BI-1 Regulates Endoplasmic Reticulum Ca²⁺ Homeostasis Downstream of Bcl-2 Family Proteins^,

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-X_L, 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-X_L fails to reduce [Ca²⁺]_er, indicating that BI-1 functions downstream of Bcl-X_L. In bax^-/-bak^-/- double knock-out cells, both BI-1 and Bcl-X_L retained their ability to reduce [Ca²⁺]_er, suggesting that BI-1 and Bcl-X_L 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 (1).

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-X_L 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)4 (2-8). Conversely, overexpression of proapoptotic Bax protein or knocking down the level of Bcl-2 by antisense increases ([Ca²⁺]_er), and augments the amount of releasable ER Ca²⁺ (9, 10). The molecular mechanism by which Bcl-2/Bax family proteins modulate ER Ca²⁺ is unknown but could be due either to the intrinsic channel-forming properties of these proteins (11-13) or to interactions with ER Ca²⁺ channel proteins, such as SERCA (14-16) or IP3R1 (10, 17-20).

BI-1 (Bax inhibitor 1), also known as TEGT (21), was identified as a suppressor of Bax-induced cell death in a yeast-based screen for mammalian cytoprotective proteins (22). BI-1 is an evolutionarily conserved, multitransmembrane protein that resides in the ER in both animal and plant cells (22-25) and that displays cytoprotective activity when overexpressed in animal or plant cells (23, 26-31). Antisense-mediated knockdown of BI-1 expression increases sensitivity of animal and plant cells to certain stress-induced forms of cell death (22, 26). 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 (32, 33), whereas ablation of the gene encoding BI-1 in plants increases sensitivity to several biotic and abiotic stimulators of cell death (34). 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 (32, 35) and subsequently confirmed in plant cells (25). Moreover, BI-1 has been reported to associate with Bcl-2 and Bcl-X_L, based on co-immunoprecipitation experiments (22), 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.

EXPERIMENTAL PROCEDURES

Plasmids—The plasmids, pcDNA3 BI-1 HA, pcDNA3 myc-Bcl-X_L, pcDNA3 SERCA2b, ER-cameleon, and pMito-YC2, have been described (8, 32, 36-38). 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 (6). Immortalized bi-1^-/- and bi-1^+/+ MEFs (32) 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-X_L.

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 (8). To deduce [Ca²⁺]_er, R_min was obtained by treating the cells with 3 mm EGTA and 2 μm ionomycin, and R_max was determined by treating the cells with 25 μm digitonin, followed by 5-10 mm Ca²⁺, 1 mm ATP, and 1 mm Mg²⁺. The R_min and R_max values were then used to convert cell data to [Ca²⁺]_er as described previously (8).

Fura-2-based imaging of cytosolic Ca²⁺ was performed as described previously (8). 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, R_min was obtained by treating the cells with 8 μm ionomycin and 10 mm EGTA in Ca²⁺-deficient HBSS, and R_max 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 (39).

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 (40), anti-Bcl-2, anti-Bcl-X_L (41), 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.

RESULTS

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 (32). Westphalen et al. (35) 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) (8). 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 (8). 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-X_L on ER Ca²⁺ dynamics (8), 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 (8, 42, 43). 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 (8). Recognizing that accurate calibrations of the maximum YFP/CFP ratio (R_max) are difficult to obtain due to the toxic effects of permeabilizing cells in the presence of high extracellular Ca²⁺ (reviewed in Ref. 44), 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 (8, 35, 45). 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.

FIGURE 1.

BI-1 modulates basal ER [Ca²⁺]. A and B, BI-1-overexpressing cells have lower resting [Ca²⁺]_er compared with control cells. HeLa cells were co-transfected with either pcDNA3-BI-1-HA or control vector pcDNA3 and ER-cameleon-encoding plasmid at a 12:1 ratio. A, lysates were prepared from cells after imaging, normalized for total protein content, and analyzed using antibodies directed against various proteins as indicated. B, cameleon-expressing cells were imaged 2-3 days after transfection to determine the yellow/cyan ratio. Data for individual cells are represented by triangles. YFP/CFP ratios (mean ± S.E.) for BI-1 and control cells were 1.85 ± 0.02 and 2.0 ± 0.02, respectively (n = 24; p < 0.01 by Student's t test). Data are representative of three independent experiments. C and D, BI-1-deficient cells have increased resting [Ca²⁺]_er compared with wild-type cells. HeLa cells were transfected with plasmids encoding BI-1 shRNA or control vector, together with ER-cameleon-encoding plasmid. C, cells were lysed for RNA recovery, and relative levels of BI-1 and CPH-1 (control) mRNA were evaluated by RT-PCR. D, cells were imaged 2-3 days after transfection to determine yellow/cyan ratio. Data for individual cells are represented by triangles. YFP/CFP ratios (mean ± S.E.) for BI-1 knockdown and control cells were 2.16 ± 0.02 and 2.04 ± 0.02, respectively (n = 24; p < 0.01 by Student's t test). Data are representative of three independent experiments.

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, Bcl-2, Bcl-X_L, Grp94, calreticulin, and IP3Rs in HeLa cells (Fig. 2).

FIGURE 2.

BI-1 does not alter expression of ER proteins. Total protein was isolated from HeLa cells (from left to right: control shRNA, BI-1 shRNA, pcDNA3 vector, pcDNA3-BI-1-HA) homogenized in detergent-containing buffer, normalized for protein content (50 μg/sample), and analyzed by SDS-PAGE (8-16% gels) and immunoblotting using various antibodies as indicated. Protein levels were assessed by scanning densitometry, normalized using tubulin as internal control. N/A, not applicable.

The shRNA-mediated reduction in BI-1 expression rendered HeLa cells more sensitive to TG-induced cell killing (Fig. 3), as expected (32). Similar conclusions were reached whether measuring the percentage of annexin V-positive/propidium iodide-negative cells by fluorescence-activated cell sorting (Fig. 3), the percentage of dead cells failing to exclude propidium iodide dye (not shown), or the percentage of cells with apoptotic morphology based on microscopic examination of Hoechst dye-stained cells (not shown). Also, consistent with our prior report (29), BI-1-deficient MEF cells exhibited greater sensitivity to apoptosis induction by TG but not TRAIL (Fig. 3), showing selective vulnerability to ER stress-induced cell death.

FIGURE 3.

BI-1 protects cell from apoptosis under ER stress. HeLa and MEF cell lines were treated with 2 μm TG or 1.0 μg/ml TRAIL for 12 h to induce apoptosis. Cells were then collected and labeled using annexin V-fluorescein isothiocyanate and propidium iodide. 10⁴ cells from each condition were analyzed by fluorescence-activated cell sorting, and the percentage of annexin-positive/propidium iodide-negative cells was calculated. BI-1-deficient cells show a significantly higher percentage of apoptosis compared with the control cells under ER stress (TG) but not following challenge with TRAIL (p < 0.05 by unpaired t test). Data shown are the average ± S.D. of three independent assays. UT, untreated control; TG, thapsigargin.

BI-1 Overexpression Increases Ca²⁺ Leakage Rate of ER—The altered [Ca²⁺]_er seen in BI-1-overexpressing cells could be due to either decreased uptake of Ca²⁺ into ER or increased passive Ca²⁺ leakage from ER. Bcl-2/Bax family proteins have been shown to regulate the Ca²⁺ leakage rate (8, 35, 45). If a functional relation exists between BI-1 and Bcl-2/Bax family proteins with respect to [Ca²⁺]_er regulation, therefore, we predict that BI-1 also should increase the leakage rate.

To answer this question, we used TG to inhibit SERCA-mediated uptake of Ca²⁺ into the ER, comparing the rate of Ca²⁺ loss from this organelle in BI-1-overexpressing and control-transfected cells, based on cameleon fluorescence measurements (Fig. 4). Measurements were performed for individual cells in Ca²⁺-containing versus Ca²⁺-deficient medium, the latter intended to exclude the effects of capacitative Ca²⁺ entry, which refills ER Ca²⁺ from import of extracellular Ca²⁺ (2, 5). In both control and BI-1-overexpressing cells, TG induced loss of Ca²⁺ from ER. Although BI-1-overexpressing cells started with approximately half the [Ca²⁺]_er of control cells, TG induced emptying of ER Ca²⁺ to similar extents, resulting in similar final [Ca²⁺]_er in control and BI-1-overexpressing cells in both Ca²⁺-containing and Ca²⁺-deficient medium (Fig. 4 and supplemental Fig. S3).

FIGURE 4.

Effects of TG on [Ca²⁺]_er in BI-1 overexpressing cells. HeLa cells were transfected 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. After 2-3 days, cells were imaged in either Ca²⁺-containing (A and B) or Ca²⁺-free (C and D) HBSS and treated with 2 μm (A and B) or 4 μm (C and D) TG (t₀ = time of TG addition). Once [Ca²⁺]_er reached a plateau, 3 mm EGTA and 2 μm ionomycin was added to obtain the R_min for each individual cell (A and B). Data present yellow/cyan ratios (mean ± S.E.; n > 6 cells) versus time (A and C) or calculated mean [Ca²⁺]_er (B and D) versus time, based on calibration methods as described (8).

As a control, we also performed experiments in which SERCA was co-expressed with BI-1, reasoning that enhanced SERCA-mediated pumping of Ca²⁺ into the ER should overcome the effects of BI-1. Indeed, resting [Ca²⁺]_er levels in cells co-expressing SERCA and BI-1 were restored to the concentrations found in control-transfected cells (Fig. 4, A and B, and supplemental Fig. S3), excluding nonspecific effects of BI-1 overexpression on the cameleon reporter system.

Inhibiting SERCA with TG causes depletion of [Ca²⁺]_er due to passive leak of Ca²⁺ from ER (37). Thus, when cells are placed into Ca²⁺-deficient medium to exclude uptake from extracellular sources, the rate of Ca²⁺ leakage from the ER can be calculated from the first order derivative of the [Ca²⁺]_er change over time (d[Ca²⁺]/dt) following TG treatment. Accordingly, we compared the leakage rates of vector control and BI-1-overexpressing cells, using the ER-targeted cameleon reporter method as in Fig. 4, C and D. BI-1-overexpressing cells were thus determined to have a higher ER Ca²⁺ leakage rate compared with vector control cells (Fig. 5), suggesting that the lower resting [Ca²⁺]_er observed in BI-1-overexpressing cells is due at least in part to increased leakage of Ca²⁺ from ER, as reported recently for Bcl-2/Bax family proteins (2, 5, 10, 46). Although we cannot exclude the possibility that reduced import of Ca²⁺ into ER also plays a role in the reduced [Ca²⁺]_er of BI-1-overexpressing cells seen under steady-state conditions when cells are cultured in Ca²⁺-containing medium, these data indicate that BI-1 shares with Bcl-2/Bcl-X_L the ability to increase the passive leakage rate of Ca²⁺ from ER.

FIGURE 5.

BI-1 increases ER Ca²⁺ leakage rate. HeLa cells transfected with control (“vector”) (black circles) or BI-1-encoding (white circles) plasmids, together with ER-cameleon plasmid (12:1 ratio), were imaged in Ca²⁺-free HBSS. TG was added, the yellow/cyan ratio was determined for individual cells, and then data were converted to [Ca²⁺]_er by calibration. The leak rate (d[Ca²⁺]/dt) was plotted against [Ca²⁺]_er.

BI-1 reduces Ca²⁺ uptake by mitochondria. The reduced [Ca²⁺]_er of BI-1-overexpressing cells would be predicted to result in less Ca²⁺ efflux into the cytosol, and consequently less uptake into mitochondria, when cells are treated with TG. Indeed, in experiments in which Fura-2 was used for simultaneously measuring cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i), along with cameleon-based measurements of [Ca²⁺]_er, we observed that the TG-induced maximum [Ca²⁺]_i was typically ∼50% lower in BI-1-overexpressing compared with control cells (Fig. 6A). Note that the time course of TG-induced Ca²⁺ efflux into cytosol showed a quick “spike” (∼200 s) after TG stimulation, whereas it takes about ∼1200 s for the TG-induced Ca²⁺ leak from ER to reach steady-state, probably due to the efflux of cytosolic Ca²⁺ out of cells or its sequestration into non-ER storage pools, such as mitochondria.

FIGURE 6.

BI-1 reduces TG-induced entry of Ca²⁺ into cytosol and mitochondria. A, HeLa cells transfected with control (vector) (blue) or BI-1-encoding (red) plasmids, together with ER-cameleon plasmid (12:1 ratio), were loaded with Fura-2 and treated with 4 μm TG (at time = 0 s), and cameleon-expressing cells were imaged in Ca²⁺-free HBSS. The Fura-2 350/380 ratio was then converted to [Ca²⁺]_i after standard calibration (8). B, HeLa cells were transfected with pcDNA3 (Vector) or BI-1-encoding plasmids, in combination with mitochondria-targeted yellow cameleon-encoding plasmid (pMito-YC2) at a 6:1 ratio. Cameleon-expressing cells were imaged 3 days after transfection. The YFP/CFP ratio was monitored after treatment with 2 μm TG (t₀ = time of TG addition). Data represent mean (n > 8 cells imaged).

Next, we compared TG-induced increases in mitochondrial Ca²⁺ using an alternative cameleon protein (pMito-YC2) that was designed for measuring mitochondrial Ca²⁺ concentrations and that was targeted to the mitochondrial matrix using duplicated mitochondrial targeting sequence of subunit VIII of human COX (38) (supplemental Fig. S1). Ratiometric determinations demonstrated that BI-1 overexpression reduces the accumulation of Ca²⁺ in mitochondria (Fig. 6B). These findings thus independently corroborate prior measurements of mitochondrial Ca²⁺ obtained using mitochondria-targeted aequorin protein (35).

BI-1 Regulates [Ca²⁺]_er Downstream of Bcl-X_L—Since BI-1, Bcl-2, and Bcl-X_L all regulate basal [Ca²⁺]_er, and all increase ER Ca²⁺ leakage rate, we studied the functional relations between BI-1 and Bcl-X_L using genetically engineered cells. First, we examined the ability of overexpressed Bcl-X_L to regulate [Ca²⁺]_er in HeLa cells in which BI-1 was knocked down using shRNA. Using the ER-targeted, Ca²⁺-sensitive cameleon reporter, we observed that Bcl-X_L reduced [Ca²⁺]_er in control-transfected but not in BI-1-deficient HeLa cells (Fig. 7A). Second, we used mouse fibroblasts from bi-1^-/- versus bi-1^+/+ embryos (MEFs), transfecting Bcl-X_L into these cells along with the ER-targeted cameleon reporter. In wild-type MEFs, Bcl-X_L significantly reduced [Ca²⁺]_er (Fig. 7B, columns 1 and 2: YFP/CFP ratio Bcl-X_L = 2.00 ± 0.02 versus vector = 2.07 ± 0.02; n = 30; p < 0.01 by unpaired t test). In contrast, in BI-1-deficient MEFs, Bcl-X_L failed to reduce [Ca²⁺]_er and actually appeared to produce an increase rather than a decrease in [Ca²⁺]_er (Fig. 7B). Immunoblot analysis confirmed equivalent levels of Bcl-X_L protein production in transfected bi-1^-/- and bi-1^+/+ cells (data not shown).

FIGURE 7.

BI-1 is required for Bcl-X_L-mediated modulation of [Ca²⁺]_er. A, stable HeLa cell lines containing BI-1 shRNA or control shRNA were transiently transfected with plasmids encoding Bcl-X_L or a control plasmid, as indicated, together with ER-cameleon plasmid. After 2 days, cells were imaged to determine the YFP/CFP ratio. Ratios (mean ± S.E.) for columns 1-4 are 2.04 ± 0.02, 1.94 ± 0.02, 2.18 ± 0.02, and 2, 16 ± 0.02, respectively (n = 30). The difference between column 1 and 2 is significant (p < 0.01 by unpaired t test). B, bi-1^+/+ or bi-1^-/- MEFs were co-transfected with ER-cameleon plasmid and either pcDNA3 or Bcl-X_L-encoding plasmid. After 2 days, cells were imaged to determine the YFP/CFP ratio. Ratios (mean ± S.E.) for columns 1-4 are 2.07 ± 0.02, 2.00 ± 0.02, 2.04 ± 0.02, and 2.17 ± 0.02, respectively (n = 30). The difference between columns 1 and 2 is significant (p < 0.01 by unpaired t test).

BI-1 deficiency renders both HeLa and MEF cells more sensitive to ER stress-induced apoptosis (Fig. 3). Note that BI-1 deficiency in MEFs appears to have only a slight effect on resting [Ca²⁺]_er, as measured using the ER-targeted cameleon reporter (YFP/CFP ratio = 2.04 ± 0.02 for bi-1^-/- versus 2.07 + 0.02 for bi-1^+/+; n = 30), although prior cytosolic Ca²⁺ imaging studies detected reduced TG-releasable intracellular Ca²⁺ pools in these cells (32), perhaps reflecting a sensitivity difference in these methods or implying that BI-1 makes less of a contribution to regulating basal [Ca²⁺]_er in these cells compared with HeLa cells, at least under nonstressed conditions. Attempts to restore bi-1 expression in bi-1^-/- MEFs using either BI-1 protein-encoding plasmid or recombinant retrovirus were only modestly successful due to poor levels of expression (supplemental Fig. S4), making it difficult to contrast the effects of Bcl-X_L in control and reconstituted bi-1^-/- MEFs. Nevertheless, the observation that Bcl-X_L fails to reduce [Ca²⁺]_er in both HeLa and MEFs with BI-1 deficiency demonstrates that this Bcl-2 family member requires BI-1 for regulating [Ca²⁺]_er.

Bax and Bak Are Not Required for BI-1-mediated [Ca²⁺]_er Regulation—The effects of BI-1 and Bcl-X_L were examined in cells deficient in both Bax and Bak, since prior data have suggested that these proapoptotic proteins operate downstream of Bcl-2 and Bcl-X_L (10) and because simultaneous deficiency of Bax and Bak has been reported to lower [Ca²⁺]_er (6). Using ER-targeted cameleon, we confirmed that [Ca²⁺]_er is slightly lower in bax^-/-bak^-/- double knock-out cells compared with wild-type cells (average YFP/CFP ratio = 2.03 ± 0.01 for wild-type versus 1.96 ± 0.02 for double knock-out; p < 0.01 by unpaired t test), corroborating data obtained by alternative Ca²⁺ measurement methods (6). Despite Bax/Bak deficiency, both BI-1 and Bcl-X_L retained the ability to lower average [Ca²⁺]_er (Fig. 8), demonstrating that Bax and Bak are not required for the ER Ca²⁺-modulating activity of either BI-1 or Bcl-X_L and implying that these antiapoptotic proteins operate downstream of or parallel to Bax and Bak, at least with respect to [Ca²⁺]_er regulation.

FIGURE 8.

BI-1 and Bcl-X_L lower [Ca²⁺]_er in Bax/Bak double knock-out cells. bax^-/-bak^-/- double knock-out (DKO) cells co-transfected with ER-cameleon plasmid and pcDNA3 (vector) or plasmids encoding BI-1 or Bcl-X_L as indicated. Cells were imaged 2 days later to determine yellow/cyan emission ratio. YFP/CFP ratios (mean ± S.E., n = 40) were 1.97 ± 0.02, 1.82 ± 0.02, and 1.82 ± 0.02, respectively. Differences between vector control and BI-1 or Bcl-X_L were significant (p < 0.01 by unpaired t test).

DISCUSSION

Using an ER-targeted Ca²⁺-sensitive fluorescent protein (cameleon), we extended recent studies (32, 35) about the [Ca²⁺]_er-regulating activity of BI-1, demonstrating here that BI-1 is similar to antiapoptotic Bcl-2 family proteins in its ability to regulate [Ca²⁺]_er. In common with Bcl-2 and Bcl-X_L, BI-1 overexpression lowers resting [Ca²⁺]_er and increases the rate of passive Ca²⁺ leakage from ER. Conversely, as shown here, BI-1 deficiency causes higher resting [Ca²⁺]_er. One consequence of lower [Ca²⁺]_er caused by BI-1 overexpression is reduced entry of Ca²⁺ into the cytosol upon discharge from the ER and reduced accumulation of Ca²⁺ in mitochondria, compared with control cells. Since mitochondrial Ca²⁺ accumulation can precipitate mitochondrial permeability transition (MPT) (reviewed in Refs. 47 and 48), resulting in release of apoptogenic mitochondrial proteins, such as cytochrome c and consequent caspase activation, we speculate that the effects of BI-1 on Ca²⁺ dynamics could account at least in part for the previously reported ability of BI-1 to inhibit downstream activation of the mitochondrial pathway for cell death following exposure of cells to TG and other inducers of ER stress (32). It should be noted, however, that [Ca²⁺]_er affects many other events directly and indirectly, besides its role in regulating MPT, including the function of ER-resident chaperones (Grp78 and Grp94) that control signaling by several ER proteins (PERK, Ire1, and ATF6) (reviewed in Ref. 49). Indeed, tissues from bi-1^-/- mice show evidence of increased signaling via the Ire1 and ATF6 pathways (33), which results in changes in the activities of stress kinases and several transcription factors (reviewed in Ref. 49). Also, with respect to MPT, because Bcl-2-mediated apoptosis regulation has been dissociated from MPT based on experiments using cyclophilin D knock-outs (50, 51), it is also possible that BI-1 regulates cell death through MPT-independent mechanisms.

In this regard, though BI-1-mediated changes in [Ca²⁺]_er correlate with previously demonstrated alterations in resistance to certain types of cell death stimuli (32), it remains to be determined whether the ability of BI-1 to modulate [Ca²⁺]_er accounts for the cytoprotective activity of this protein. Bcl-2 family proteins also regulate [Ca²⁺]_er, correlating with their effects on cell death, but direct cause and effect data linking [Ca²⁺]_er regulation to cell death control are presently lacking. However, consistent with an important role for [Ca²⁺]_er, experimental manipulations that independently regulate [Ca²⁺]_er, such as overexpression of SERCA to increase [Ca²⁺]_er and expression of calreticulin to sequester luminal ER Ca²⁺ and thereby lower [Ca²⁺]_er, have been shown to alter cellular sensitivity to several types of cell death stimuli and to mimic or negate the actions of Bcl-2/Bax family proteins (4, 52-54). Still, the mechanisms connecting altered [Ca²⁺]_er to cell death responses are unclear and may be multifactorial.

By using cells genetically engineered to produced BI-1 deficiency, we determined that BI-1 is required for the [Ca²⁺]_er-lowering activity of Bcl-X_L, suggesting that BI-1 operates downstream of Bcl-X_L. Conversely, Bcl-X_L retained its ability to lower [Ca²⁺]_er in cells lacking both Bax and Bak, consistent with a recent report (10, 17-20). Taken together, these findings suggest a hierarchical pathway for regulation of [Ca²⁺]_er in which Bax and Bak operate upstream of or parallel to Bcl-X_L and in which Bcl-X_L is upstream of BI-1. The placing of BI-1 as a distal component of this proposed hierarchical pathway is consistent with data indicating a more ancient function for BI-1 as a protector against cellular stress in both plants and animals, compared with Bcl-2/Bax family proteins, which exist only in animal species. Since plants contain no recognizable homologs of Bcl-2/Bax family proteins based on sequence comparisons, it is possible that they possess functional analogs that regulate BI-1 without sharing overt sequence homology.

How BI-1 regulates [Ca²⁺]_er remains to be determined. The multiple membrane-spanning domains of the BI-1 protein are suggestive of intrinsic ion channel activity, but BI-1 may indirectly control [Ca²⁺]_er through interactions with other ER Ca²⁺ channel proteins, such as IP3Rs, as recently suggested for Bcl-2 family proteins (10). In yeast, however, the ability of BI-1 to protect against Bax-induced cell death was recently reported to be dependent on genes encoding homologs of SERCA (25), indicating a functional link between BI-1 and ER Ca²⁺ ATPases. If BI-1 does indirectly regulate the activity of ER Ca²⁺ channels, then we surmise that these channel proteins must be conserved in yeast, plants, and animals, because BI-1 overexpression has been shown to block cell death induced by ectopic expression of Bax in such diverse eukaryotic organisms. However, BI-1 may possess other functions besides [Ca²⁺]_er modulation that account for its cytoprotective activity.