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J. Biol. Chem., Vol. 278, Issue 31, 28856-28864, August 1, 2003
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From the
Department of Cardiology, Wales Heart
Research Institute, University of Wales College of Medicine, Heath Park,
Cardiff CF14 4XN, United Kingdom and the
¶Department of Biochemistry, University College
Cork, Cork, Ireland
Received for publication, December 6, 2002 , and in revised form, April 29, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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In Ca2+-driven cell death, the Ca2+ storage status of the endoplasmic reticulum (ER)1 and the levels of cytoplasmic free Ca2+ ([Ca2+]c) are proposed as the main determinants of cell fate, but the relative contribution of these in triggering cell death pathways is widely debated. Augmented ER Ca2+ load in COS cells caused cell death (10), whereas depletion of ER Ca2+ was toxic in SH-SY5Y neurons (11). Similarly, depletion of ER Ca2+ appeared to have a protective function for HeLa cells (12), in contrast to pancreatic B-cells where increased ER Ca2+ storage protected from nitric oxide-induced apoptosis (13). Studies in hippocampal neurons showed that increased [Ca2+]c was sufficient to mediate cell death (14) and in astrocytes similar elevation in [Ca2+]c is achieved solely from influx of extracellular Ca2+ with negligible involvement of intracellular Ca2+ stores (15). Conversely, it has been shown that a moderate and sustained elevation of cytoplasmic Ca2+ in neurons is protective (16).
Other cellular elements contribute to modulation of the intracellular Ca2+ signal. The close apposition of mitochondria with the ER is implicated in shaping cellular Ca2+ phenomena (17) and mitochondrial function appears crucial in both cell survival (18) and destruction (9). The spatial organization of Ca2+ release channels, ryanodine receptors (RyR), or inositol 1,4,5-trisphosphate receptors (IP3R) cells appears to be of key importance in determining cellular fate. Increased density of IP3R and RyR triggers cell death (19–21), and the finding that both IP3R and RyR can directly sense cytoplasmic and luminal Ca2+ environments (22–24) suggests that Ca2+ itself is a key transducer in the activation of programmed cell death.
We have investigated the specific role of RyR in mediating altered cellular
phenotype. RyRs are large homotetrameric channels (molecular mass, 
2.3
MDa) that mediate Ca2+ release from sarcoplasmic
reticulum (SR) in skeletal and cardiac muscle. Recent evidence predicts that
in situ RyRs do not exist as discrete entities but act as scaffolding
complexes that integrate a multitude of regulatory signals from accessory
proteins, including calmodulin, FK506-binding protein (FKBP), protein kinase
A, and protein phosphatases
(25,
26). It has been demonstrated
that altered stoichiometry between RyR and interacting accessory proteins
results in pathogenic Ca2+ signaling. For example,
dissociation of the RyR2·FKBP12.6 complex induces an aberrant leak of
Ca2+ from the SR, which is implicated in the
pathogenesis of heart failure
(27,
28).
We have generated CHO cells stably expressing discrete levels of recombinant human cardiac RyR (CHOhRyR2) and showed that a selective interaction occurred between hRyR2 and co-expressed FKBP12.6, which critically modulated the Ca2+ release activity of hRyR2 in situ (29). This study investigates the empirical observation that CHOhRyR2 exhibit increased cellular toxicity and decreased proliferation when compared with wild-type CHO cells. We demonstrate that these phenotypic changes are underscored by perturbations in cellular Ca2+ handling specifically mediated by hRyR2. Normal cellular phenotype was restored by modulating hRyR2-mediated Ca2+ release via co-expression of FKBP12.6, which highlighted the role of FKBP12.6 in homeostatic Ca2+ signaling and cell function. Our data indicate that expression of hRyR2 in an FKBP12.6-deficient context leads to increased cytoplasmic Ca2+ flux, without an accompanying change in mean cytoplasmic Ca2+ levels, which is deleterious to cell survival.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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Cell Culture and Analysis of Recombinant Protein Expression—Chinese hamster ovary (CHOhRyR2) cells stably expressing discrete levels of hRyR2 (29) were routinely cultured in supplemented nutrient Ham's F-12 medium (containing fetal calf serum (10% (v/v), amphotericin B (2.5 µg/ml), and G418 sulfate (500 µg/ml)). CHOWT cells were cultured in supplemented nutrient Ham's F-12 medium lacking G418 sulfate. In some experiments, cells were transfected with plasmid pCR3 (Invitrogen, UK) encoding human FKBP12 or FKBP12.6 using LipofectAMINE2000 in a ratio of 0.8 µg:1 µg (DNA:lipid) according to the manufacturer's protocol, and transfection efficiencies of >85% were routinely achieved. FKBP-hRyR2 interaction was disrupted following incubation of cells with rapamycin (5 µM).
Immunoblotting analysis of recombinant hRyR2 in post-nuclear supernatants obtained from wild-type CHO (CHOWT) and CHOhRyR2 was performed as described using an RyR2-specific antibody, pAb129 (30). The cellular expression of endogenous and recombinant FKBP12 and FKBP12.6 isoforms was determined using a rabbit polyclonal anti-FKBP12 antibody (see below) in immunoblotting analysis of post-nuclear supernatants (100 µg) obtained from CHOWT and CHOhRyR2 following separation of proteins on 20% (v/v) acrylamide SDS-PAGE (29).
Immunofluorescent localization of endogenous IP3R and recombinant hRyR2 in CHOhRyR2 cells was performed using pAb-40 (anti-IP3R, 1:250 dilution) (31) and pAbN-19 (anti-RyR, 1:500 dilution) (Santa Cruz Biotechnology), respectively, followed by subsequent detection using donkey secondary antibodies conjugated to Alexa488 (IP3R) and Alexa546 (RyR) as previously described (29). The extent of co-localization between IP3R and RyR signals was quantified using Scanware software (Leica). DiOC6 (3) (0.5 µM) was used to visualize the ER in RyR-deficient CHOWT cells.
Production of Anti-FKBP12 Polyclonal Antiserum—Recombinant human FKBP12-glutathione S-transferase (hFKBP12-GST) fusion protein was generated as previously described (32). hFKBP12-GST (250 µg), complexed with glutathione-Sepharose beads (Amersham Biosciences) and resuspended in PBS (250 µl) after extensive washing in PBS, was emulsified with an equal volume of Freund's Complete adjuvant (first immunization) or Freund's Incomplete adjuvant (second and third immunizations) prior to subcutaneous injection into adult New Zealand White female rabbits (University College Cork Biological Services Unit). Purified hFKBP12 (50 µg) (Sigma) emulsified in Freund's Incomplete adjuvant as above was used as the final immunogen. Antiserum was isolated from whole blood following clotting and was stored in sodium azide (0.02% (w/v)).
Determination of Cell Death Modality and Cell Proliferation—Cell viability was determined 36 h post-seeding using Trypan blue (0.4% (w/v) in PBS (NaCl, 140 mM; KCl, 2.7 mM; Na2HPO4, 10 mM; NaH2PO4, 2 mM; pH 7.4) and propidium iodide (PI; 1 µg/ml in PBS) permeability assays (33). The plasma membranes of viable cells are impermeable to Trypan blue and PI, and only cells that were stained positively by both Trypan blue and PI were confirmed to be non-viable. Cells were visualized using phase (Trypan blue) and fluorescent microscopy (PI; Emmax > 620 nm), respectively. All assays were performed on adherent cells, and there was negligible detachment of cells from the culture surface throughout these experiments as determined by hemocytometric measurement of aspirated culture medium (data not shown).
The extent of apoptotic cell death was quantified using a fluorometric TdT-mediated dUTP nick-end labeling (TUNEL) assay (DeadEndTM, Promega, UK). Briefly, resting cells or cells preincubated with modulators of intracellular Ca2+ handling (see below) were fixed in paraformaldehyde (3% (v/v) in PBS), permeabilized in Triton X-100 (0.2% (v/v) in PBS), and incubated in buffer containing terminal deoxynucleotidyl transferase enzyme (25 units) for1hat37 °C to incorporate fluorescein-12-dUTP into caspase-fragmented DNA, a phenotypic marker of apoptosis. Cells were washed in 2x SSC (300 mM NaCl, 30 mM sodium citrate; pH 7.0), and apoptotic cells were identified by the presence of highly fluorescent nuclei (Emmax, 511 nm).
Determination of Cellular Proliferation—The proliferative capacity of sub-confluent CHOWT and CHOhRyR2 cells was determined on the same cell populations at 24-h intervals for 96 h. Cells were incubated with medium containing AlamarBlue (10% (v/v)) for 5 h prior to fluorometric analysis of aspirated medium (LS50B, PerkinElmer Life Sciences). Cellular metabolic activity reduces AlamarBlue from a nonfluorescent (blue) form into a fluorescent (red) form (Exmax, 560 nm; Emmax, 590 nm). The oxidized and reduced forms of AlamarBlue are freely cell permeable and do not contribute to cellular toxicity (34). The seeding density of cells was adjusted to ensure that the cells were sub-confluent throughout these procedures. The data obtained using AlamarBlue cell viability assays was corroborated by hemocytometric measurement of cell number performed in parallel experiments.
Measurement of [Ca2+]c, ER Ca2+ Load, and Intracellular Ca2+ flux—The Ca2+-dependent fluorescence of fluo3 was calibrated in situ in streptolysin O (200 units/ml)-permeabilized cells (35) loaded with fluo3-AM (15 µM), which were incubated with extracellular solutions containing known [Ca2+] (17 nM to 39 µM) to clamp [Ca2+]c (Molecular Probes). The apparent Kd (Kd,app) of 580 nM was generated using Graph-Pad Prism software. [Ca2+]c was calculated from fluorescent data using: [Ca2+] = Kd,app (F – Fmin)/(Fmax – F) (36). Fmax and Fmin were determined following the addition of ionomycin (1 µM) and EGTA (5 mM), respectively, at the end of experiments. Cells grown on coverslips were loaded with fluo3-AM (15 µM) for 1 h at 23 °C in Krebs-Ringer-Hepes medium (KRH; 120 mM NaCl, 25 mM Hepes, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgCl2, 5.5 mM glucose, 1.3 mM CaCl2; pH 7.4), and immediately prior to experiments, cells were transferred to KRH containing nominal free Ca2+ (KRH-Ca2+, where EGTA (1 mM) replaced CaCl2). Resting and agonist-stimulated [Ca2+]c was measured in cells using a confocal microscope (SP2, Leica, Heidelberg, Germany) in bidirectional scan mode (512- x 64-pixel resolution) controlled with Leica software.
To specifically trigger Ca2+ release through hRyR2, 4-chloro-m-cresol (4-CMC; 0.5 mM) was added to cells, and Ca2+ release through endogenously expressed IP3R (37) was triggered by the intracellular generation of IP3 from PIP2 via the activation of purinergic (P2Y) receptors using ATP (1 mM) (38). To investigate the effects of blocking RyR-mediated Ca2+ release, cells were exposed to ryanodine (1 mM) for 15 h or for 2 h prior to agonist-stimulated Ca2+ release.
IP3R-mediated Ca2+ release was inhibited
by cytoplasmic loading of heparin using a hypo-osmotic pinocytic lysis
procedure (39). Briefly, cells
were incubated in Leibowitz L-15 medium containing sucrose (0.5
M), polyethylene glycol 1000 (10%, v/v), and heparin (5 mg/ml) for
5 min to form heparin-containing pinocytic vesicles. Cells were exposed to
hypo-osmotic media (6 parts L-15 medium to 4 parts water) for 2 min
to promote pinocytic lysis and the distribution of heparin throughout the
cytoplasm, prior to rinsing and further culturing in normal nutrient F-12
media. This procedure, which resulted in the loading of 95% cells as
determined by cytoplasmic localization of fluorescent fluorescein
isothiocyanate-dextran, did not contribute to cell toxicity (data not shown).
In some experiments, cells were loaded with BAPTA-AM (0.1 µM), a
cytoplasmic Ca2+ chelator, for 15 h prior to
experiments. The ER Ca2+ load status was estimated from
the measurement of peak Ca2+ release following addition
of thapsigargin (5 µM) to CHOWT and
CHOhRyR2 cells maintained in KRH-Ca2+
(29). Thapsigargin-induced
Ca2+ release in CHOWT cells was designated as
100%.
The variability in the intracellular Ca2+ flux in
resting (non-stimulated) CHOhRyR2, when compared with
CHOWT, was estimated following calculation of the F ratio
(40). The F ratio,
defined as
(where S.D.a is the standard deviation of
[Ca2+]c values determined in
CHOhRyR2 cells, and S.D.b is the standard deviation of
[Ca2+]c values calculated in CHOWT
cells for each experimental condition), numerically describes the amplitude
variability in resting cytoplasmic Ca2+ flux. All
experiments were performed in medium containing nominal free
Ca2+ to negate the contribution of extracellular
Ca2+ influx.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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560 kDa) was CHOH > CHOL >>
CHOWT (RyR-deficient) (Fig.
1A). The normal morphology of CHOWT cells is
shown with typically 2–4% of cells exhibiting altered morphology
(Fig. 1B, panel a,
arrowed). In contrast, CHOH, a cell line stably expressing
high levels of hRyR2, were characterized by a dramatically increased
prevalence of small, morphologically altered cells
(Fig. 1B, panel b,
arrowed), a small proportion of which exhibited significant cell surface
membrane blebbing (Fig.
1B, panel c), a phenotypic marker of apoptotic
cell death. CHOL cells, which express lower levels of hRyR2
(Fig. 1A), also
exhibited pronounced cellular toxicity
(Fig. 1B, panel d,
arrowed) with evidence of apoptosis
(Fig. 1B, panel
e). We determined a marked reduction in the viability of resting
CHOhRyR2 when compared with CHOWT, which was
proportional to the expression levels of hRyR2, i.e. viability of
CHOH < CHOL << CHOWT
(Fig. 1C, panel
b). The normal modulation of RyR2 requires a multitude of accessory
proteins, some of which are not endogenously expressed in CHO cells
(21,
29) and thus our
CHOhRyR2 cell model provides a powerful platform to determine the
impact of defective hRyR2 Ca2+ signaling on cell
function.
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Inhibition of hRyR2 Ca2+ release by incubation of cells with ryanodine (1 mM) fully restored the viability of CHOhRyR2 cells to levels measured in CHOWT (Fig. 1C, panel b), indicating that the increased cell death measured in CHOhRyR2 was mediated by the expression of functional hRyR2. Analysis of the mode of cell death in resting CHOhRyR2 identified significantly elevated levels of necrotic cell death, which was prevented following ryanodine-mediated inhibition of hRyR2 (Fig. 1C, panel a). These data are in broad agreement with findings that heterologous expression of RyR produces marked phenotypic changes when compared with wild-type host cell lines (21, 41, 42), but our results extend these findings by demonstrating that cell death in resting (non-stimulated) cells expressing functional hRyR2 predominantly occurs via necrosis. However, the precise mechanistic basis of necrosis is unknown in these cells, and thus throughout this study the term "necrosis" refers to a currently undefined mechanism of cell death that is distinct from apoptosis.
Increased Cytoplasmic Ca2+ Flux via hRyR2 Mediates Cell Necrosis—Because the resting [Ca2+]c in CHOhRyR2 was effectively constant (Fig. 2A), the decreased viability of CHOhRyR2 did not arise from sustained elevations in cytoplasmic Ca2+. Despite similar resting [Ca2+]c in CHOWT and CHOhRyR2 (Fig. 2B, panel a), we observed that increased expression of hRyR2 was associated with a concordant increase in "noise" (flux) in resting Ca2+ signals recorded in CHOhRyR2 cells (Fig. 2A, "control" traces). We hypothesized that the increased amplitude of Ca2+ flux measured in CHOhRyR2, which is clearly apparent in Fig. 2A, arose as a consequence of aberrant Ca2+ efflux from the ER via hRyR2 and that steady-state resting [Ca2+]c was achieved by a concordant increase in Ca2+ sequestration via hRyR2-dependent up-regulation of the SR/ER Ca2+-ATPase in CHOhRyR2 cells (29). We estimated the variability in the intracellular Ca2+ flux in resting (non-stimulated) CHOhRyR2 by calculating the F ratio (40). The F ratio (as defined under "Experimental Procedures") numerically described the amplitude variability in resting cytoplasmic Ca2+ flux. Quantifying this amplitude variability of [Ca2+]c (Fig. 2B, panel b (40)) identified CHOWT and CHOhRyR2 to have very distinct F ratios, despite similar mean [Ca2+]c (Fig. 2B, panel a). Thus, similar resting [Ca2+]c masked the degree of variability in intracellular Ca2+ flux CHOhRyR2 cells. Furthermore, the magnitude of cytoplasmic Ca2+ flux correlated with the expression levels of hRyR2 and the rank order of variation in intracellular Ca2+ flux was CHOH > CHOL >> CHOWT (Fig. 2B, panel b).
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To investigate more precisely a causative role of hRyR2 in mediating the
increased cytoplasmic Ca2+ flux in CHOhRyR2
cells, we co-expressed FKBP12.6, a potent modulator of RyR2 function
(27,
29) and determined its effects
on Ca2+ handling and cellular phenotype in
CHOhRyR2 cells. Polyclonal antiserum raised against FKBP12, a
homologue of FKBP12.6 that does not functionally interact with RyR2
(29,
43,
44), cross-reacted with
FKBP12.6 owing to a high degree of sequence homology (85%)
(Fig. 2C). FKBP12
exhibits an extensive tissue distribution
(43) and was endogenously
expressed in CHOWT and CHOhRyR2 cells. In contrast,
FKBP12.6 is expressed at far lower levels in tissues
(45) and was not detected in
post-nuclear fractions obtained from these cells
(Fig. 2C, left
panel). High level expression of recombinant FKBP12.6 in
CHOhRyR2 cells (Fig.
2C, middle panel) was associated with
significantly decreased intracellular Ca2+ flux
(Fig. 2, A and B,
panel b) and a concordant restoration of CHOhRyR2
viability (Fig. 2D).
In contrast, co-expression of FKBP12 (Fig.
2C, right panel) had little effect on the
magnitude of cytoplasmic Ca2+ flux
(Fig. 2, A and
B, panel b) and did not affect cell viability
(Fig. 2C) consistent
with the absence of FKBP12-hRyR2 interaction. However, in complete agreement
with ryanodine inhibition of hRyR2 (Fig.
1), FKBP12.6 expression was associated with a reduced necrosis in
the few non-viable cells (5% of the total population), which persisted
(Fig. 2D, panel
a). Disruption of specific protein-protein interaction between
FKBP12.6-hRyR2 using rapamycin
(46) reversed the inhibitory
effect of FKBP12.6 on hRyR2-mediated cytoplasmic Ca2+
flux (Fig. 2, A and
B, panel b) and abolished the pro-survival
effects of FKBP12.6 in CHOhRyR2
(Fig. 2D).
Interestingly, the restoration of cell viability following co-expression of
FKBP12.6 in CHOhRyR2 was associated with a marked potentiation of
the ER Ca2+ store
(Fig. 2B, panel
c) suggesting that super-filling ER Ca2+ stores did
not directly contribute to CHOhRyR2 cell death. However, there was
close correlation between the magnitude of cytoplasmic
Ca2+ flux and the extent of cell toxicity
(R2 = 0.937), strongly indicating that increased
cytoplasmic Ca2+ flux, independent of resting
[Ca2+]c or ER Ca2+ load
status, is a key determinant of CHOhRyR2 cell viability.
Furthermore, the finding that FKBP12.6 expression had a marked pro-survival
effect on CHOhRyR2 by inhibiting the increased cytoplasmic
Ca2+ flux through hRyR2 may go some way to providing a
mechanistic basis for the use of FKBP12.6-mediated strategies as a candidate
therapy in preventing the abnormal SR Ca2+ efflux
underpinning heart failure
(47). Although it is widely
accepted and clearly demonstrated in the present work that hRyR2
Ca2+ release is modulated by interaction with FKBP12.6,
controversy exists as to the precise nature of RyR2-FKBP12 interaction
(48,
49). In the present context of
our CHOhRyR2 cell system, we show that FKBP12 does not interact
functionally with hRyR2.
We next investigated the effect of biochemical modulation of hRyR2-mediated cytoplasmic Ca2+ flux on cell phenotype. In agreement with the data obtained following co-expression of FKBP12.6 in CHOhRyR2 cells, ryanodine-mediated inhibition of recombinant hRyR2 did not affect [Ca2+]c (Fig. 3B, panel a), yet significantly reduced cytoplasmic Ca2+ flux (Fig. 3, A and B, panel b) and restored cellular viability (Fig. 3B, panel b) by prevention of necrotic cell death (Fig. 3B, panel a). This prosurvival effect of ryanodine in CHOhRyR2 was associated with augmentation of the ER Ca2+ capacity (Fig. 3B, panel c). We used BAPTA to directly investigate the phenotypic effects of suppressing cytoplasmic Ca2+ flux in CHOhRyR2 and CHOWT cells. However, because normal cell processes are dependent on coordinated fluctuations in [Ca2+]c, BAPTA treatment (>0.5 µM) was associated with a dose-dependent toxicity (data not shown), and thus lower concentrations were used (0.1 µM), because this adequately suppressed cytoplasmic Ca2+ fluctuations (Fig. 3A) yet did not adversely affect the cell viability during these experiments. Incubation of cells with BAPTA-AM (0.1 µM, 15 h), a potent inhibitor of apoptosis (50) (Fig. 3C, panel a), decreased resting [Ca2+]c and ER Ca2+ load (Fig. 3B, panels a and c, respectively), markedly suppressed intracellular Ca2+ flux (Fig. 3, A and B, panel b) and restored CHOhRyR2 cell viability (Fig. 3C, panel b). Inhibition of endogenously expressed IP3R following cytoplasmic loading of heparin had little effect on resting [Ca2+]c, cytoplasmic Ca2+ flux, and ER Ca2+ load (Fig. 3B, panels a–c, respectively), and accordingly, CHOhRyR2 viability remained low (Fig. 3C). Taken together, our findings do not predict a clear role for mean resting [Ca2+]c or altered ER Ca2+ load in the decreased cell viability of CHOhRyR2 but strongly indicate that CHOhRyR2 toxicity is underscored by increased hRyR2-mediated cytoplasmic Ca2+ flux. Cells are tolerant of moderate cytoplasmic Ca2+ fluxes, which encrypt homeostatic signaling events (7, 8), and the dramatic loss of cell viability in CHOhRyR2 highlights the extent to which expression of dysregulated hRyR2 perturbs the normal mode of Ca2+ signaling.
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Apoptosis Is Triggered following Agonist Activation of hRyR2 or via Functional Interaction with Endogenous IP3R—Our results point to a pivotal role of increased hRyR2 Ca2+ release in initiating necrotic cell death in resting CHOhRyR2, and hence we investigated the phenotype resulting from activation of hRyR2 in agonist-stimulated cells. Addition of 4-CMC (0.5 mM), a potent and specific activator of hRyR2 Ca2+ release (51), increased [Ca2+]c in CHOhRyR2, and the magnitude of Ca2+ release was proportional to the levels of hRyR2 expressed in each cell type (CHOH > CHOL) (Fig. 4A). No Ca2+ was released in CHOWT following addition of 4-CMC (Fig. 4A), confirming that CHOWT does not express functional levels of RyR, and CHOWT cell viability remained high (Fig. 4C). Ryanodine treatment abolished 4-CMC-induced Ca2+ release in CHOhRyR2 populations, whereas inhibition of endogenous IP3R using heparin had no effect on Ca2+ release triggered by 4-CMC (Fig. 4A). This moderate increase in [Ca2+]c upon 4-CMC addition to CHOH and CHOL cells (0.32 ± 0.04 and 0.17 ± 0.03 µM, respectively) was associated with a profound decrease in cellular viability via apoptosis (Fig. 4B). Ryanodine inhibition of hRyR2 prevented apoptosis, whereas heparin had no effect on 4-CMC-induced cellular toxicity (Fig. 4B, gray bars) highlighting a role for hRyR2 Ca2+ release in determining the extent and mode of cell death in CHOhRyR2. The initiation of apoptotic pathways following the activation of skeletal muscle RyR (RyR1) in heterologous expression systems has been reported (21), but our data highlight a fundamental switch in cell death mechanisms upon agonist-induced Ca2+ mobilization. Although apoptosis and necrosis are proposed to share common initiating programs (50, 52), when taken together our data provide strong evidence that different modes of Ca2+ release in resting (increased cytoplasmic Ca2+ flux) and activated cells ("global" Ca2+ mobilization) initiate distinct cell death pathways (necrosis and apoptosis, respectively) in CHOhRyR2 cells.
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It was necessary to determine whether the profound loss of CHOhRyR2 viability following 4-CMC addition was due to (a) the mode of Ca2+ mobilization or (b) the magnitude of Ca2+ release from intracellular stores. To address this issue, ATP (1 mM) was used to activate IP3R-mediated Ca2+ release via the purinergic (P2Y) receptor generation of IP3 (38). Unlike 4-CMC, which only elicited Ca2+ release in CHOhRyR2 cells, ATP elicited a large [Ca2+]c increase in CHOWT (0.71 ± 0.08 µM), CHOH (0.92 ± 0.11 µM), and CHOL (0.88 ± 0.09 µM) (Fig. 4C). Note that the ATP-induced Ca2+ release was augmented in CHOhRyR2 cells when compared with CHOWT cells, in agreement with the finding that the expression of hRyR2 augments ER Ca2+ load (Fig. 2B, panel c). Importantly, in CHOWT cells maintained in nominal free extracellular Ca2+, ATP addition did not decrease cell viability, whereas a similar magnitude of Ca2+ release in CHOhRyR2 produced significant cell death via apoptosis, the extent of which was proportional to the expression levels of hRyR2 (Fig. 4D). Cytoplasmic loading of heparin abolished IP3R-dependent Ca2+ release in all cells (Fig. 4C) and prevented cell death in CHOhRyR2 (Fig. 4D) indicating that the ATP-induced toxicity in CHOhRyR2 cells required the activation of endogenous IP3R. Importantly, following incubation of CHOhRyR2 cells with ryanodine, the magnitude of ATP-induced Ca2+ release was decreased (Fig. 4C) and resulted in a remarkable preservation of cell viability (Fig. 4D). These results raise the intriguing possibility that the pronounced loss of viability in CHOhRyR2 following IP3R-activation is specifically mediated by Ca2+ release from ryanodine-sensitive stores via hRyR2. In accord with this hypothesis, this loss did not occur in RyR-deficient CHOWT cells where viability remained intact following 4-CMC or ATP addition (Fig. 4, A–D). There was extensive intracellular co-localization between recombinant hRyR2 and endogenous IP3R in CHOH and CHOL cells (90 ± 7% and 94 ± 5% coincidence (yellow), respectively (n = 6)) with both Ca2+ release channels displaying a perinuclear lattice-like distribution characteristic of the ER network (Fig. 5). It has been shown that spatially coincident IP3R and RyR Ca2+ release channels participate in functional "cross-talk" (53, 54), and indeed, our results suggest that activation of IP3R (heparin-sensitive) triggered Ca2+ release via recombinant hRyR2 (ryanodine-sensitive), which critically modulated the CHOhRyR2 phenotype. These data also provided compelling evidence that the loss of cell viability in agonist-stimulated CHOhRyR2 was underpinned by the mode of Ca2+ release (i.e. via hRyR2) rather than the magnitude of Ca2+ release. We currently do not know the precise mechanism by which activation of IP3R triggers hRyR2 Ca2+ release in these cells, or conversely, how activation of recombinant hRyR2 fails to trigger Ca2+ release via IP3R, but it is likely that cellular machinery required to permit functional interaction between these Ca2+ release channels (25, 53, 54) are present in CHOhRyR2. Clearly, a more refined analysis of sub-cellular Ca2+ mobilization is required to more fully elucidate the molecular basis of this phenomenon.
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We demonstrated a profound effect of FKBP12.6, but not FKBP12, in restoring the viability of resting CHOhRyR2 cells via the specific modulation of hRyR2 Ca2+ release (Fig. 2), and thus we investigated whether the beneficial effects of FKBP12.6 persisted in agonist-stimulated CHOhRyR2 cells. In CHOWT, co-expression of FKBP12.6 did not affect Ca2+ release triggered by 4-CMC or ATP (Fig. 6, A and C, respectively), confirming that FKBP12.6 did not functionally interact with endogenous IP3R and had no impact on viability or mode of cell death (Fig. 6B, panels b and a, respectively). In contrast, FKBP12.6 inhibited 4-CMC or ATP-triggered Ca2+ release in CHOhRyR2 cells (Fig. 6, A and C, respectively) and preserved CHOhRyR2 cell viability by inhibiting apoptosis (Fig. 6, B and D). Rapamycin antagonized the beneficial effects of FKBP12.6 on intracellular Ca2+ release (Fig. 6, A and C) and CHOhRyR2 viability (Fig. 6, B and D) further emphasizing that the effects of FKBP12.6 on cell phenotype were mediated by specific protein-protein interactions with hRyR2.
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Abnormal Intracellular Ca2+ Handling Mediates Decreased CHOhRyR2 Proliferation—Finally, we investigated the effects of the altered Ca2+ handling in CHOhRyR2 on cellular function by measuring their proliferative capacity. Increased expression levels of hRyR2 were associated with decreased cellular proliferation (CHOH, 37 ± 14%; CHOL, 59 ± 10% when compared with CHOWT (100%) during the first 72 h) (Fig. 7A). However, although cells did not reach confluency during the experiment, the rate of proliferation slowed as the cell density increased (days 1–4) (Fig. 7A, panels a–d), suggesting that the proliferation of CHOWT and CHOhRyR2 was sensitive to the ambient cellular environment. Modulation of hRyR2 by ryanodine (Fig. 7A, panel b) and FKBP12.6 (Fig. 7A, panel d) restored CHOhRyR2 cellular proliferation rates to those measured in CHOWT cells (Fig. 7B). Expression of FKBP12 had no effect on the cell growth (Fig. 7A, panel c). Incubation of cells with heparin or BAPTA (even at 0.1 µM) for the duration of the experiments resulted in total cell death (data not shown), presumably due to the sustained inhibition of normal homeostatic signaling events, which are dependent on endogenous IP3R Ca2+ release (1). These results indicate that biochemical (ryanodine) or physiological (FKBP12.6) modulation of hRyR2, which inhibits pathological fluxes in cytoplasmic Ca2+ (Figs. 2–3), restored normal cell proliferation in populations of CHOhRyR2. Thus, an important demonstration in the present study is that the co-expression of recombinant FKBP12.6 was necessary and sufficient to regulate RyR2, whereas FKBP12 did not interact with RyR2 and had no discernible effect on cellular phenotype in our model.
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Although we cannot exclude the possibility that the decreased proliferation of CHOhRyR2 reflected decreased ATP availability due to the energy demands of continuously pumping Ca2+ from the cytoplasm back into the ER, we propose that ATP depletion is not causative of abnormal phenotype. The ratio of AlamarBlue fluorescence, a robust indicator of cellular metabolism to actual cell number was similar in all cells at all time points directly indicating that the expression of hRyR2 (Fig. 7B, panel a) or following its modulation of hRyR2 using ryanodine (Fig. 7B, panel b) or co-expression of FKBP12 or FKBP12.6 (Fig. 7B, panels c and d, respectively) did not compromise the metabolic activities of CHOH and CHOL cells, which were indistinguishable from those of CHOWT (Fig. 7B, panels a–d). Second, expression-profiling analysis of CHOH demonstrated that increased Ca2+ flux mediates precise Ca2+-dependent (ryanodine-sensitive) transcriptional alterations in intracellular signaling networks (55). In view of our data, we propose that increased Ca2+ flux results in the specific modulation of signaling pathways, which are directly causative of the CHOhRyR2 phenotype.
The finding that the expression levels of hRyR2 determined the extent of intracellular Ca2+ dysregulation and the degree of phenotypic modulation is an important conclusion of the present work. The CHOhRyR2 cell model represents a powerful platform to study the role of RyR2 on cellular function in an FKBP12.6-deficient context. Furthermore, our demonstration that reconstitution of the RyR2·FKBP12.6 complex in intact cells inhibited aberrant ER-cytoplasmic Ca2+ flux and resulted in complete restoration of normal cellular phenotype represents a significant advance in the characterization of RyR-mediated signaling in cell function. It would be interesting to determine whether similar phenotypes are established as a consequence of defective functionality of other Ca2+ regulatory molecules or whether those elucidated in this study represent RyR-specific alterations in cellular phenotype. However, in the present context, our data suggest that increased Ca2+ flux in resting CHOhRyR2 precisely modulates signaling programs involved in cell viability and proliferation, and our cell model could enable the determination of the impact of defective RyR-driven Ca2+ signaling on other diverse cellular processes.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 44-292-074-4431; Fax:
44-292-074-3500; E-mail:
georgech{at}cf.ac.uk.
1 The abbreviations used are: ER, endoplasmic reticulum;
[Ca2+]c, cytoplasmic
Ca2+; RyR, ryanodine receptor; hRyR, human RyR;
IP3, inositol 1,4,5-trisphosphate; IP3R, IP3
receptor; SR, sarcoplasmic reticulum; FKBP, FK506-binding protein; CHO,
Chinese hamster ovary cells; BAPTA-AM,
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid tetrakis (acetoxymethyl ester); WT, wild-type; GST, glutathione
S-transferase; PBS, phosphate-buffered saline; PI, propidium iodide;
KRH, Krebs-Ringer-Hepes medium; 4-CMC, 4-chloro-m-cresol;
Emmax, wavelength for maximum emission;
Exmax, wavelength for maximum excitation;
DiOC6, 3,3'-dihexyloxacarbocyanine iodide.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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