmer A.E. Tsien R.Y. Nat. Protoc. 2006; 1: 1057-1065Crossref PubMed Scopus (360) Google Scholar), our values of resting ER [Ca2+] in control HeLa cells nevertheless agree well with prior reports (supplemental Fig. S2), based on methods using ER-targeted green fluorescent protein-based cameleons or aequorin (8Palmer A.E. Jin C. Reed J.C. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17404-17409Crossref PubMed Scopus (521) Google Scholar, 35Westphalen B.C. Wessig J. Leypoldt F. Arnold S. Methner A. Cell Death Differ. 2005; 12: 304-306Crossref PubMed Scopus (46) Google Scholar, 45Bassik M.C. Scorrano L. Oakes S.A. Pozzan T. Korsmeyer S.J. EMBO J. 2004; 23: 1207-1216Crossref PubMed Scopus (242) Google Scholar). Also, it bears noting that whereas the absolute YFP/CFP ratio values varied, depending on which Ca2+ imaging system (microscope) was used, the relative differences in ER-targeted cameleon fluorescence seen for these genetically engineered cells were reproducible. Next, we knocked down BI-1 expression in HeLa cells using shRNA targeting BI-1 mRNA and confirmed reduced BI-1 mRNA by reverse transcription-PCR (Fig. 1C). (Note that antibodies that detect endogenous BI-1 have not been attainable to date, despite considerable effort, thus precluding analysis of endogenous BI-1 protein.) We used three different shRNA sequences to target BI-1, all three of which were comparably effective (not shown). Comparison of resting [Ca2+]er in control- and shRNA-transfected HeLa cells revealed that average resting [Ca2+]er was significantly higher in BI-1-deficient cells, with BI-1 knockdown cells having an average YFP/CFP ratio of 2.16 ± 0.02 (n = 24) compared with control-transfected cells with an average YFP/CFP ratio of 2.04 ± 0.02 (n = 24) (p < 0.01 by unpaired t test) (Fig. 1D). Additional immunoblotting studies demonstrated that BI-1 overexpression and BI-1 knockdown do not alter the levels of proteins known to modulate [Ca2+]er, including SERCA2, Bax," @default.
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• W2014294890 title "BI-1 Regulates Endoplasmic Reticulum Ca2+ Homeostasis Downstream of Bcl-2 Family Proteins" @default.
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