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Address correspondence to Clark W. Distelhorst, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4937. Tel.: (216) 368-4546. Fax: (216) 368-1166. email: cwd{at}case.edu
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Abstract |
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Key Words: InsP3 receptor; calcium signaling; apoptosis; calcium channel; T cell receptor
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Introduction |
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Bcl-2 is the founding member of a large family of proteins that regulate apoptosis from critical control points on both mitochondria and the ER (Cory and Adams, 2002; Danial and Korsmeyer, 2004). Bcl-2 is an integral membrane protein that localizes to both the ER and mitochondria (Kaufmann et al., 2003). Although a significant proportion of Bcl-2 is on the ER (Kaufmann et al., 2003), its role in inhibiting cytochrome c release from mitochondria has been emphasized (Green and Reed, 1998; Gross et al., 1999). Recent findings indicate that Bcl-2 localized specifically on the ER also inhibits cytochrome c release and apoptosis in response to a variety of signals (Zhu et al., 1996; Hacki et al., 2000; Wang et al., 2001; Thomenius et al., 2003).
Previous studies have shown that changes in cellular calcium signaling can dramatically modulate the induction of apoptosis (for review see Hajnoczky et al., 2003; Orrenius et al., 2003). It has been proposed that the action of Bcl-2 is mediated in part due to its ability to regulate cytosolic and mitochondrial calcium changes. For example, it was demonstrated a decade ago that Bcl-2 dampens calcium oscillations and prevents redistribution of calcium from ER to mitochondria after growth factor withdrawal (Baffy et al., 1993; Magnelli et al., 1994). However, the absolute effect of Bcl-2 is unclear, with changes in cytosolic, ER, and mitochondrial calcium signaling being reported in different studies (Lam et al., 1994; Zornig et al., 1995; Marin et al., 1996; Ichimiya et al., 1998; Zhu et al., 1999; Williams et al., 2000; Pinton et al., 2001). Furthermore, the mechanism by which Bcl-2 interacts with calcium signaling systems is controversial. This is exemplified by the conflicting reports about Bcl-2's effect on ER luminal calcium. We, and others, have provided evidence that Bcl-2 preserves luminal calcium, whereas several recent papers suggest that Bcl-2 increases leakage of calcium from the ER and decreases luminal calcium (for review see Distelhorst and Shore, 2004).
The present study was undertaken to test the hypothesis that Bcl-2 on the ER regulates InsP3-linked calcium signals that mediate cell cycle entry and apoptosis. We report that Bcl-2 inhibits InsP3-induced calcium release from the ER by impeding calcium release through InsP3Rs. This action of Bcl-2 was not due to an alteration in InsP3R expression or luminal calcium concentration, but was mediated through a functional interaction of Bcl-2 with InsP3Rs that inhibited their channel opening in response to InsP3.
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Results |
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Luminal calcium concentration was measured directly using the low affinity calcium-sensitive dye Fura-2FF AM. Optimal conditions for loading Fura-2FF AM into the ER were determined in preliminary experiments. The organelle distribution of Fura-2FF fluorescence was in a reticular pattern distinct from the punctate mitochondrial pattern detected with the potentiometric dye tetramethylrhodamine ethyl ester (TMRE; Fig. 4 A). To quantify the relative amount of Fura-2FF localized to the ER lumen, the plasma membrane was permeabilized with digitonin and cells were perfused with intracellular buffer (ICB) supplemented with an ATP-regenerating system, 10 µM InsP3, and 100 µM MnCl2 (Fig. 4 B). The initial decrease in the emission intensity at both 340 and 380 nm excitation (at 80 s) signifies the point at which the plasma membrane is permeabilized. The subsequent decrease in 340 and 380 nm emission (at
190 s) is due to InsP3-induced opening of InsP3Rs on the ER, allowing MnCl2 to enter the ER lumen. Fura-2FF has high affinity for MnCl2, which in turn quenches the dye. In multiple experiments, more than 90% of the fluorescence remaining after digitonin permeabilization was quenched by perfusing cells with 10 µM InsP3 and 100 µM MnCl2. Using this assay system, ER luminal calcium concentration was compared in Bcl-2negative and positive clones. A typical continuous single cell tracing is shown in Fig. 4 C. Cells were initially perfused with extracellular buffer (ECB) and then with ICB supplemented with an ATP regenerating system and digitonin. The 340:380 fluorescence emission ratio increased dramatically when cells were permeabilized, reflecting a typically high ER luminal calcium concentration. The calcium concentration was calculated from the steady-state ratio in numerous experiments (Fig. 4 D). Two sets of experiments were performed. The first consisted of a direct comparison of N1 versus B27 cells, and the second set consisted of a direct comparison of N2 versus B17 cells. Based on these findings, there was no detectable difference between Bcl-2positive and negative cells in terms of free calcium concentration within the ER lumen. In these experiments, luminal calcium was allowed to come to steady-state, provided with ATP by an ATP-regenerating system. In a different experimental approach, cells were perfused with 10 µg/ml digitonin in ECB and luminal calcium concentration was based on the 340:380 ratio at the time point where cells were initially permeabilized. This alternative experimental approach confirmed no difference in ER luminal calcium concentration between Bcl-2positive and negative clones (unpublished data). These findings indicate that the inhibitory effect of Bcl-2 on anti-CD3induced calcium elevation was not due to a Bcl-2imposed decrease in the mobilizable ER calcium pool.
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Bcl-2 targets the InsP3R to inhibit calcium release
To exclude the first possibility, the signal transduction pathway mediating the response to anti-CD3 antibody was bypassed by measuring cytoplasmic calcium elevation after addition of a cell-permeant InsP3 ester, D-myo InsP3 hexakisbutyryloxymethyl ester (D-myo InsP3BM), to intact Bcl-2positive and negative cells. After a brief delay required for de-esterification, D-myo InsP3BM induced an elevation in cytosolic calcium that had a shorter latency period, a more rapid rate of increase, and a higher peak amplitude in Bcl-2negative cells compared with Bcl-2positive cells (Fig. 5). These findings indicated that Bcl-2 acts at the level of the ER to inhibit anti-CD3induced calcium elevation, rather than interfering with upstream components of the signal transduction pathway initiated by TCR activation.
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Discussion |
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Although an inhibitory action of Bcl-2 on InsP3-mediated calcium release has not been reported previously, an inhibitory effect of Bcl-2 on calcium-mediated signaling pathways has been reported, including the induction of the transcription factor c-fos (Qi et al., 1997). Significantly, Linette et al. (1996) demonstrated that Bcl-2 inhibits anti-CD3/TCRmediated activation of NFATc and induction of interleukin-2 (IL-2) expression, thereby inhibiting cell cycle entry by delaying Go/G1 transition into S phase and also inhibiting TCR activation-mediated apoptosis. Active NFATc is generated by calcineurin, which binds to and dephosphorylates NFATc in the cytoplasm, permitting NFATc to enter the nucleus. It has been suggested that Bcl-2 inhibits NFATc activation by sequestering calcineurin to intracellular membranes (Shibasaki et al., 1997). Our findings suggest that Bcl-2 may inhibit calcineurin activation by inhibiting InsP3-mediated calcium release from the ER. In T cells, calcium/calcineurin-mediated activation of NFATc increases IL-2 expression, which in turn stimulates dual pathways, one leading to cell death and the other leading to cell survival. IL-2 induces cell death via Stat2-mediated induction of the death receptor ligand Fas, whereas IL-2 promotes cell survival via Akt-mediated induction of Bcl-2 expression (Parijs et al., 1999). The findings of the present paper raise the possibility that increased expression of Bcl-2 may form a feedback loop that dampens InsP3-mediated calcium signals, thereby controlling T cell proliferation while maintaining cell survival.
InsP3Rs are known to associate with several factors that regulate InsP3-gated channel activity (Mackrill, 1999; Roderick and Bootman, 2003). This paper is the first to suggest that Bcl-2 may interact with InsP3Rs and regulate their functional activity. Although the full functional significance of the inhibitory effect of Bcl-2 on InsP3R channel activity is as yet unknown, this action of Bcl-2 may contribute to the established inhibitory effects of Bcl-2 on cell cycle entry and/or apoptosis induction. In view of the wide range of cellular functions mediated by InsP3-induced calcium signals (Berridge et al., 2003), it will be interesting in future studies to determine if the function of Bcl-2 goes beyond that of regulating cell cycle entry and apoptosis.
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Materials and methods |
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Cell culture and transfection procedures
WEHI7.2 and S49.A2 cells were cultured in DME supplemented with 10% serum, L-glutamine, and nonessential amino acids. Human Bcl-2 cDNA from the pB4 plasmid (American Type Culture Collection) was cloned into the pSFFV-Neo vector. Transfection and cloning were performed as described previously (Dieken and Miesfeld, 1992). Flag-tagged Bcl-2 was selectively localized to the ER by exchanging the COOH-terminal transmembrane sequence of Bcl-2 for the ER-targeting sequence of cytochrome b5, as described previously (Wang et al., 2001). Bcl-2 expression was monitored by flow cytometry of fixed cells using antiBcl-2 antibody (BD Biosciences; 15131A) at a 1:500 dilution and Alexa Fluor 488 goat antihamster IgG (H+L) conjugate (Molecular Probes; A-21110) as the secondary antibody at a dilution of 1:500.
Calcium fluorometry
Cells (10 ml volume, 1 million per milliliter) were incubated with 1 µM Fura-2 AM for 45 min at 25°C in ECB (130 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 25 mM Hepes, pH 7.5, 1 mg/ml BSA, and 5 mM glucose), after which they were pelleted and resuspended in ECB for an additional incubation at 25°C for 30 min to permit dye de-esterification. Fluorescence was continuously recorded at 37°C (alternating 340 and 380 nm excitation, 510 nm emission) in a fluorometer (Photon Technology Inc.). EGTA (final concentration 10 mM) was added to chelate extracellular calcium immediately before adding 100 nM TG, anti-CD3 antibody (BD Biosciences; 1:150 dilution), or 25 µM D-myo InsP3BM. In experiments using D-myo InsP3BM, the volume of cell suspensions was scaled down to 250 µl in a 300 µl cuvette. All measurements were performed in triplicate. Rmax and Rmin values were determined in each experiment by cell permeabilization with digitonin, followed by sequential addition of calcium and EGTA/Tris. Calcium concentration was calculated, based on the published Kd for Fura-2 of 220 nM, by the equation of Grynkiewicz et al. (1985) using Felix Software (Photon Technologies Inc.).
Calcium imaging
Cells adhered to poly-L-lysinecoated coverslips (15 mm) were loaded with 1 µM Fura-2 AM (Molecular Probes) as described in Calcium fluorometry. Coverslips were placed in a recording/perfusion chamber (model RC-25F; Warner Instruments) mounted on the stage of an inverted microscope (model Diaphot; Nikon) equipped with a 20x Fluor objective. Excitation light was alternated between 340 and 380 nm by a filter wheel (Sutter Instrument Co.) and emitted light was filtered at >510 nm and collected with an intensified charge-coupled device camera (model UltraPix; PerkinElmer). The video signal was digitized using Ultraview software (PerkinElmer) and subsequently processed using Microsoft Excel. Cells were perfused with ECB at 25°C and stock solutions of both anti-CD3 antibody (1:40 dilution) and 25 µM D-myo InsP3BM were diluted in ECB immediately before addition to the perfusion chamber. To determine Rmin, cells were perfused with ECB deficient in calcium and supplemented with 4 mM EGTA and 10 µM ionomycin. Rmax was obtained by perfusing cells with ECB supplemented with 4 mM CaCl2 and 10 µM ionomycin. Calcium concentration was calculated as described in Calcium fluorometry.
ER calcium measurement
Luminal calcium measurement was modified after that of Hofer (1999). Cells adhered to poly-L-lysinecoated coverslips were incubated with 1 µM Fura-2FF AM for 30 min at 37°C in ECB, after which the buffer was replaced with fresh ECB and the incubation continued for 45 min at 25°C to permit de-esterification. Cell imaging was performed as described in Calcium imaging. Cells were initially perfused with ECB at 25°C, and then with ICB (125 mM KCl, 25 mM NaCl, 10 mM Hepes, 0.5 mM Na2ATP, 200 µM CaCl2, and 500 µM EGTA, pH 7.25), supplemented with an ATP-regenerating system consisting of 10 µg/ml creatine phosphokinase Type I and 10 mM phosphocreatine. As soon as cells were permeabilized, the perfusion buffer was switched to ICB plus ATP-regenerating system. After the fluorescence ratio reached steady-state, cells were perfused with the same buffer supplemented with D-myo-InsP3 at concentrations given in the text and accompanying figures. Stock solutions of D-myo-InsP3 and L-myo-InsP3 were prepared at a concentration of 3 mM in calcium-free water and stored at 20°C until use. After cell permeabilization with digitonin, the proportion of Fura-2FF located in the ER was determined by quenching luminal dye by perfusing with 10 µM D-myo-InsP3 in ICB supplemented with 100 µM MnCl2. To determine Rmin, permeabilized cells were perfused with ECB deficient in calcium and supplemented with 4 mM EGTA and 10 µM ionomycin. Rmax was obtained by perfusing permeabilized cells with ECB supplemented with 4 mM CaCl2.
The InsP3 dose response relationship was determined by sequential addition of D-myo-InsP3 to digitonin-permeabilized cells while continuously monitoring the Fura-2FF 340:380 ratio. The InsP3-mediated change in the 340:380 ratio was plotted as a function of InsP3 concentration by nonlinear least squares best fit of the data to the Langmuir isotherm equation using Origin 6.0 (Microcal).
Organelle imaging
Confocal Z-series stacks of TMRE-loaded (0.1 µM, 10 min) cells were acquired after permeabilization with digitonin in ICB at RT using an Ultra View LCI (PerkinElmer) with a microscope (model TE300; Nikon), x100/1.3 objective (Nikon), and Orca ER camera (Hamamatsu) at excitation 568 nm, emission 600 ± 40 nm. The laser intensity was kept at a minimum to prevent irradiation-induced mitochondrial damage. Image acquisition was by UltraView software (PerkinElmer). Nonconfocal Z-series stacks of Fura-2FF loaded cells were obtained after permeabilization with digitonin in ICB at RT using an Ultra View LCI with a microscope, x100/1.3 objective, epifluorescence arc-lamp as a light source (excitation 330380 nm, dichroic 400LP, emission 420LP), and Orca ER camera. Subsequent image restoration was achieved with the deconvolution software AutoDeblur (Autoquant).
Western blotting
Cell lysates containing 4060 µg of protein were separated by SDS-PAGE (15% for Bcl-2 and 415% linear gradient for InsP3Rs) followed by electrophoretic transfer onto Immobilon-P PVDF membranes (Millipore). The following antibodies and their respective dilutions were used: antihuman Bcl-2 (BD Biosciences; 15131A, 1:2000), antimouse Bcl-2 (BD Biosciences; 554218, 1:1000), anti-actin (Santa Cruz Biotechnology, Inc.; sc-8432, 1:1000), anti-InsP3R Type I (Calbiochem; 407144, 1:2000), anti-InsP3R Type II (Chemicon; AB3000, 1:100), anti-InsP3R Type III (BD Biosciences; 610312, 1:2000), and anti-SERCA3 ATPase (Affinity BioReagents, Inc.; PA1-910, 1:500).
BN-PAGE
ER membranes were isolated from WEHI7.2 cells as described previously (Krajewski et al., 1993). In brief, cells (110 x 108) were washed twice with ice-cold PBS (Invitrogen), pH 7.2, and resuspended in 1.5 ml of homogenization buffer (20 mM Hepes, pH 7.4, 2.5 mM EDTA, 250 mM sucrose, and protease inhibitor cocktail). After homogenization for 1525 strokes with a Dounce homogenizer, samples were centrifuged at 3,000 g for 15 min at 4°C and 1 ml of supernatant was layered onto an 11-ml sucrose gradient (0.75 to 1.9 M sucrose in 20 mM Hepes, pH 7.4, and 2.5 mM EGTA) and centrifuged at 110,000 g for 2 h at 4°C. Sequential fractions of 0.8 ml were collected from the gradient and the distribution of ER and mitochondria was determined by Western blotting. Calnexin (anti-calnexin antibody, SPA-8600; StressGen Biotechnologies) and Cox IV (anti IV COX, A-6403; Molecular Probes) were used as markers of ER and mitochondria, respectively. Fractions containing purified ER membranes were pooled and mixed with three volumes of 20 mM Hepes, pH 7.4. The sample was centrifuged at 105,000 g for 1.5 h at 4°C and the resulting membrane pellet was used for BN-PAGE, performed as described previously (Schagger et al., 1994). The ER membrane was solubilized with 750 mM 6-aminocaproic acid, 50 mM BisTris, pH 7.0, and 1% dodecyl maltoside (5 µg total protein per microliter), and centrifuged at 100,000 g for 15 min at 4°C. Before starting BN-PAGE, Coomassie blue was added to the resulting supernatant to 0.25%. 100 µg of protein was added to each well of 410% linear gradient BN-PAGE. Single bands were excised from the blue native gel and applied to a 420% linear gradient SDS-PAGE with sample buffer, followed by Western blotting. Sizes of molecular complexes were estimated using a high molecular mass calibration kit for native electrophoresis (170445-01; Amersham Biosciences). After completion of electrophoresis, gels were subjected to Western blotting using antibodies to InsP3Rs and human Bcl-2 as described above.
Immunoprecipitation
108 cells were washed twice with PBS and incubated on ice 30 min with 1 ml lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM EDTA, 1% CHAPS wt/vol, 50 mM NaF, 200 nM okadaic acid, 1 mM Na3VO4, and protease inhibitor cocktail [Complete Mini; Roche Applied Science]). Cell lysates were centrifuged at 20,000 g for 15 min at 4°C. The resulting supernatant was rotated with 100 µl of 50% protein A or protein G agarose beads at 4°C for 2 h. After removing the beads, the supernatant was incubated with one of the following antibodies: antihuman Bcl-2 (hamster 1:250), antimouse Bcl-2 (1:250), anti-InsP3R antibody (anti-Type I, 1:200; anti-Type II, 1:150; anti-Type III, 1:200; or antibody recognizing all three subtypes [Calbiochem; 407143]) overnight at 4°C, followed by rotation with 50 µl protein A or G agarose for 2 h at 4°C. Nonspecific control antibodies used were rabbit serum (1:200 dilution; Life Technologies) and hamster IgG (5 µg/ml; BD Biosciences). The beads were washed four times with lysis buffer and boiled for 5 min in 50 µl sample buffer. The eluted proteins were resolved by SDS-PAGE and analyzed by Western blotting.
InsP3 binding
Specific binding of radiolabeled InsP3 to microsomes was measured as described previously (Riley et al., 2002). Microsomes (50 µg) were added in duplicate to 100 µl binding buffer (2 nM 3H-InsP3 [Dupont NET-911], 2 mM Tris, pH 9.0, 1 mM EDTA, and 1 mg/ml albumin, with or without a range of concentrations of unlabeled InsP3) and incubated on ice for 20 min. The mixture was centrifuged for 15 min at 20,000 g at 4°C. Pellets were solubilized in 100 µl water and added to 10 ml ACS scintillation cocktail (Amersham Biosciences) and their activity was determined by liquid scintillation counting. Nonspecific binding was determined using an excess of unlabeled InsP3. The Kd was calculated by Scatchard analysis.
Planar lipid bilayer analysis of InsP3R channel activity
Full-length human Bcl-2 was purified from Escherichia coli M-15 (pRep-4) cells transformed with a pProex-1/hBcl-2 using methods described previously (Lam et al., 1998). Type I InsP3Rs were purified from microsomes isolated from COS-1 cells transfected with pInsP3R1-DT1-ALT plasmid as described previously (Mignery et al., 1989, 1990). Gradient fractions containing InsP3R protein were then identified by immunoblotting with Type 1 receptor antibody and reconstituted into proteoliposomes as previously described (Mignery et al., 1992; Perez et al., 1997). Planar lipid bilayers were formed across a 150-µm diameter aperture in the wall of a Delrin partition as described previously (Perez et al., 1997). Proteoliposomes were added to the solution on one side of the bilayer (defined as the cis-chamber). The other side was defined as the trans-chamber. Standard solutions contained 220 mM CsCH3SO3 cis (20 mM trans), 20 mM Hepes, pH 7.4, and 1 mM EGTA ([Ca2+]Free = 250 nM). A custom current/voltage conversion amplifier was used to optimize single-channel recording. Acquisition software (pClamp; Axon Instruments, Inc.), an IBM compatible 486 computer, and a 12-bit A/D-D/A converter (Axon Instruments, Inc.) were used. Single channel data were digitized at 510 KHz and filtered at 1 KHz. Channel sidedness was determined by InsP3 sensitivity. The orientation of the channels studied was such that the InsP3 sensitive side (i.e., cytoplasmic side) was in the cis compartment.
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Acknowledgments |
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This work was supported by National Institutes of Health grants CA85804 and CA42755 (C.W. Distelhorst), by an International Union Against Cancer Yamagiwa-Yoshida Memorial International Cancer Study grant (C.W. Distelhorst), and by Fondecyt 1020927 and 7020927 (P. Velez). We also thank the Biotechnology and Biological Sciences Research Council and Human Frontier Science Programme (M.D. Bootman and H.L. Roderick) for financial support and the Engineering and Physical Sciences Research Council Mass Spectrometry Service for mass spectra.
Submitted: 24 September 2003
Accepted: 2 June 2004
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