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Originally published In Press as doi:10.1074/jbc.M500800200 on April 4, 2005

J. Biol. Chem., Vol. 280, Issue 25, 23829-23836, June 24, 2005
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BH3-only BIK Regulates BAX,BAK-dependent Release of Ca2+ from Endoplasmic Reticulum Stores and Mitochondrial Apoptosis during Stress-induced Cell Death*

Jaigi P. Mathai{ddagger}§, Marc Germain{ddagger}§, and Gordon C. Shore{ddagger}||

From the {ddagger}Department of Biochemistry and McGill Cancer Center, McGill University, Montreal, Quebec H3G 146, Canada

Received for publication, January 21, 2005 , and in revised form, March 30, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
BIK, a pro-apoptotic BH3-only member of the BCL-2 family, targets the membrane of the endoplasmic reticulum (ER). It is induced in human cells in response to several stress stimuli, including genotoxic stress (radiation, doxorubicin) and overexpression of E1A or p53 but not by ER stress pathways resulting from protein malfolding. BIK initiates an early release of Ca2+ from ER upstream of the activation of effector caspases. Release of the mobile ER Ca2+ stores in baby mouse kidney cells doubly deficient in BAX and BAK, on the other hand, is resistant to BIK but is sensitive to ectopic BAK. Over-expression of p53 stimulates recruitment of BAK to the ER, and both its recruitment and assembly into higher order structures is inhibited by BIK small interfering RNA. Employing small interfering RNA knockdowns, we also demonstrated that release of ER Ca2+ and mitochondrial apoptosis in human epithelial cells requires BIK and that a Ca2+-regulated target, the dynamin-related GTPase DRP1, is involved in p53-induced mitochondrial fission and release of cytochrome c to the cytosol. Endogenous cellular BIK, therefore, regulates a BAX,BAK-dependent ER pathway that contributes to mitochondrial apoptosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Utilizing a DNA microarray analysis of genes that are stimulated by the oncogenic E1A protein of adenovirus, we previously identified BH3-only BIK as a strong responder in human KB epithelial cells. E1A is a potent inducer of both BIK protein and apoptosis in human epithelial cells, dependent on its ability to up-regulate the levels of p53 (1). Moreover, overexpression of p53 in p53-null lung H1299 cells also induced BIK mRNA and protein with kinetics very similar to the induction of p21WAF1, which is a rapid response protein in this pathway. Additionally, BIK is induced in estrogen-dependent MCF7 breast cancer cells in response to inhibition of estrogen signaling (2), and induction of BIK contributes to the apoptotic selection of mature B lymphocytes (3). The fact that BIK is primarily regulated through induction of BIK protein is consistent with studies indicating that BIK is a constitutively active proapoptotic protein.

The murine ortholog of BIK, Blk, is largely restricted to hematopoietic and endothelial cells and, in contrast to BIK, is not induced by genotoxic stress (4). Moreover gene deletion had little if any effect on the sensitivity of murine cells to genotoxic stress, and animals developed normally (4). In contrast to most BH3-only proteins in mouse and man, which exhibit a high degree of amino acid sequence identity (5, 6), the human and mouse orthologs of BIK are only 42.5% identical, despite having very similar gene structures (7, 8). Consistent with the findings reported by Coultas et al. (4), we also have found no evidence that Blk mRNA or protein is induced by either genotoxic stress or p53 overexpression in a variety of mouse cell lines and primary cell cultures.1 Remarkably, therefore, murine Blk and human BIK respond differently to stress stimuli. Consistent with the findings that human BIK may contribute to tumor suppression, there is reported evidence that mutation of the BIK gene is a frequent feature of B-cell lymphomas (9), and the chromatin locus 22q13.3, which contains BIK, exhibits deletions in human breast and colorectal cancers (10). To better understand the contribution of BIK induction to apoptosis in human epithelial cells, we utilized BIK RNA interference.

BH3-only BIK interacts with the multi-BH domain anti-apoptotic members of the BCL-2 family but not with pro-apoptotic BAX and BAK (11–13). It contains a single transmembrane segment at its extreme COOH terminus, but in contrast to most BH3-only proteins, which target mitochondria, BIK is integrated almost exclusively in the membrane of the endoplasmic reticulum (ER)2 (1, 14). Although other members of the BCL-2 family, including anti-apoptotic BCL-2 itself and the multidomain BAX and BAK pro-apoptotic effector molecules, also target the ER (reviewed in Ref. 33), the role of the ER in supporting the mitochondrial apoptosis pathway is only now beginning to emerge (15–17). In the Fas death pathway, for example, cleavage of BAP31 at the ER membrane causes an early release of ER Ca2+ stores and concomitant uptake of Ca2+ by mitochondria, which triggers the recruitment of a dynamin-related GTPase, DRP1, to the organelle surface followed by mitochondrial fission (18). DRP1 is responsible for scission of the outer membrane during normal mitochondrial fission and fusion, and in the absence of fusion, converts the tubular "worm-like" network of steady-state mitochondria into punctiform fragments (19, 20). Such DRP1-dependent mitochondrial fragmentation is an early event in several apoptotic pathways (21), and in these pathways, DRP1 appears to be necessary for effective egress of cytochrome c from the organelle to the cytosol (15, 21). Cytoplasmic cytochrome c, in turn, becomes a critical constituent of the apoptosome, which processes and activates effector procaspases (22, 23).



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  		FIG. 1.
Induction of BIK mRNA and protein expression. A, BIK is induced by the oncogene E1A. H1299 lung carcinoma and KB oral epithelial cell lines stably expressing or not expressing HA-BCL-2 were infected for the indicated periods of time with either Ad p53 or Ad E1A vectors. Expression of the indicated genes was determined by Northern blot analysis using corresponding cDNA probes (see "Experimental Procedures"). The bands corresponding to 26 and 18 S ribosomal RNA are indicated and provide gel loading controls. B, activation of endogenous p53 results in BIK expression. KB cell lines expressing HA-BCL-2 were exposed to 25 gray of {gamma} radiation or treated with 0.4 µg/ml doxorubicin (Dox). The cells were harvested at the indicated times and BIK expression analyzed by Western blot. C, ER stress does not induce BIK expression. Protein levels of binding protein, BIK, and actin from H1299 cells lines stably expressing HA-BCL-2, treated with either 2 µM thapsigargin or infected with Ad p53 for the indicated times. Gy, gray; wt, wild type; BiP, binding protein.

 
Recent work has established that BAX,BAK regulates ER Ca2+ homeostasis (24, 25). Here we employed an adenovirus containing p53 as a tool to induce endogenous BIK in p53-null H1299 human lung epithelial cells. Utilizing BIK siRNAs, we demonstrated that BIK induction in this system is required to initiate early release of Ca2+ from the ER, mitochondrial fragmentation, and activation of the mitochondrial cytochrome c release pathway. BIK initiates the release of Ca2+ from ER stores by a pathway dependent on BAX,BAK.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Cell Culture, Plasmids, and Reagents—Stable human KB oral epithelial and H1299 lung carcinoma cell lines, either expressing or not expressing ectopic HA-BCL-2 (26), were cultured in {alpha}-minimal essential medium supplemented with 10% fetal bovine serum and 100 mg/ml streptomycin and penicillin. Transformed baby mouse kidney epithelial cells and baby mouse kidney cells derived from BAX,BAK doubly deficient (DKO) mice (36) were cultured in {alpha}-minimal essential medium supplemented with 10% fetal bovine serum and 100 mg/ml streptomycin and penicillin.

Plasmids encoding CFP fused to the NH2 terminus of DRP1(K38E) were gifts from H. McBride (Ottawa Heart institute, Ottawa, Ontario, Canada). pGL3-CMV and pRL-CMV plasmids were from Promega. Carbobenzoxy-valvy-alanyl-aspartylmethyl ester-fluoromethyl ketone (Z-VAD-fmk) was purchased from Enzyme System Products, Fura-2/AM was from Calbiochem, and doxorubicin and thapsigargin were purchased from Sigma. All transfections were performed using LipofectamineTM Plus (Invitrogen) according to the manufacturer's protocols.

Cloning of NOXA cDNA and Northern Blots—NOXA cDNA was cloned as described in Ref. 1 utilizing the sequences deposited in Gen-BankTM (accession number D90070 [GenBank] ). The primers used for the cloning of NOXA were 5'-TTGGATCCCTCCAGTTGGAGGCTGAGGTT-3' and 5'-CGGAATTCCTTGAAGGAGTCCCCTCATGC-3'. Northern blots were performed as described in Ref. 1 using 30 µg of total RNA extracted from H1299 cells or KB cells, either expressing or not expressing ectopic HA-BCL-2.

RNA Interference of BIK and Viral Infection—The following siRNA duplexes, with a 3'-end dTT overhang and corresponding to two separate regions within the BIK RNA sequence, were purchased from Dharmacon Research (Lafayette, CO) (numbers are in relation to the start site nucleotide for translation): siRNA BIK145, 5'-AUGCAUGGAGGGCAGUGAC-3'; siRNA BIK315 5'-GUUUCAUGGACGGUUUCAC-3'. Double-stranded siRNA duplex 5'-CUUACGCUGAGUACUUCGA-3' with a 3'-end dTT overhang corresponding to a region within the luciferase gene of the pGL3 plasmid (designated siRNA-LUC) was also purchased for use as a control. The final concentration of siRNA used/transfection was 60 nM. Adenoviral infection of cells was performed ~12 h after transfection with siRNA as described previously (27), using 100 plaque-forming units/cell of virus.

Antibodies, Immunoblots, Immunofluorescence, and Microscopy— The following antibodies were utilized: goat anti-BIK (Santa-Cruz Biotechnology), mouse anti-actin (ICN Biomedical), rabbit anti-TOM20 (described in Ref. 42), monoclonal anti-p53 (Pharmingen), rabbit anti-calnexin and rabbit anti-binding protein (gift from J. Bergeron), mouse anti-cytochrome c (Pharmingen), rabbit anti-BAX (Santa-Cruz Biotechnology), and monoclonal anti-BAK (Oncogene Research Products). SDS-PAGE of whole cell lysates, transfer of proteins to nitrocellulose filters, development of blots with antibodies, and detection by enhanced chemiluminescence have been documented in earlier publications (1, 15). For immunofluorescence, cells were plated onto coverslips at ~50% confluency for transfection and adenoviral infection. After the indicated infection times, the cells were treated and visualized as previously described (15). In experiments for Fig. 5C, all cells were treated with 5 µM nocodozol for 20 min prior to PFA fixing to aid in the visualization of fission events (28).

Luciferase Assays—The firefly luciferase vector pGL3-CMV was transfected with Renilla luciferase vector pRL along with siRNA-LUC using Lipofectamine Plus according to the manufacturer's protocol. The cells well harvested 24 h later and lysates assessed for luciferase activity using a Lumat LB 9507 luminometer and the Dual Luciferase reporter assay system (Promega) according to the manufacturer's instruction.

Ca2+ Measurements—Thapsigargin-releasable ER calcium was calculated as the difference in cytoplasmic calcium measured before and after the addition of 2 µM thapsigargin to cells in Ca2+-free buffer (15, 29). In brief, 2 x 106 cells were harvested and washed in Ca2+-free buffer (20 mM HEPES, pH 7.4, 143 mM NaCl, 6 mM KCl, 1 mM MgSO4, 0.1% glucose, 0.1% bovine serum albumin, 250 mM sulfipyrazone). The cells were resuspended in 200 µl of calcium-free buffer containing 0.02% pluronic acid and subsequently loaded with the cell-permeable fluorescent indicator Fura-2/AM at 3 mM for 30 min at 37 C. After a final wash, the cells were resuspended in Ca2+-free buffer and a 340/380-nm excitation ratio at a 510-nm emission wavelength were obtained using a LS 50B PerkinElmer Life Sciences luminescence spectrophotometer. For Fig. 6, the cells were grown and treated on poly-L-lysine-treated coverslips and loaded by adding 100 µM Fura-2/AM to the culture medium for 30 min. Coverslips were washed with Hanks' buffer (3 mM Na2HPO4, 5.4 mM KCl, 0.4 mM KH2PO4, 1.3 mM CaCl2, 0.5 mM MgCl2, 0.8 mM MgSO4, 5 mM HEPES, 10 mM glucose, 137 mM NaCl, 4.2 mM NaHCO3) followed by two washes with Ca2+-free Hanks' Buffer (3 mM Na2HPO4, 5.4 mM KCl, 0.4 mM KH2PO4, 0.68 mM NaCl, 0.5 mM MgCl2, 0.8 mM MgSO4, 5 mM HEPES, 10 mM glucose, 137 mM NaCl, 4.2 mM NaHCO3). Images were obtained as previously described (30) using an intensified charge-coupled device camera (IC200) and PTI (Photon Technology International Inc., Princeton, NJ) software at a single emission wavelength (510 nm) with a double excitatory wavelength (340 and 380 nm). Fluorescence ratio (340/380) was measured in cells treated with 2 µM thapsigargin and the Fura-2 ratio values converted to [Ca2+] according to the formula of Grynkiewicz et al. (31). The peak thapsigargin-releasable [Ca2+]cyto was calculated as the difference in cytoplasmic calcium measured before and after the addition of 2 µM thapsigargin to cells in Ca2+-free Hanks' buffer.



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  		FIG. 2.
siRNA-BIK315 specifically inhibits BIK expression. A, H1299 cells were transfected with an expression plasmid containing HA-BIK L61G along with siRNAs as indicated. Cells were harvested after 24 h and total cell lysates analyzed by SDS-PAGE and immunoblotting. B, the plasmids pGL3-CMV and pRL-CMV (internal control) containing different luciferase reporter genes were co-transfected into H1299 cells along with siRNA-LUC or siRNA-BIK315. The cells were collected after 24 h and luciferase activity measured. Shown are mean ± S.D. of three independent experiments. C, H1299 cells were transfected with the indicated siRNAs and infected with either Ad p53 or control Ad rtTa. The cell lysates were collected and analyzed for BIK expression by SDS-PAGE and immunoblotting using the indicated antibodies. LUC, luciferase.

 

   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
BIK Expression Is Induced by Oncogenic and Genotoxic but Not ER Stress—We have previously shown that BIK is induced by adenovirus E1A in a p53-dependent manner. The resulting BIK protein accumulates to especially high levels in cells expressing BCL-2, because BIK is induced upstream of BCL-2 and does not decay in these BCL-2-protected cells (1). In Fig. 1A, expression of BIK mRNA, together with that of another p53-inducible gene product, NOXA, was assessed by Northern blots of total RNA following infection of KB epithelial cells (p53 wild type) with Ad E1A, which elicits a strong pro-apoptotic stress stimulus. The adenoviral vector Ad5 dl520E1B was used for this purpose (32), which delivers only the pro-apoptotic 243-amino acid E1A12S oncoprotein, with no E1B products, which are protective against cell death agonists. Pro-apoptotic cell stress can also be initiated by overexpressing p53 itself in p53-null cells (1). For reference, the p53–/–human lung carcinoma cell line H1299 was infected with an adenoviral vector encoding wild-type human p53 (Ad p53). BIK mRNA was undetectable prior to delivery of Ad E1A or Ad p53 (time 0, Fig. 1A). The subsequent increase of BIK mRNA in response to these inducers, however, was robust. In contrast to BIK protein levels (1), BCL-2 did not strongly influence BIK mRNA levels. Because BIK protein is induced by E1A in a p53-dependent manner (1), we also examined stimuli that up-regulate endogenous p53 in KB cells. As shown in Fig. 1B, genotoxic damage conferred by exposure of the cells to 25 gray of {gamma} radiation or treatment with 0.4 µg/ml topoisomerase inhibitor doxorubicin also stimulated BIK protein induction in parallel with the accumulation of p53. Because BIK is strongly concentrated at the ER from where it is able to exert its pro-apoptotic function independent of a mitochondrial association (1, 14, 33), we also sought to determine whether BIK induction might occur in response to ER stress stimuli. To that end, we treated H1299 cells overexpressing BCL-2 with either Ad p53 or the ER stressor thapsigargin for the indicated times. Thapsigargin inhibits the sarcoplasmic/endoplasmic reticulum calcium ATPase pump, thereby preventing normal Ca2+ uptake into the ER from the cytosol and causing depletion of releasable ER Ca2+ by passive leak. Over time, this leads to induction of unfolded protein response proteins, such as the chaperone binding protein (Fig. 1C) and ultimately apoptosis. Even after 48 h, however, no evidence of BIK induction was observed despite the observed induction of binding protein by 24 h. Similar observations were made with another ER stressor, tunicamycin, and with different cell lines. Moreover, the times at which the presence of BIK was examined overlapped with the appearance of dying cells (data not shown). BIK regulation, therefore, is not sensitive to ER stress.



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  		FIG. 3.
BIK knockdown prevents p53-induced morphological changes and caspase activation. A, time course of BIK induction by p53. H1299 cells were infected with Ad p53 for the indicated times, and the expression of BIK and p53 protein were assessed by Western blot analysis of cell lysates. B, H1299 cells were transfected with either siRNA-BIK315 or siRNA-LUC, followed by infection with Ad p53 or control Ad rtTa for 16 h. Cells were visualized by phase contrast light microscopy. C, the detached cells from the culture medium in B were collected and counted. The remaining adherent cells were trypsinized, counted, and the percentage of detached cells from the total was calculated. Shown is a representative of five independent experiments. D, as in B, caspase-3 like protease activity was measured by the ability of cell lysates to hydrolyze the fluorogenic caspase substrate DEVD-7-amino-4-methylcoumarin. Data presented are means ± S.D. for three independent experiments and are expressed as the fold increase in DEVDase activity compared with mock-transfected rtTa-infected cells. Cell extracts from p53-infected cells were analyzed by Western blotting to assess the extent of BIK knockdown (gel insert). LUC, luciferase; RFU, relative fluorescence units.

 



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  		FIG. 4.
p53-regulated ER calcium sensitivity is diminished by BIK knockdown. A, H1299 cells were infected with Ad BIK in the presence (open circle) or absence (closed square) of 50 µM Z-VAD-fmk, and cell viability was measured as the percentage of cells that excluded trypan blue at the indicated times. B, p53-induced ER calcium release is reduced by BIK knockdown. H1299 cells were transfected with siRNA-BIK315 or siRNA-LUC, followed by infection with either Ad p53, Ad BIK, or control Ad rtTa for 14 h. The cells were then loaded with Fura-2/AM, and peak cytosolic Ca2+ concentrations were measured as the difference in Fura-2 fluorescence recorded before and after the addition of 2 µM thapsigargin. Data is presented relative to that of untreated cells. Shown are the mean ± S.D. of five independent experiments. LUC, luciferase.

 



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  		FIG. 5.
BIK promotes BAK localization and oligomerization at the ER. A, p53 induces ER BAK localization and oligomerization diminished by siRNA-BIK315. H1299 cells were transfected with siRNA-BIK315 or siRNA-LUC and infected with either Ad p53 or Ad rtTa in the presence of Z-VAD-fmk. Light membrane (LM) and mitochondrial fractions were isolated 13 h after infection, treated with 0.5 mM bismaleimidohexane (BMH) or Me2SO, (DMSO), and the fractions analyzed by SDS-PAGE and probed with the indicated antibodies. B, BIK induces BAK ER localization and oligomerization attenuated by BCL-2. H1299 cells or H1299 stably expressing BCL-2 were infected with either Ad-BIK or Ad-rtTa in the presence of 50 µM Z-VAD-fmk for 13 h. ER and mitochondrial fractions were isolated and treated as in A. HM, heavy membrane.

 



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  		FIG. 6.
BIK-induced ER Ca2+ release is dependent on BAX and BAK. A, representative trace of [Ca2+]cyto from Fura-2-loaded wild-type (Wt) and DKO cells treated with 2 µM thapsigargin. B, wild-type or DKO baby mouse kidney cells were infected with BAK at a multiplicity of infection of the 20 and with BIK or rtTa at a multiplicity of infection of 100 adenoviral vectors for 11, 14, and 15 h, respectively, in the presence of 50 µM Z-VAD-fmk. Peak [Ca2+]cyto of Fura-2-loaded cells was obtained upon the addition of 2 µM thapsigargin. Shown is the mean ± S.D. of three independent experiments measuring 10–20 individual cells/experiment.

 
Knockdown of BIK by siRNA—To investigate the role of BIK in situations where it is induced, we employed RNA interference (RNAi). To this end, we designed the small interfering ribonucleic acid (siRNA) duplexes siRNA-BIK145 and siRNA-BIK315, which are homologous to regions within the BIK coding sequence initiating at nucleotides 145 and 315 relative to the start site of translation, respectively. An HA-BIK mutant harboring a disabling point mutation within its BH3 region (L61G), which permits high accumulation of the protein (1), was co-transfected with siRNAs BIK315 or BIK145 or a control siRNA targeting the luciferase gene within the pGL3-CMV vector (designated siRNA-LUC, Fig. 2A). siRNA-BIK315 exhibited a strong inhibition of BIK accumulation, whereas siRNA-BIK145 was a weaker inhibitor, and siRNA-LUC showed no effect on BIK expression. The endogenous protein levels of actin were also not significantly affected by any of the siRNA duplexes. To further confirm the specificity of siRNAi-BIK315, the vector pRL-CMV, which encodes the gene for Renilla luciferase, was co-transfected with the pGL3-CMV plasmid, which contains the gene for firefly luciferase. siRNA-BIK315 or control siRNA-LUC were also included in the transfection, and the luciferase activity quantified after 24 h. As shown in Fig. 2B, the siRNA-LUC inhibited nearly all firefly luciferase activity, whereas siRNA-BIK315 had no effect on activity as compared with the control. Thus siRNA-BIK315 is both a strong and specific inhibitor of BIK protein expression. In Fig. 2C, the siRNAs were analyzed for their ability to knock down endogenous BIK in H1299 cells infected with Ad p53. Again, siRNA-BIK315 strongly inhibited Ad p53-induced BIK expression, whereas siRNA-BIK145 also inhibited but to a lesser extent, and siRNA-LUC had no effect. The ability of siRNA-BIK145 to inhibit induction of endogenous BIK (Fig. 2C) more effectively than its ability to counter the large amount of BIK(L61G) generated in BIK-transfected cells (Fig. 2A) is consistent with siRNA-BIK145 exhibiting intermediate effectiveness against its target. Thus, siRNA-BIK315 serves as an effective means to specifically knockdown expression of endogenous BIK, with siRNA-BIK145 as a potential intermediate inhibitor and siRNA-LUC as a negative control molecule.

BIK Is Required for Activation of Caspases in Response to Ad p53—Fig. 3A shows the time course of appearance of BIK and p53 proteins following infection of p53-null H1299 cells with Ad p53; both proteins were detectable by 9 h post-infection. By 16 h of infection with Ad p53, H1299 cells typically exhibit classical changes characteristic of the apoptotic phenotype, such as cell rounding, membrane blebbing, and activation of caspases (1) (Fig. 3B). Transfection with siRNA-BIK315 inhibited these p53-induced morphological transformations from occurring at 16 h. In the presence of siRNA-BIK315, Ad p53-infected cells looked similar to those infected with control adenovirus vector encoding reverse tet transactivating protein (Ad rtTa) (Fig. 3B), with over three times the number of cells remaining adherent to cell culture plates compared with that of the siRNA-LUC control (Fig. 3C). Activation of effector caspases (DEVDase activity) was optimally detected by 16 h post-infection with Ad p53 (not shown). This was also attenuated by knock down by siRNA-BIK315 of both endogenous BIK induced by Ad p53 and ectopic BIK expressed by Ad BIK (Fig. 3D). As expected, infection with control Ad rtTa vector did not result in activation of effector caspase activity. Of note, although siRNA-BIK145 was capable of knocking down a significant fraction of the endogenous BIK that was induced by p53 (Fig. 3D, gel insert), substantial effector caspase activity was still observed, although lower than that of cells transfected with control siRNA-LUC. This is in contrast to cells in which p53-induced BIK expression was nearly completely knocked down by siRNA315 (Fig. 3D, gel insert), where the corresponding caspase activity was more strongly inhibited. Thus, there is a dose-dependent inhibition of caspase activation in response to the extent of BIK knockdown, which further validates the specificity of the BIK siRNA and confirms that BIK plays an important role in the stress-induced apoptosis elicited by overexpression of p53.

BIK Medites Early Ca2+ Release from ER—Emerging evidence suggests that Ca2+ signaling by the ER contributes to the mitochondrial apoptosis pathway (15, 21). Because these ER-mediated events occur upstream of activation of effector caspases (15), we focused our analysis at earlier times (14 h post-infection) following infection of cells with Ad p53. Moreover, we included 50 µM Z-VAD-fmk in all subsequent assays, because this inhibitor effectively blocks the activation of caspases (11, 20) and loss of cell viability (Fig. 4A) that can result from exposure of cells to BIK over an extended time period.

Consistent with a role for BIK in this ER calcium signaling, we found that infection of H1299 cells with Ad BIK in the presence of Z-VAD-fmk induced early and robust release of Ca2+ from ER stores, whereas the control adenovirus vector, Ad rtTa, did not (Fig. 4B, right). The loss of ER Ca2+ was measured by loading cells with the cytosolic Ca2+-sensitive dye Fura-2 in the absence of extracellular Ca2+ and determining the difference in peak [Ca2+]cyto before and after the addition of thapsigargin, which causes immediate depletion of ER calcium stores. Similar to Ad BIK, Ad p53 also induced an early loss of ER Ca2+ (14 h post-infection) to an extent similar to that seen for Ad BIK, and importantly, this response to Ad p53 was strongly inhibited by siRNA-BIK315 (Fig. 4B, left). As expected (14), the pancaspase inhibitor Z-VAD-fmk (50 µM) was without effect on either BIK- or p53-induced release of ER Ca2+, suggesting that Ca2+ release from ER in response to Ad p53 was upstream of effector caspases.

BIK Triggers Recruitment and Oligomerization of BAK at the ER—Recent evidence has indicated that, in addition to targeting mitochondria, a relatively small fraction of total cellular BAX and BAK can also reside at the ER, where they undergo oligomerization in response to stress stimuli (25, 34, 35). It has been further suggested that BAK and BAX regulate ER Ca2+ stores and, through this mechanism, influence multiple apoptotic signals (25). Because oligomerization of BAK and BAX typically involves BH3-only proteins, we investigated whether BIK might influence BAK oligomerization at the ER. H1299 cells were transfected with siRNA-BIK315 or siRNA-LUC followed by infection with Ad p53 or control Ad rtTa for 13 h in the presence of 50 µM Z-VAD-fmk. Light membranes (LM, enriched in ER membranes) and heavy membranes (enriched in mitochondria) were collected and incubated with the sulfhydryl-reactive chemical cross-linking agent bismaleimidohexane to cross-link oligomerized proteins. In the absence of cross-linker, we observed a strong recruitment of endogenous BAK to the LM after infection with Ad p53 (Fig. 5A). Moreover, bismaleimidohexane treatment of LM resulted in the appearance of higher order BAK oligomers following p53 expression (Fig. 5A). The LM was not contaminated with mitochondria, as indicated by the absence of the mitochondrial outer membrane-resident protein TOM 20. In Fig. 5, the exposure time of the blots was selected to optimize BAK resolution; in fact, the amount of BAK that distributes to the LM following p53 stimulation is small (10–15%) relative to the pool that is in the heavy membrane fraction. Of note, the ability of Ad p53 to induce localization and oligomerization of BAK at the ER were retarded by siRNA-BIK315 compared with control siRNA-LUC (Fig. 5A, lanes 1 and 2). To examine the ability of BIK on its own to initiate these events, control H1299 cells or H1299 cells stably overexpressing BCL-2 (1) were infected for 12 h with an Ad BIK or control Ad rtTa, in the presence of 50 µM Z-VAD-fmk. Fig. 5B shows that, similar to Ad p53, Ad BIK was also able to trigger BAK ER recruitment and oligomerization. As expected for a BH3-only protein, these BIK-induced events were inhibited by the overexpression of BCL-2.

Effects of BAX,BAK Gene Deletion—The ability of Ad BIK to release mobile stores of Ca2+ from the ER was then examined in transformed baby kidney epithelial cells derived from BAX,BAK doubly deficient (DKO) mice (36). As previously documented for embryonic fibroblast cells (25), [Ca2+]ER is somewhat lower in DKO epithelial cells compared with wild type (Fig. A). Of note, however, strong release of ER Ca2+ was observed upon overexpression of ectopic BAK in these DKO cells in the presence of 50 µM Z-VAD-fmk (Fig. 6B), indicating that, as in wild-type cells, these ER stores of Ca2+ can indeed be mobilized in response to this pro-apoptotic stimulus. In contrast, DKO cells were strongly resistant to the ability of Ad BIK to stimulate the release of ER Ca2+, whereas wild-type cells were responsive (Fig. 6B). Release of ER Ca2+ in response to BIK, therefore, is dependent on the pro-apoptotic BAX,BAK setpoint.



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  		FIG. 7.
BIK knockdown inhibits p53-induced fission of mitochondria, cytochrome c release, and BAX/BAK activation. A, BIK knockdown mitigates p53-induced mitochondrial fission. H1299 cells were transfected with either siRNA-BIK315 or siRNA-LUC, followed by infection with Ad p53 or control Ad rtTa for 14 h in the presence of 50 µM Z-VAD-fmk. The cells were then fixed and stained with anti-TOM 20 antibody (representative images are shown). B, the percentage of cells from A with mitochondrial fission was scored. Shown is the mean ± S.D. of four independent experiments. C, CFP-DRP1(K38E) prevents p53-induced cytochrome c release. H1299 cells were transiently transfected with CFP or CFP-DRP1(K38E) and subsequently infected with either Ad p53 or control Ad rtTa in the presence of Z-VAD-fmk. 13 h post-infection, the cells were fixed, stained with anti-cytochrome c antibody, and immunofluorescence microscopy was used to assess the distribution of cytochrome c in cells positive for CFP. Shown is the mean ± S.D. of three independent experiments. D, BIK knockdown diminishes p53-induced cytochrome c (Cyt. c) release and BAX,BAK activation. Transfection was done as in A, except the coverslips were fixed at 16 h after infection with Ad p53 or control Ad rtTa and stained with either the anti-cytochrome c antibody or active conformation-specific anti-BAX or anti-BAK (not shown) antibodies. Representative images are shown. E, the cells in D were scored for BAX and BAK activation, as well as cytochrome c release. Shown is the mean ± S.D. of three independent experiments.

 
Requirement of BIK for Mitochondrial Fragmentation and Release of Cytochrome c to the Cytosol in Response to Ad p53—As previously documented, release of Ca2+ from the ER activates a pathway leading to recruitment of DRP1 to tubular mitochondria, causing fragmentation of the organelle and sensitization to stimuli that cause release of cytochrome c to the cytosol (18). Fragmentation can be inhibited by a dominant negative active site mutant of the enzyme DRP1(K38E) (20, 28, 37). DRP1(K38E) also inhibits cytochrome c release from mitochondria in response to diverse stimuli (15, 21, 38). Thus we examined the influence of CFP-DRP1(K38E)-transfected cells at early stages of Ad p53-induced apoptosis (13 h), compared with control CFP-transfected cells. Again, 50 µM Z-VAD-fmk was included to prevent the potential influence of feedback stimulation by caspases, and the location of cytochrome c in Ad p53- or Ad rtTa-infected cells that were marked by CFP expression was determined by immunofluorescence. As shown in Fig. 7A, CFP-DRP1(K38E) inhibited the release of cytochrome c from mitochondria. Furthermore, H1299 cells treated with siRNA-315 prior to Ad p53 infection retained the extended mitochondrial tubular network that is typically seen in untreated cells. This is in contrast to the fragmented mitochondria seen in those cells transfected with control siRNA (Fig. 7, A and B), indicating that BIK is required for fission of mitochondria in this pathway. Moreover, BIK knockdown inhibited direct manifestations of mitochondrial apoptosis, including conformational changes associated with activation of mitochondrial BAX/BAK and release of cytochrome c to the cytoplasm. This is shown in Fig. 7, D and E, in which H1299 cells were transfected with siRNA-BIK315 and infected with Ad p53 for 16 h in the presence of 50 µM Z-VAD-fmk. Quantification by immunofluorescence showed a marked decrease in the amount of p53-induced cytochrome c release to the cytoplasm compared with control Ad rtTa infection or siRNA-LUC controls (Fig. 7D, top panel, and Fig. 7E, left). The use of conformation-specific antibodies, which recognize an exposed NH2-terminal epitope associated with the active forms of mitochondrial BAX (39, 40) and BAK (41), also revealed that this activation in response to p53 was mitigated by BIK knockdown, because, similar to cytochrome c release to the cytosol, these conformational changes were strongly inhibited by siRNA-BIK315 (Fig. 7D, bottom panel, and Fig. 7E, right).

In summary, BIK protein is induced in response to select cell stress stimuli, including DNA damaging agents and overexpression of E1A or p53 but not by stress agents that cause protein malfolding in the ER. Of note, however, BIK is located at the ER from where it elicits pro-apoptotic signals and, given sufficient time, these signals lead to cell death by pathway(s) that are strongly inhibited by the wide-spectrum caspase inhibitor Z-VAD-fmk (1, 14). To investigate the initiating events associated with BIK expression, therefore, we focused on the early effects of BIK that still occur in the presence of Z-VAD-fmk, and determined whether these BIK-initiated events contribute to the stress pathway elicited by overexpression of p53 in p53-null human epithelial cells, as judged by BIK knockdown by siRNA. Altogether, the results indicate that BIK induction by Ad p53 is critical for subsequent activation of the mitochondrial apoptosis pathway, with Ca2+ release from the ER and DRP1-regulated egress of cytochrome c from mitochondria representing early steps in this process. Employing transformed kidney epithelial cells derived from wild-type and BAX-,BAK DKO mice, we showed that both cell types maintain a mobile pool of ER Ca2+, because in both cases, significant release of these pools to the cytosol was achieved by expressing ectopic BAK. The fact that BIK, on the other hand, initiated release of ER Ca2+ in wild-type but not DKO cells indicates that BIK achieves calcium release through a BAX,BAK-regulated mechanism. As previously documented, such apoptotic release of ER Ca2+ contributes to DRP1-dependent fragmentation of mitochondria and release of cytochrome c (18), and as shown here, cytochrome c egress from mitochondria in response to Ad p53 is also dependent on DRP1. Thus BIK, by initiating BAX,BAK-dependent release of Ca2+ from the ER, can contribute to the activation of mitochondrial apoptosis in stress pathways in which BIK protein is induced.


   FOOTNOTES
 
* This work was supported by the Canadian Institutes of Health Research and the National Cancer Institute of Canada through funds provided by the Canadian Cancer Society. 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

§ Recipients of the Canadian Institutes of Health Research Doctoral Research award. Back

|| To whom correspondence should be addressed: McIntyre Medical Sciences Bldg., McGill University, Montréal, Québec, Canada H3G 1Y6. Tel.: 514-398-7282; Fax: 514-398-7384; E-mail: gordon.shore{at}mcgill.ca.

1 J. P. Mathai, M. Germain, and G. C. Shore, unpublished data. Back

2 The abbreviations used are: ER, endoplasmic reticulum; siRNA, small interfering ribonucleic acid; HA, hemagglutinin; LM, light membrane; rtTa, reverse tet transactivating protein; RNAi, RNA interference; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; DKO, double knock-out. Back


   ACKNOWLEDGMENTS
 
We thank Eileen White for providing the BAK/BAK DKO and control cells, as well as Mai Nguyen and Imed Gallouzi for helpful discussions. We are grateful to James Martin for the use of apparatus for single-cell Ca2+ measurements and Barbara Tolloczko for guidance and generous assistance.



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 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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