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Originally published In Press as doi:10.1074/jbc.M705183200 on September 17, 2007

J. Biol. Chem., Vol. 282, Issue 45, 32983-32990, November 9, 2007
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Role of Inositol 1,4,5-Trisphosphate Receptors in Apoptosis in DT40 Lymphocytes*Formula

M. Tariq Khan, Cunnigaiper D. Bhanumathy, Zachary T. Schug, and Suresh K. Joseph1

From the Department of Pathology and Cell Biology, Thomas Jefferson University School of Medicine, Philadelphia, Pennsylvania 19107

Received for publication, June 25, 2007 , and in revised form, August 23, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The role of inositol 1,4,5-trisphosphate receptors (IP3R) in caspase-3 activation and cell death was investigated in DT40 chicken B-lymphocytes stably expressing various IP3R constructs. Both full-length type-I IP3R and a truncated construct corresponding to the caspase-3 cleaved "channel-only" fragment were able to support staurosporine (STS)-induced caspase-3 activation and cell death even when the IP3R construct harbored a mutation that inactivates the pore of the Ca2+ channel (D2550A). However, a full-length wild-type IP3R did not promote caspase-3 activation when the 159-amino acid cytosol-exposed C-terminal tail was deleted. STS caused an increase in cytosolic free Ca2+ in DT40 cells expressing wild-type or pore-dead IP3R mutants. However, in the latter case all the Ca2+ increase originated from Ca2+ entry across the plasma membrane. Caspase-3 activation of pore-dead DT40 cells was also more sensitive to extracellular Ca2+ chelation when compared with wild-type cells. STS-mediated release of cytochrome c into the cytosol and mitochondrial membrane potential depolarization could also be observed in DT40 cells lacking IP3Rs or containing the pore-dead mutant. We conclude that nonfunctional IP3Rs can sustain apoptosis in DT40 lymphocytes, because they facilitate Ca2+ entry mechanisms across the plasma membrane. Although the intrinsic ion-channel function of IP3Rs is dispensable for apoptosis induced by STS, the C-terminal tail of IP3Rs appears to be essential, possibly reflecting key protein-protein interactions with this domain.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis is an essential process required for normal development and tissue homeostasis and can be activated by diverse stimuli, including cytotoxic drugs, DNA damage, irradiation, withdrawal of growth factors, and activation of death receptors by ligands such as tumor necrosis factor or Fas. A requirement for elevated levels of Ca2+ has been implicated in many of these models of apoptosis (1–3). Several key enzymes activated during apoptosis such as endonucleases (4), phospholipase-A2 (5), and calpains (6) are known to be stimulated by Ca2+. However, the exact steps in apoptotic cascades that are affected by Ca2+ and the mechanisms resulting in the perturbation of Ca2+ homeostasis are poorly understood.

Elevations of cytosolic Ca2+ ([Ca2+]i) are achieved in cells by mobilization of intracellular Ca2+ stores and/or enhancement of Ca2+ entry across the plasma membrane. Inositol 1,4,5-trisphosphate receptors (IP3Rs)2 are a family of three intracellular Ca2+-release channels, mainly located in the ER membrane, which are primarily responsible for the agonist-mediated release of Ca2+ from intracellular stores. A number of different experimental approaches have implicated IP3Rs as pro-apoptotic regulators. The targeted deletion of all three IP3R isoforms in the chicken DT-40 lymphocyte cell line renders the cells more resistant to apoptotic stimuli induced by anti-IgM or staurosporine (STS) (7, 8). Decreasing the expression of the type-III (9) or the type-I (10) IP3R isoforms has been shown to confer resistance to various apoptotic stimuli in Jurkat T-cell lines. A selective role for the type-III IP3R in apoptosis was also reported in a study using small interference RNA to suppress expression of individual IP3R isoforms (11). However, mice deficient in both type-II and III IP3R isoforms do not show defects in developmental apoptosis suggesting the presence of significant redundancy in the requirement for individual IP3R isoforms (12).

The intrinsic pathway of apoptosis involves the release of cytochrome c (Cyt c) from the mitochondrial intermembrane space and the initiation of a cascade of events that ultimately leads to the activation of caspase-3. Although the exact transport pathway utilized for Cyt c release during apoptosis or necrosis is controversial, it is well established that enhanced accumulation of Ca2+ by the mitochondria sensitizes the Cyt c release pathway to stimulation by apoptotic agents (3, 13). The close proximity of ER to mitochondria suggests that the Ca2+ channel function of IP3Rs could be important in modulating the kinetics and sensitivity of apoptotic pathways (3, 13, 14). Further evidence for the pro-apoptotic role of IP3Rs is indicated by the finding that a number of key components of apoptotic cascades appear to interact with and regulate IP3R function. The anti-apoptotic proteins Bcl-2 and Bcl-xL have been shown to bind IP3Rs, but the studies differ as to whether this results is an inhibition or activation of IP3R channel activity (15, 16). Mouse embryonic fibroblast cell lines obtained from Bax/Bak double knock-out mice show an increased phosphorylation of the IP3R and an enhanced Ca2+ leak across the ER membrane (17). This has been attributed to the effect of unrestrained Bcl-2 on IP3Rs (17). The type-I IP3R isoform contains a consensus caspase-3 cleavage site and has been shown to be a substrate for this enzyme in various models of apoptosis (8, 18–20). The C-terminal "channel-only" fragment produced from caspase-3 cleavage has been reported to form a constitutively open channel and to contribute to the Ca2+ leak pathway across ER membranes (8, 21). Cyt c released from the mitochondria has been found to bind and activate IP3Rs by decreasing the feedback inhibition of these channels by Ca2+ (22). Finally, IP3Rs have been found to be substrates for the important anti-apoptotic regulatory protein Akt kinase (23). The highly conserved phosphorylation site for Akt kinase, as well as the binding sites for Bcl-xL and Cyt c, are all localized to the short, cytosol-exposed C-terminal tail of the receptor (16, 23, 24).

With the exception of Akt kinase phosphorylation, a common feature of all the aforementioned modes of IP3R regulation during apoptotic signaling is that they are considered to modulate the Ca2+ channel function of the intact or caspase-cleaved IP3R ion channel. To better understand the role of IP3Rs in regulating apoptosis we have examined the effect of introducing an ion channel-defective IP3R mutant into the IP3R DT-40 triple knock-out lymphocytes. Surprisingly, this cell line continued to respond to cytotoxic apoptotic stimuli in a similar manner as wild-type DT-40 cells. Our studies reveal an ion-channel independent role of IP3Rs in apoptosis, which may involve protein-protein interactions, possibly with the C-terminal tail of the receptor.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Pfu polymerase was from Stratagene (Madison, WI). Protogel-stabilized acrylamide solution was from National Diagnostic (Atlanta, GA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Amersham Biosciences. Enhanced chemiluminescent substrate was obtained from Pierce. Mouse anti-chicken IgM (clone-M4) was from Southern Biotech (Birmingham, AL). RPMI 1640 culture media and G418 sulfate (Geneticin) were from Mediatech (Herndon, VA). Staurosporine, cyclosporine, FK506, and CHAPS were purchased from Sigma. DEVD-7-aminomethyl coumarin was purchased from Alexis (San Diego, CA). For cell viability measurements we used the CellTitre 96 Aqueous One solution cell proliferation assay purchased from Promega (Madison, VI).

Antibodies—The Ab against the C terminus of the type-I IP3R (CT-1 Ab) has been described previously (25). The antibody was further affinity-purified using the peptide coupled to Ultralink beads as described by the manufacturer (Pierce). Anti-cytochrome c (Cyt c) mAb was from Zymed Labs (San Francisco, CA) and anti-HA mAb was purchased from Roche Applied Science. Anti-tubulin mAb was from Invitrogen-Molecular Probes (Eugene, OR).

DNA Constructs—The rat type I IP3R construct containing a Kozac sequence and subcloned into pcDNA 3.1 has been described previously (26). The splice variants used in this study were SI(-), SII(+), and SIII(-). The channel-only (aa 1892–2749) portion of IP3R was generated using PCR. The forward and reverse primers used were 5'-TCGAATTCCCACCATGCGGGATGCCCCATCCCGAAAG-3' and 5'-GCTTATGGTTTCTAGATTCGCG-3'. These primers encoded EcoR1/XbaI sites (underlined), which were utilized to clone the PCR product into similarly digested pcDNA 3.1 vector. The product was confirmed by automated sequencing (Nucleic acid Facility, Thomas Jefferson University, Philadelphia, PA). The S2681A and S2681E point mutants in full-length IP3R were constructed as described earlier (23). The S2681A, S2681E, and the D2550A pore mutation in full-length receptors were transferred to the channel-only IP3R construct using BstbI/XbaI restriction sites. Constructs encoding deletions in the C-terminal tail were generated as described by Schug and Joseph (27).

Cells and Stable Transfection—DT40 cells (wild-type and triple knock-out (TKO) of IP3R isoforms) were a kind Gift of Dr. T. Kurosaki (Kansai Medical University, Moriguchi, Japan). Stable DT40 cells expressing the rat type I IP3R were a gift from Dr. Kevin Foskett (University of Pennsylvania, PA). DT40 cells were grown in RPMI 1640 media supplemented with 10% fetal bovine serum, 1% chicken serum, and 100 units/ml penicillin, 100 µg/ml streptomycin, and maintained at 37 °C in 5% CO2 atmosphere. The stable cell lines expressing various IP3R mutants were prepared by electroporation of 0.5 ml of DT40 TKO cells (106 cells/ml) with 40 µg of XbaI linearized DNA using a Gene pulsar apparatus (Bio-Rad, 340 V and 950 microfarads). The cells were grown in 30 ml of RPMI for 24 h and were then serially diluted in a volume of 1:10, 1:100, and 1:1000 in RPMI containing 1.5 mg/ml G418. The diluted cells were transferred to 96-well plates and incubated for 1–2 weeks in a CO2-incubator at 37 °C. Positive clones were identified by screening for expression of IP3Rs by immunoblotting.

Measurement of Caspase Activation in DT40 Cells—For fluorometric assays, DT40 cells were collected and lysed in a buffer containing 20 mM Tris/Hepes, pH 7.4, 0.1% CHAPS, 5 mM EDTA. Lysates containing 10 µg of protein were incubated with 25 µM DEVD-aminomethyl coumarin as substrate for 30 min at 37 °C. Fluorescence of the aminomethyl coumarin product was measured at 380 nm excitation and 460 nm emission wavelength. Flow cytometry detection of caspase-3 was carried out according to manufacturer's instructions using a caspase-3 reagent (FLICA, Invitrogen-Molecular Probes). After apoptosis induction, the DT40 cells were analyzed on a Flow cytometer with 488 nm excitation using 530 nm band pass and 670 nm long pass emission filters.

Cell Viability Assays—The assay using CellTitre 96 Aqueous One reagent from Promega was done as described by the manufacturer with some modifications. Briefly, 0.5 x 106 DT40 cells were grown in 1 ml of media (in 24-well plates). The cells were treated with STS for 6 h, 20 µl of CellTitre reagent was added to each well, and the mixture was incubated for another 2 h. For a blank, 20 µl of CellTitre reagent was added to 1 ml of RPMI media. The supernatants were transferred in Eppendorf tubes and centrifuged on a microcentrifuge for 2 min. The supernatant was read at 490 nm against the media blank.


Figure 1
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  		FIGURE 1.
Domain organization and protein-protein associations in the C-terminal tail of IP3Rs. A, the domain organization of IP3Rs with the N-terminal binding domain, C-terminal channel domain, and intervening regulatory domain. The small region at the N terminus, which acts to suppress IP3 binding, and the predominant site of caspase-3 cleavage is also shown. B, shows in more detail the 159-amino acid, cytosol-exposed, C-terminal tail, which contains interaction sites for several proteins that are key regulators of apoptosis. CC, coiled-coil domain; TM6, transmembrane domain 6; PP-1{alpha}, protein phosphatase-1{alpha}; HAP1, huntingtin-associated protein-1; Httexp, huntington with a polyQ expansion. Other abbreviations are defined in the text. The location of several deletion and point mutations used in subsequent studies are also shown. The numbering of the amino acids corresponds to the rat type I IP3R isoform.

 
Measurement of [Ca2+]i in DT40 Cells—Changes in cytosolic [Ca2+] in individual DT40 cells plated on coverslips coated with poly-D-lysine was measured by digital imaging fluorescence microscopy as previously described (28). The cells were loaded with Fura-2 by incubating with 2 µM Fura-2AM in Hepes buffer, pH 7.4 (10 mM Hepes, 120 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 6 mM glucose, and 1 mM glutamine) for 20 min at room temperature. The coverslips were washed with the same buffer for 10 min to de-esterify Fura-2 and were transferred to a chamber with 1 ml of HEPES buffer and mounted onto the stage of an inverted microscope thermostatically maintained at 37 °C. Fluorescence images with a 4-s delay were recorded alternately at excitation wavelengths of 340 and 380 nm with an emission wavelength of 460–600 nm using a charge-coupled device imaging system. Changes in cytosolic calcium were measured in response to STS or anti-IgM in the presence or absence of extracellular calcium and are expressed as the ratio of Fura-2 fluorescence at 340 nm and 380 nm (F340/F380). Typically 50–60 cells were monitored per experiment, and traces represent the average of three different experiments.

Cyt c Release, Annexin V Assays, and Measurement of Mitochondrial Membrane Potential—Cyt c release into the cytosol was measured in DT40 cells after fractionation using the Apoalert cell fractionation kit (Clontech, Mountain View, CA). Western blotting of lysates was carried out on 15% SDS-PAGE gels with immunoblotting for Cyt c using a monoclonal Ab from Zymed Laboratories Inc. Annexin V fluorescein isothiocyanate conjugate (BioSource, Camarillo, CA) was added together with propidium iodide (5 µg/ml), and staining was analyzed on a flow cytometer. Changes in mitochondrial membrane potential were assessed using the MitoProbeTM DiIC1 (5) assay kit as described by the manufacturer (Invitrogen-Molecular Probes). The stain intensity decreases when reagents disrupt mitochondrial membrane potential. The cells were analyzed on a flow cytometer with 633 nm excitation using emission filters for far red (658 nm). Histograms were analyzed using WinMDI Software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
STS-induced Caspase-3 Activation and Cell Death in DT40 Cell Lines Containing Functional and Non-functional IP3Rs—DT40 cells containing a targeted deletion of all 3IP3R isoforms (TKO) were transfected with a number of IP3R mutants and stable cells lines were established. Fig. 1A shows the basic architecture of IP3 receptors with an N-terminal ligand-binding domain, a C-terminal channel domain, and an intervening regulatory domain. Fig. 1B shows a detail of the 159-amino acid C-terminal tail of the receptor that projects into the cytosol together with the binding sites for known proteins that interact with this region. The location of the deletion mutants (channel only, 1TM, tail-less) and point mutants (Asp-2550 and Ser-2618) used in this study are also indicated in the figure. Fig. 2A shows the expression of these mutant constructs detected by immunoblotting with an Ab to the C-terminal 18 amino acids of the type-I IP3R (CT-1) or, in the case of the tail-less mutants, with a C-terminal HA tag Ab. The mutants encoding the caspase-3-cleaved fragment, referred to as "channel-only" (CO) mutants (8), were expressed as a doublet of bands. These could correspond to glycosylated and non-glycosylated bands (cf. Refs. 21 and 29). In initial experiments we measured the time course of cell death induced by 0.5 µM STS in wild-type and TKO cells and found that the biggest differences were evident at earlier time points (<6 h) (data not shown). The time course of caspase-3 activation over this period is shown in Fig. 2B. In agreement with published data (8), caspase-3 activation was substantially slowed in TKO cells. However, 80% of the caspase-3 activation of wild-type cells could be rescued by the D2550A IP3R pore mutant, which has previously been shown to inactivate the channel (30). The absence of Ca2+ mobilization in response to cross-linking of cell surface IgM receptors with anti-IgM Ab in D2550A cells is shown in Fig. 2B (inset). Several other studies have confirmed that this mutant is functionally inactive (31–33). Data on caspase-3 activation in several additional mutant cell lines is summarized in Fig. 2C and corresponding measurements of cell viability are shown in Fig. 2D. The wild-type DT40 cells contain three chicken IP3R isoforms, whereas the mutant cells contain only one IP3R isoform made in the background of the rat type-I IP3R. For comparison, we have also used a stable DT40 cell line expressing the wild-type rat type-I IP3R as a control (kindly given by Kevin Foskett). These cells showed a somewhat higher caspase-3 activation, but their cell death response was the same as wild-type DT40 cells.


Figure 2
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  		FIGURE 2.
STS-induced changes in caspase-3 activity and cell viability in mutant IP3R DT40 cell lines. A, immunoblots of cell lysates prepared from the indicated DT40 cell lines. The reason why the CO constructs express as a doublet is not known but may be due to differential glycosylation. B, a time course of caspase-3 activation in the indicated DT40 cell lines in response to 0.5 µM STS measured fluorometrically in cell lysates. The inset shows cytosolic free Ca2+ changes measured in WT and D2550A Fura-2-loaded cells in response to 5 µg/ml {alpha}-IgM. C, the cumulative data of caspase-3 activation in response to 0.5 µM STS for 6 h measured in the indicated cell lines and expressed relative to the activation in WT cells. Data are mean ± S.E. (n = 3–6). *, p < 0.05 relative to WT cells. In Panel D, conditions were as described in C except that cell viability was measured with a Promega 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay kit.

 
Two non-functional IP3R mutants have previously been shown to support STS and anti-IgM induced apoptosis in DT40 cells (8). One of these mutants was deleted in the N-terminal 225 amino acids corresponding to the suppressor domain (Fig. 1A). The other was the CO mutant encoding the C-terminal fragment formed from the receptor after caspase-3 cleavage. The results were interpreted as indicating that the caspase-3-cleaved fragment from both inactive mutants could still induce aCa2+ leak from the ER that was necessary for sustaining apoptosis (8). In agreement with this previous study, the cells expressing the CO mutant were sensitive to STS-induced cell death (Fig. 2, C and D). However, the same result was obtained with the CO mutant encoding the pore inactivating D2550A mutant. It is possible that the CO mutant when functioning in an ER Ca2+ leak pathway has completely different pore architecture than a full-length wild-type receptor. Nevertheless, the data also raise the possibility that full-length receptors (or their caspase-3-cleaved fragments) do not contribute directly to ER Ca2+ leak pathways and that they have some other role in regulating apoptosis.

The 1TM construct (amino acids 2253–2749) encodes the segment of the receptor that contains the C-terminal channel domain. This construct was also able to support STS-induced caspase-3 activation and cell death (Fig. 2, C and D) indicating that the large segment of the receptor from the caspase-3 cleavage site at amino acids 1892–2253 is not required for apoptosis. However, deletion of 141 or 159 amino acids of the C-terminal cytosolic tail from full-length receptors was sufficient to suppress caspase-3 activation and cell death to the levels observed in TKO cells (Fig. 2, C and D). In independent experiments we have established that the TL-5 and TL-6 mutants are functionally inactive as IP3-gated channels (27). Because channel-inactive mutants (e.g. D2550A or CO) can still support cell death, this implies that the C-terminal tail has a unique function in regulating apoptosis induced by STS. Similar findings were made when using anti-IgM as an inducer of apoptosis, notably, caspase-3 activation was supported by the D2550A pore-inactive full-length IP3R or the D2550A CO mutant but not the IP3R lacking the C-terminal tail (supplemental Fig. S1).

We have previously shown that IP3Rs are phosphorylated by Akt kinase in the C-terminal tail at serine 2618 without altering IP3-dependent channel activity (23). The functional role of this phosphorylation is unknown. However, we noted that the ability of IP3Rs to support STS-induced caspase-3 activation was enhanced when the Akt phosphorylation site was inactivated by mutation to alanine (S2618A) as compared with a phosphomimic mutant (S2618E). It was suggested that perhaps important functional responses to Akt kinase phosphorylation could be expressed after the receptor had been cleaved by caspase-3 (23). However, the data in Fig. 2 (C and D) show that the CO construct containing the S2618A or S2618E mutant did not show significant differences in their ability to support STS-induced caspase-3 activation or cell death, suggesting that the functional effects of Akt kinase phosphorylation are exerted at the level of full-length receptors.

Cyt c Release and Mitochondrial Membrane Potential Depolarization in IP3R-defective DT40 Cell Lines—The release of Cyt c into the cytosol is a mandatory step in most models of apoptosis activated by the intrinsic pathway. We therefore measured Cyt c release in DT40 cell lines containing wild-type and mutant IP3Rs employing a crude subcellular fractionation procedure and using immunoblotting for Cyt c detection. Surprisingly, the TKO DT40 cell line released Cyt c into the cytosol with indistinguishable kinetics from the wild-type DT40 cells (Figs. 3, A and B). Although some variability was observed in the amount of basal Cyt c released, all the nonfunctional IP3R mutant cell lines also released Cyt c in response to STS. This included the D2550A, TL-5, and TL-6 mutants (Fig. 3C). A loss of mitochondrial membrane potential invariably accompanies Cyt c release (34). We measured membrane potential using a fluorescent cationic carbocyanine dye, and the data are shown in Fig. 4. In agreement with our data on Cyt c release, both the wild-type and TKO cells showed a mitochondrial depolarization response when treated with STS. With the exception of TL-6, which showed a smaller response, all the non-functional IP3R mutants displayed a response that was not statistically significant from the wild-type DT40 cells. The reason for the unique membrane potential response of the TL-6 mutant is unknown and was not investigated further. The finding that TL-6 DT40 cells did not show a smaller Cyt c release could indicate that the two parameters do not have a quantitatively linear relationship and/or that the two processes are not necessarily tightly coupled (35, 36).


Figure 3
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  		FIGURE 3.
STS-induced Cyt c release. A, WT and TKO DT40 cells were treated with 0. 5 µM STS for various time periods and were fractionated as described under "Experimental Procedures." 10 µg of protein was electrophoresed on a 15% SDS-PAGE gel, transferred to nitrocellulose, and immunoblotted with monoclonal anti-Cyt c antibody. The data shown are from a representative experiment. B, cumulative data (mean ± S.E.) showing the densitometric quantitation of blots from three experiments as described in A. C, representative Western blots of Cyt c release from various mutant DT40 cells were carried out as described in A, with and without treatment with 0.5 µM STS for 6 h. The nitrocellulose membranes were subsequently stripped and probed for tubulin as a loading control.

 
Ca2+ Changes Mediated by STS in Wild-type and Mutant IP3R Cell Lines—To explore the Ca2+ dependence of apoptosis induced by STS we initially measured cytosolic [Ca2+] changes in Fura-2-loaded DT40 cells. STS elicited a Ca2+ signal (Fig. 5A), which is in agreement with a previous study in DT40 cells (8), and observations in other cell types (22). In the presence of extracellular Ca2+ the wild-type DT40 cells gave the largest Ca2+ response, the TL-6 and TKO cells generated much smaller responses, and the TL-5 and pore mutant cell line produced an intermediate-sized Ca2+ signal (Fig. 5B). When extracellular Ca2+ was removed the STS response of wild-type cells was partially inhibited indicating that a component of the Ca2+ signal originated from intracellular Ca2+ release in these cells. However, in the absence of extracellular Ca2+ the response of the pore-mutant, TKO, TL-5 and TL-6 cells were almost eliminated (Fig. 5C). The data suggest that the Ca2+ signal seen in response to STS in the pore-defective IP3R mutant is entirely derived from Ca2+ entry across the plasma membrane. A maintained coupling of the pore-defective IP3R to Ca2+ entry could explain the residual apoptosis seen in the pore-defective DT40 cells. The dependence of STS-induced caspase-3 activation on extracellular Ca2+ chelation in selected cell lines that maintain high (wild-type, pore-mutant, CO) and low (TKO cells) caspase-3 activation is shown in Fig. 5D. Chelation of extracellular Ca2+ had a partial inhibitory effect (40%) on caspase-3 activation induced by STS in wild-type cells, which is in agreement with previous findings in DT40 cells in which apoptosis was quantified in a different manner (37). By contrast extracellular Ca2+ chelation inhibited caspase-3 activation in the pore-mutant by 80%. This is consistent with our hypothesis that the pore-defective cell line relies to a greater extent on extracellular Ca2+ to supply the Ca2+ required for apoptosis. The CO cell line behaved similarly to wild-type cells, although the inhibitory effect of extracellular Ca2+ chelation was somewhat larger (60%). The blockade of intracellular Ca2+ mobilization (and coupled Ca2+ entry processes) with BAPTA-AM totally suppressed caspase-3 activation in all cell lines.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Many studies using different experimental models have concluded that IP3Rs enhance the rate of apoptosis (reviewed in Ref. 14). The key conclusion that emerges from the present study is that IP3Rs do not necessarily have to function as intracellular Ca2+ release channels to fulfill this role. This conclusion is based on studies with a DT40 cell line containing defective IP3R mutants and using STS as the apoptosis-inducing stimulus. The results do not imply that intracellular Ca2+ release from functional IP3Rs have no role in apoptosis induced in wild-type cells. STS-induced caspase-3 activation in both wild-type and pore-defective mutant cells lines is Ca2+-dependent, as shown by its blockade by BAPTA loading in both types of cells (Fig. 5D). STS induces an increase of cytosolic Ca2+ in the pore-mutant cells, but this increase is entirely prevented by chelation of extracellular Ca2+ (Fig. 5C). Therefore, we suggest that the Ca2+ requirements for apoptosis in the pore-mutant cells is met exclusively by Ca2+ entry across the plasma membrane, whereas the wild-type cells could potentially use both intracellular Ca2+ release or Ca2+ entry mechanisms. Van Rossum et al. (31) have shown that agonist-mediated Ca2+ entry can be activated in DT40 cells containing the D2550A IP3R pore-defective mutant and have suggested that IP3 binding to a pore-defective channel is sufficient to activate a Ca2+ entry mechanism in the plasma membrane. The activation of this type of Ca2+ entry mechanism by STS could account for the continued caspase-3 activation and cell death responses observed in the pore-defective mutant cells. The exact Ca2+ entry channels involved during apoptosis of DT40 cells are not known. Vasquez et al. (38) have shown that IP3R expression is required to measure the activity of endogenous TRPC7 channels in isolated patches derived from DT40 cells. However, the necessity for IP3R channel function for this effect was not tested. There have also been reports of IP3Rs operating directly as Ca2+ entry channels in the plasma membrane of DT40 cells, but these are unlikely to be involved in the apoptotic responses being examined here, because these channels are inactivated by pore mutations (33). Interestingly, functionally inactive mutant ryanodine receptor channels also remain coupled to a Ca2+ entry pathway in dyspedic myotubes (39), and the presence of ryanodine receptors has been documented in DT40 cells (40). A possible role of ryanodine receptors in coupling to apoptotic pathways in IP3R-inactive DT40 cells lines remains to be explored.


Figure 4
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  		FIGURE 4.
Measurements of mitochondrial membrane potential depolarization. A, DT40 cells were incubated with STS (0. 5 µM) for 6 h to induce apoptosis. 50 nM Mitoprobe (DiIC1 (5)) was added for 15 min. The cells were centrifuged and washed twice in PBS before analysis on a flow cytometer as described under "Experimental Procedures." The first panel shows complete mitochondrial uncoupling induced by 0.5 µM CCCP for 5 min. The second and third panels show the data for WT and TKO cells treated with STS. B, the number of cells having fluorescence values in the range observed for carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treated cells was considered to have depolarized mitochondria. This was quantitated from the flow cytometry data in three experiments (mean ± S.E.) for the various DT40 cell lines after treatment with 0.5 µM STS for 6 h. *, p < 0.05; significantly different from WT DT40 cells.

 
Assefa et al. (8) reported that a non-functional IP3R mutant missing 225 amino acids from the N terminus could still support STS or anti-IgM-induced apoptosis in DT40 cells. Because this mutant can be cleaved by caspase-3 to generate the CO fragment, the result was interpreted as indicating an important role for this fragment in supplying the Ca2+ required for apoptosis. We found no evidence that the CO cell lines encoded constitutively leaky ER channels based on measurements of thapsigargin-releasable Ca2+ (data not shown). Furthermore, a CO domain containing the inactivating pore mutation was equally effective in supporting STS-induced apoptosis (Fig. 2). This suggests that the IP3R CO fragment is unlikely to be directly involved in promoting an ER Ca2+ leak, but does not exclude the possibility that the proteolytic fragment could regulate a separate leak mechanism. Our data differ in one additional respect from the study of Assefa et al. (8). This study noted that STS induced an increase of cytosolic Ca2+ in both wild-type cells and non-functional IP3R 1–225 deletion mutant cells. In both types of cells these increases were derived from intracellular Ca2+ release and were blocked by pre-treatment with the general caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone (8), consistent with the idea that the Ca2+ changes are secondary to the generation of the CO fragment. In our experiments, the STS-induced increase in cytosolic Ca2+ seen in the pore-defective mutant cells was derived exclusively from extracellular Ca2+ and was not blocked by the specific caspase-3 inhibitor DEVD-CHO at concentrations that inhibit STS-induced caspase-3 activation (data not shown). The reasons for these discrepancies are presently not clear, although the use of different non-functional IP3R mutants and other methodological differences may be involved. Further work is necessary to clarify the issue of whether the caspase-3 cleavage fragment of type-I IP3Rs plays an important primary role in apoptotic signaling.

The release of Cyt c from the mitochondrial intermembrane space is a key step involved in the downstream activation of effector caspases. Although, the exact mitochondrial mechanism involved in Cyt c release during apoptosis is controversial, it is generally accepted that the process is stimulated by elevations of cytosolic Ca2+ in the local environment of the mitochondria (3, 34). Such an environment would be provided at the open mouth of either IP3R or Ca2+ influx channels (41, 42). The finding that Cyt c binds to the C-terminal tail of IP3Rs and prevents Ca2+ inhibition of the channel has suggested an additional amplification mechanism by which IP3R channels could potentiate Cyt c release (22, 24). However, our studies show that STS-induced Cyt c release and mitochondrial membrane depolarization occur in TKO cells, as well as in cell lines containing non-functional IP3Rs. Indeed, the kinetics of Cyt c release is not markedly different between STS-stimulated wild-type and TKO cells. Cyt c release has previously been observed in TKO cells stimulated with anti-IgM (22). This suggests that IP3Rs are not essential to elicit or sustain Cyt c release in DT40 cells. In the absence of IP3Rs, even small local elevations of cytosolic Ca2+ induced by Ca2+ entry may be sufficient to trigger mitochondrial membrane depolarization and Cyt c release. Interestingly, recent studies have shown that Ca2+ entry may actually direct the movement of mitochondria to the cell periphery in Jurkat T-lymphocytes (43).


Figure 5
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  		FIGURE 5.
Cytosolic calcium changes induced by staurosporine. A, WT DT40 cells were incubated with the indicated concentrations of STS in the presence of 2. 5 mM extracellular Ca2+. Cytosolic free Ca2+ was measured in Fura-2-loaded cells as described under "Experimental Procedures." The averaged STS (1 µM) responses of the indicated cell lines in the presence of extracellular Ca2+ (B) and in the absence of extracellular Ca2+ (2.5 mM EGTA added 1 min prior to STS; C) are shown. D, the indicated DT40 cell lines were incubated with 0.5 µM STS for 6 h in the presence of 2.5 mM EGTA to chelate extracellular Ca2+ or were pre-loaded with 10 µM BAPTA-AM for 30 min prior to STS stimulation. Caspase-3 activity was measured fluorometrically in cell lysates.

 
Our data suggest that the impaired caspase-3 activation seen in TKO and tail-less IP3R mutant cell lines occurs at step(s) distal to mitochondrial membrane potential depolarization and Cyt c release. The nature of this site and how it is regulated by IP3Rs remains a matter of speculation. Our working hypothesis is that IP3Rs serve at least two critical functions in the apoptotic pathway. The first is to provide elevated cytosolic Ca2+ either directly from intracellular stores and/or by coupling to Ca2+ entry pathways. The latter mechanism obviously predominates in cell lines expressing only non-functional IP3Rs. A second role appears to be intimately linked to the activation mechanism of caspase-3 itself. There are no indications in the literature that physiological increases of Ca2+ regulate key steps in caspase activation, such as apoptosome assembly or the catalytic activity of caspases. The experimental data would also suggest that Ca2+ elevation alone is not the primary regulatory factor, because the TL-5 and TL-6 mutant cell lines have different STS-mediated Ca2+ signals but are both deficient in caspase-3 activation (Fig. 2). Removal of the C-terminal tail suppresses caspase-3 activation induced by STS and is known to interact with a number of proteins, including Cyt c (24), Bcl-XL (16), and 4.1N (44). The C-terminal tail also contains the phosphorylation site for Akt kinase, and mutations of this site also modulate caspase-3 activity in DT-40 cells (23). One hypothesis is that this portion of the IP3R represents a major binding site for a protein that regulates caspase-3 activation in the cytosol. Loss of this binding site would alter the partition of this regulator between ER membranes and the cytosol. Preliminary studies indicate that a deletion of 60 amino acids, which removes the coiled-coil domain and putative binding sites for Bcl-2 (Fig. 1), produces cells that retain wild-type caspase-3 responses to STS (data not shown). Additional mutagenesis to localize the required regions of the C-terminal tail is presently being carried out. Finally, it should be noted that there are precedents for membrane-bound regulators of cytosolic caspases in the literature, including the proteins BRUCE/Apollon (45) and the integral ER protein ARMER, which inhibit the catalytic activity of caspase-9 (46).

Our studies lend weight to the argument that IP3Rs may do more than act as Ca2+ channels in cells (47, 48). More than 50 different proteins have been documented to interact with IP3Rs (47, 49), suggesting that there may be considerable heterogeneity in the types of IP3R complexes present in intact cells. Mutant DT40 cell lines containing only non-functional IP3Rs may survive by "rewiring" their Ca2+ transport pathways to supply their biological requirements. We suggest that even wild-type cells may contain pools of inactive IP3R complexes that subserve specific functions, such as coupling to Ca2+ entry pathways and regulation of caspase-3 activity. Further studies on the role of non-functional IP3R complexes may lead to pharmacological approaches that allow a more selective approach to interfering with the biological effects regulated by IP3Rs.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants R01-DK34804 and R29-NS051822 (to S. K. J.) and Training Grant T32-AA07463 (to Z. T. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 To whom correspondence should be addressed: Dept. of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 1020 Locust St., JAH 230A, Philadelphia, PA 19107. Tel.: 215-503-1222; Fax: 215-923-6813; E-mail: suresh.joseph{at}mail.tju.edu.

2 The abbreviations used are: IP3R, inositol 1,4,5-trisphosphate receptor; ER, endoplasmic reticulum; CHAPS, (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; Cyt c, cytochrome c; aa, amino acid(s); Ab, antibody; mAb, monoclonal antibody; STS, staurosporine; HA, hemagglutinin; TKO, triple knock-out; CO, channel-only; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester). Back


   ACKNOWLEDGMENTS
 
We thank Drs. Gyorgy Hajnoczky and Darren Boehning for reading draft versions of the manuscript.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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Z. T. Schug, P. C. A. da Fonseca, C. D. Bhanumathy, L. Wagner II, X. Zhang, B. Bailey, E. P. Morris, D. I. Yule, and S. K. Joseph
Molecular Characterization of the Inositol 1,4,5-Trisphosphate Receptor Pore-forming Segment
J. Biol. Chem., February 1, 2008; 283(5): 2939 - 2948.
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