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Originally published In Press as doi:10.1074/jbc.M312467200 on February 27, 2004

J. Biol. Chem., Vol. 279, Issue 20, 21312-21317, May 14, 2004
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Translation Initiation Factor 4E Blocks Endoplasmic Reticulum-mediated Apoptosis*

Shunan Li, David M. Perlman, Mark S. Peterson, David Burrichter, Svetlana Avdulov, Vitaly A. Polunovsky, and Peter B. Bitterman{ddagger}

From the Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota 55455

Received for publication, November 14, 2003 , and in revised form, February 5, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Eukaryotic translation initiation factor 4E (eIF4E) is the mRNA cap-binding protein required for translation of cellular mRNAs utilizing the 5' cap structure. The rate-limiting factor for mRNA recruitment to ribosomes, eIF4E is a major target for regulation of translation by growth factors, hormones, and other extracellular stimuli. When overexpressed, eIF4E exerts profound effects on cell growth and survival, leading to suppression of oncogene-dependent apoptosis, causing malignant transformation and conferring tumors with multiple drug resistance. We found previously that overexpressed eIF4E interdicts the apoptotic pathway induced by growth factor withdrawal and cytotoxic drugs by selectively activating the expression of Bcl-XL, thus preventing mitochondrial release of cytochrome c. In this study, we examined the impact of ectopic eIF4E expression on apoptosis mediated by the endoplasmic reticulum (ER). Here we show that eIF4E rescued cells from the ER stressors brefeldin A, tunicamycin, thapsigargin, and the Ca2+ ionophore A23187 [GenBank] . In addition, we found that cells rescued from Ca2+ ionophore-triggered apoptosis did not release calcium from their ER nor did they translocate caspase-12 from the ER to the cytoplasm. These data lend strong support to the concept that eIF4E functions as a pleiotropic regulator of cell viability and that integration of critical organelle-mediated checkpoints for apoptosis can be controlled by the cap-dependent translation apparatus.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intracellular membrane-bound organelles are the principal loci where processes regulating the functions of caspases reside (1). Among these, the mitochondrion and endoplasmic reticulum (ER)1 are the best documented integrators of intrinsic and extracellular signals leading to cell death. The function of these organelles in apoptosis is regulated by the Bcl-2 family of proteins, which controls critical decision nodes in the apoptotic cascade and shares homology in up to four conserved regions termed BH domains (2, 3). The "multidomain" anti-apoptotic proteins Bcl-2 and Bcl-XL and the pro-apoptotic proteins Bax and Bak have three or four BH domains and reside in the cytosol, perinuclear membrane, outer membrane of mitochondria, and membranes of the ER (4, 5). The intramembrane balance between these opposing Bcl-2 family members controls an essential gateway for apoptotic signals (2). The pro-apoptotic "BH-3-only" members of the family, such as BID, BIM, and BAD, have sequence homology within the {alpha}-helical BH3 region and function upstream of membrane organelles. They select and connect proximal death signals to Bax and Bak by targeting cytosolic apoptotic agonists to membranes with subsequent activation by homooligomerization (6). When activated, Bax and Bak change organelle membrane permeability for a set of critical metabolites, which efflux from membrane-bound compartments, resulting in dramatic perturbations of intracellular homeostasis, activation of effector caspases, and cell death. Pro-apoptotic signals activate Bax- and Bak-driven membrane alterations in both mitochondria and the ER (4), providing a means to integrate and coordinate the role of these compartments in controlling intrinsic apoptosis.

Despite sharing these upstream signaling pathways and downstream effector molecules, apoptotic checkpoints controlled by the mitochondrion and ER require distinct categories of death stimuli and utilize upstream regulators and downstream effectors that are unique to each organelle (4). In this regard, altered permeability of the outer mitochondrial membrane results from signals mediated by BH3-only proteins. This leads to an efflux of transmembrane proteins including cytochrome c, which associates with dATP, Apaf-1, and procaspase-9 to form the apoptosome, which in turn activates downstream effector caspases (7-10). In contrast, the ER is only minimally responsive to BH3-only members (4) but is highly responsive to signals that induce ER stress and ER membrane protein misfolding, such as lipid messengers (11) or oxidative stimuli (12). Bax- and Bak-induced alterations in ER membrane permeability result in Ca2+ release from the ER lumen with subsequent activation of caspase-12 (13). Localized in the ER, caspase-12 is activated by ER stressors targeting the transport of proteins from the ER to the Golgi body (e.g. brefeldin A), inhibiting their N-glycosylation (e.g. tunicamycin), disrupting ER calcium homeostasis (e.g. thapsigargin), or augmenting cytosolic Ca2+ concentration (e.g. calcium ionophore A23187 [GenBank] ) (14). Accumulation of unfolded protein intermediates in the ER activates an integrated set of stress signals designated the unfolded protein response (15, 16). Caspase-12-deficient cells are resistant to ER stress-induced apoptosis (13), and ER stressors can activate caspase-12, -9, and -3 independently of cytochrome c and Apaf-1 (17, 18). Thus, available data suggest that caspase-12 is a specific mediator of ER stressor-triggered apoptosis (13, 17, 18).

Despite evidence documenting their unique and distinctive features, the mitochondrial and ER apoptosis checkpoints frequently function in an interconnected and integrated manner. For example, administration of exogenous ER stressors or interventions that target ectopic Bax to the ER result in the release of Ca2+ from the ER, with a subsequent increase in mitochondrial Ca2+ uptake and the enhanced efflux of cytochrome c (4, 19). A variety of apoptotic triggers including genotoxic agents and growth factor withdrawal can activate the Bax/Bad-mediated checkpoints in both mitochondria and the ER (4). This coupling of ER stress to the mitochondrial death pathway strongly implies the existence of a mechanism to coordinate control of the apoptotic functions in these organelles with proapoptotic signals from the extracellular environment. However, the mechanisms by which extracellular events modulate the organelle-specific set points of susceptibility to apoptosis are incompletely understood.

In this report, we sought to identify an apical regulator capable of coordinating the responses of mitochondria and the ER to pro- and anti-apoptotic forces in the extracellular milieu. We found previously (36) that the set point for cellular susceptibility to apoptosis through the mitochondrial checkpoint is governed by pre-translational and translational control of Bcl-XL mediated by the cap-dependent translation initiation apparatus. A trimolecular complex designated eIF4F, the initiation apparatus is required for recruitment of capped mRNAs to the 40 S ribosomal subunit. It comprises the mRNA cap-binding protein, eukaryotic translation initiation factor 4E (eIF4E), eIF4A (an ATP-dependent RNA helicase), and eIF4G (a docking protein) (20). Here we show that activation of the cap-dependent translational apparatus also rescued cells from ER stress-induced apoptosis in this case by mediating the blockade of calcium release from the ER to the cytosol and by preventing activation of caspase-12. These findings indicate that the integration of critical organelle-mediated checkpoints for apoptosis can be controlled by the cap-dependent translation apparatus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines—Parental NIH 3T3 fibroblasts and cells constitutively expressing eIF4E (3T3/4E) have been described previously (21). Both NIH 3T3 and 3T3/4E cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 4.5 g/liter glucose, 5.9 g/liter HEPES, 100 units/ml penicillin, 100 units/ml streptomycin, and 250 ng/ml amphotericin.

Analysis of Apoptosis—To trigger apoptosis, cells were treated with the ER stressors thapsigargin, brefeldin A, tunicamycin, and calcium ionophore A23187 [GenBank] in Dulbecco's modified Eagle's medium, 10% fetal calf serum. Pilot dose-response studies were carried out to determine the optimal concentration of each agent in our cell system. After treatment with the designated ER stressor, both detached and adherent cells were collected for analysis. The cells were fixed with 70% ethanol, washed with PBS, and stained with propidium iodide (50 µg/ml propidium iodide, 0.1% Triton X-100, 32 µg/ml EDTA, 2.5 µg/ml RNase in PBS) for 45 min at 37 °C. DNA content was determined by quantitative flow cytometry using the CellQuest program.

Measurement of Calcium Permeability Changes in Response to A23187 [GenBank] —Cells were seeded on glass coverslips coated with fetal calf serum and grown in Dulbecco's modified Eagle's medium, 10% fetal calf serum for 24 h. The cells were incubated at 37 °C for 1 h with 4 µM of fura-2-AM. The cells were washed with fura-out buffer (139 mM NaCl, 3 mM KCl, 10 mM HEPES, 1.8 mM CaCl2, 0.8 mM MgSO4, 10 µM glycine, and 15 µM sucrose), excited, and monitored. After adding 5 µM A23187 [GenBank] in fura-out buffer, fluorescence was measured at 340- and 380-nm wavelengths every 5 min for 1 h.

Caspase Assays—Cells were treated with 5 µM A23187 [GenBank] for the indicated time intervals, both detached and adherent cells were collected, washed with PBS, and disrupted in lysis buffer (50 mM Tris, 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 10 mM sodium pyrophosphate) supplemented with proteinase/phosphatase inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 2 µg/ml aprotinin, 1 µg/ml pepstatin, 100 µM Na3VO4, 20 mM {beta}-glycerolphosphate) by three cycles of freeze and thaw (15 min at -70 °C, 5 min at 37 °C). Lysates were centrifuged for 10 min at 10,000 x g, and resultant supernatants were centrifuged for 1 h at 100,000 x g. Protein concentration was quantified using a Pierce BCA protein assay kit. For caspase-3 activity, 16 µg of lysates were added to a fluorogenic substrate (Ac-DEVD-AMC) (Calbiochem) that contains the caspase-3 cleavage site, DEVD, for 2 h at 37 °C. The release of free AMC was measured by a fluorometer. Caspase-8 activity was detected by adding the fluorometric substrate IETD-AFC (Biovision) by a 96-well fluorescent plate reader. Caspase-9 activity was determined by cleavage of LEHD-p-nitroanilide using a caspase-9 colorimetric assay (R & D Systems).

Immunoblot—Cells were rinsed with PBS, released from the dish with trypsin, centrifuged, and resuspended in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% deoxycholate) supplemented with protease inhibitor mixture (0.1 mM phenylmethylsulfonyl fluoride and 2 µg/ml each leupeptin, aprotinin, and pepstatin A). The cells were disrupted by forcing them through a 25-gauge needle, centrifuged at 12,000 x g for 10 min, and the supernatant retained. For each sample to be analyzed, 50 µg of protein was resolved by 12% SDS-PAGE and transferred to a nitrocellulose membrane. The blots were blocked in buffer with 5% dry milk and incubated with the following antibodies: anti-caspase-12 (Cell Signaling) and anti-actin (Sigma). The blots were then washed and incubated with horseradish peroxidase-coupled secondary antibody followed by ECL development.

Caspase-12 Immunostaining—Cells were seeded at a density of 5 x 103 cells/cm2 onto fetal calf serum-coated glass coverslips and cultured for 24 h in complete medium. The cultures were continued either in complete medium or in medium containing 5 µM A23187 [GenBank] for 24 h. The cells were rinsed with PBS and fixed in PBS containing 4% paraformaldehyde. The fixed cells were incubated in blocking buffer (PBS containing 5% normal donkey serum, 1% bovine serum albumin, and 0.3% Triton X-100) for 30 min and for an additional 2 h in PBS containing 1% bovine serum albumin and 1% normal donkey serum with rabbit anti-caspase-12 antibody (1:50) (Cell Signaling). To label the ER, we also included an antibody recognizing grp78 (Bip, a chaperone protein localized to the ER) in the incubation mixture (goat anti-GRP78 antibody, Santa Cruz). The cells were washed three times in PBS and incubated for 30 min in PBS containing 1% bovine serum albumin, 1% normal donkey serum with donkey anti-rabbit-IgG cyanine-2 antibody (1:50) (Jackson ImmunoResearch, Inc., West Grove, PA) and donkey anti-goat-IgG cyanine-3 antibody (1:50) (Jackson ImmunoResearch Inc, West Grove, PA). To label the nuclei, the cells were rinsed with PBS and treated with 0.1 µg/ml DAPI for 30 min in the dark. The coverslips were mounted, and the images were acquired with a Zeiss Atto Arc HBO 110-watt upright microscope (13).

Measurement of Intracellular Calcium Level—Cells were grown in complete medium with or without 5 µM A23187 [GenBank] for the time interval indicated previously. Floating and adherent cells were collected, centrifuged at 1600 rpm for 5 min, and washed with PBS. The cells were stained with 3 µM indo-1 acetoxymethyl ester (indo-1 AM) (Molecular Probes) at 37 °C for 30 min prior to analysis. Indo-1 AM was excited by an Innova 300 UV laser with a 50 nm band-pass filter used for detection. Intracellular calcium ion concentration was assessed by calculating the 325/450 nm ratio (22).

Data Analysis—All data points represent the mean ± S.D. of three independent experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Overexpression of eIF4E Rescues Cells from Endoplasmic Reticulum Stressor-induced Apoptosis—Cellular perturbations leading to apoptosis primarily operate through two organelles, the mitochondrion and the ER. We reported previously that ectopic expression of the mRNA 5' cap-binding protein, eIF4E, rescues cells from growth factor withdrawal and cell cycle-active drugs, which are apoptogenic stimuli that activate the mitochondrial death pathway (23, 24). ER stress can be exerted by inhibiting one of its Ca2+ pumps with thapsigargin by disrupting cytosolic calcium homeostasis with Ca2+ ionophores or by triggering the unfolded protein response with the protein glycation inhibitors tunicamycin and brefeldin A. Treatment of NIH 3T3 cells with each class of ER stressor resulted in a significant increase in apoptosis, ranging from 15.35% for brefeldin A to 78.845% for A23187 [GenBank] (peak responses shown in Fig. 1A). In marked contrast, ectopic overexpression of eIF4E (3T3/4E) nearly abolished apoptosis in response to each of the ER stressors. Detailed analysis of NIH 3T3 cell apoptosis in response to calcium ionophore A23187 [GenBank] showed it to be strictly concentration- and time-dependent, whereas only minimal apoptosis was triggered in 3T3/4E cells (Fig. 1, B and C). These data indicate that eIF4E potently antagonizes ER stressor-mediated apoptosis and identify Ca2+ ionophore as the most potent ER stressor in our cell system.



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  		FIG. 1.
eIF4E rescues cells from endoplasmic reticulum stressor-induced apoptosis. NIH 3T3 and 3T3/4E cells were cultured in complete medium with or without ER stressors at optimal concentrations (10 µg/ml brefeldin, 1 µg/ml tunicamycin, 5 µM thapsigargin, or 7.5 µM A23187 [GenBank] ) (A) or with Ca2+ ionophore A23187 [GenBank] at the indicated concentrations (B) for 24 h or with 5 µM A23187 [GenBank] added at t = 0 (C). Shown are apoptotic frequencies quantified by flow cytometry.

 
Calcium Ionophore A23187 [GenBank] Has Similar Potency in Cells Expressing either Physiological or Ectopic eIF4E—Because calcium ionophore A23187 [GenBank] is thought to trigger apoptosis by disrupting intracellular calcium homeostasis (13), it was possible that A23187 [GenBank] affects calcium permeability differently in NIH 3T3 and 3T3/4E cells. To evaluate this, we treated NIH 3T3 and 3T3/4E cells with 5 µM A23187 [GenBank] for up to 60 min and measured intracellular calcium levels. Under basal conditions, the Ca2+ concentration is higher in the ER (10-100 µM) than in the cytoplasm (100-300 nM), and this gradient is maintained by an ATP-dependent pump designated SERCA (smooth ER Ca2+ ATPase) (25). To measure intracellular calcium levels, cells were loaded with 4 µM fura-2-AM, washed with buffer, and monitored every 5 min for 1 h. When the cells were treated with A23187 [GenBank] , there was a rapid increase in cytoplasmic calcium, which returned to the base line after a few minutes. The amplitude and duration of the Ca2+ elevation was comparable in both NIH 3T3 and 3T3/4E cells (Fig. 2). Thus, eIF4E rescue does not result from differential efficacy of the ionophore.



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  		FIG. 2.
Calcium ionophore A23187 [GenBank] has similar potency in cells with physiologically or ectopically expressed eIF4E. NIH 3T3 cells (A) or 3T3/4E cells (B) were cultured in complete medium for 24 h. A23187 [GenBank] (5 µM) was added to the cells for the indicated time interval, and the intracellular free Ca2+ level measured in individual cells is displayed as the 340/380 nm ratio.

 
Ectopic Expression of eIF4E Prevents A23187 [GenBank] Activation of Caspase-3 and Upstream Caspases—The cleavage of procaspase-3 (32 kDa) into its active 17-kDa form is a downstream step common to most forms of apoptosis (7, 18, 26). To investigate the impact of overexpressed eIF4E on this critical node in apoptotic signaling, we quantified caspase-3 activity in NIH 3T3 and 3T3/4E cells after imposition of an ER stress. Treatment of NIH 3T3 cells with 5 µM A23187 [GenBank] activated caspase-3 as early as 18 h, with a peak level measured at 24 h (Fig. 3). In marked contrast, the cells ectopically expressing eIF4E did not activate caspase-3 (Fig. 3).



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  		FIG. 3.
eIF-4E prevents A23187 [GenBank] activation of caspase-3. NIH 3T3 cells or 3T3/4E cells were cultured in the presence or absence of 5 µM A23187 [GenBank] for the indicated times. Shown is the caspase-3 activity (average of three independent experiments) quantified using a fluorogenic assay.

 
Caspase-8 and caspase-9 lie upstream of caspase-3. For death receptor-mediated apoptosis, oligomerization of death receptors through adaptor proteins leads to caspase-8 activation, which in turn cleaves and activates caspase-3 (27, 28). For the intrinsic apoptotic pathway, various noxious stimuli cause cytochrome c release, which promotes formation of the cytochrome·Apaf-1·procaspase-9 complex, which leads to caspase-9 activation. Activated caspase-9 in turn cleaves and activates caspase-3 (7, 10).

Even though these pathways are distinct in some cell systems, in others, cross-talk between them has been documented (29-31). Cleavage of Bid by caspase-8 in response to the activation of cell surface death receptors causes the release of cytochrome c from mitochondria and leads to apoptosis (30, 31). Recent data suggest that ER stressors do not directly activate caspase-8 but could activate caspase-9 in an Apaf-1- and cytochrome c-independent manner (17, 18). We therefore examined whether A23187 [GenBank] causes apoptosis by direct or indirect activation of caspase-8 or caspase-9 in our system rather than through the posited activation of caspase-12. When NIH 3T3 cells were treated with A23187 [GenBank] , neither caspase-8 nor caspase-9 were activated (Fig. 4), indicating that in our cell system, A23187 [GenBank] -induced apoptosis was independent of the classical death receptor or mitochondria-dependent pathways.



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  		FIG. 4.
A23187-mediated apoptosis in NIH 3T3 cells does not involve activation of caspase-9 or -8. NIH 3T3 (triangles) and 3T3/4E (circles) cells were cultured in the presence or absence of 5 µM A23187 [GenBank] for the indicated times. Shown are caspase-9 (A) and caspase-8 (B) activity.

 
Overexpressed eIF4E Prevents Activation of ER Caspase-12—Caspase-12 localizes in the ER and mediates ER-specific apoptosis (13). To determine whether caspase-12 played a role in our system, we treated cells with A23187 [GenBank] for 24 h and detected caspase-12 activation by immunoblot. Caspase-12, like other caspases, requires cleavage of its prodomain for its enzymatic function (10). We used a caspase-12 antibody, which detects both full-length and cleaved caspase-12, and found cleaved caspase-12 only in NIH 3T3 cells but not in 3T3/4E (Fig. 5A). Caspase-12 is reported to be predominantly associated with the ER (13). Consistent with this, morphological analysis demonstrated that caspase-12 was localized mainly in a punctate cytoplasmic pattern (green granules) in both NIH 3T3 and 3T3/4E cells in their basal state. The caspase-12 signal was frequently found to be co-localized (orange granules) with the ER marker grp78 (Bip) (red granules) (Fig. 5B). However, after treatment with A23187 [GenBank] , the caspase-12 signal in NIH 3T3 cells appeared in a diffuse pattern, suggesting it was translocated from the ER to the cytosol (Fig. 5B). As expected from the apoptosis data, condensed nuclei and decreased cell numbers were also apparent. In contrast, the morphological pattern of the eIF4E signal remained punctate in cells ectopically expressing eIF4E (Fig. 5B). These data indicate that caspase-12, the signature ER stress caspase, is not activated in cells rescued from calcium ionophore-induced apoptosis by eIF4E.



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  		FIG. 5.
Ectopic expression of eIF4E prevents activation of caspase-12. NIH 3T3 and 3T3/4E cells were cultured for 24 h in complete medium with or without 5 µM A23187 [GenBank] . A, immunoblot analysis of caspase-12 (with actin shown as a loading control). B, immunolocalization of caspase-12 (green, left), the ER protein grp78 (red, center), and the merging of both images (right; co-localized caspase-12 and grp78 signal appears orange). Nuclei, stained with DAPI, appear blue. Shown are NIH 3T3 cells in complete medium (a, b, c) or after treatment with 5 µM A23187 [GenBank] (d, e, f) and 3T3/4E cells in complete medium (g, h, i) or after treatment with 5 µM A23187 [GenBank] (magnification = x630).

 
Ectopic Expression of eIF4E Inhibits ER Calcium Release—Calcium release from the ER is the hallmark apoptotic event after prolonged calcium ionophore treatment and is thought to lie upstream of caspase-12 activation in the ER-mediated apoptotic pathway (13). To examine intracellular calcium concentration in our system, NIH 3T3 and 3T3/4E cells were incubated in complete medium with or without 5 µM A23187 [GenBank] for the time intervals indicated (Fig. 6). The cells were collected and stained with the calcium-binding dye indo-1, and the signal was quantified by flow cytometry. In response to A23187 [GenBank] , the intracellular calcium level increased more than 2-fold in parental NIH 3T3 cells but less than 25% in NIH 3T3 cells ectopically expressing eIF4E (Fig. 6). Thus, our data suggest that eIF4E rescues cells from calcium ionophore-induced apoptosis by mitigating calcium release from the ER.



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  		FIG. 6.
Ectopic expression of eIF4E inhibits ER calcium release. NIH 3T3 cells and 3T3/4E cells were cultured in complete medium with or without 5 µM A23187 [GenBank] for the indicated time, harvested, incubated with calcium-binding dye indo-1 AM, and analyzed by flow cytometry (ratio of 325/50 nm). The intracellular calcium level is shown as the percentage increase above control (i.e. untreated cells, in which the calcium signal remained stable for the entire 24-h incubation). Data points represent the mean ± S.D. of three independent experiments).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The ER subserves an essential role in protein synthesis, folding, and trafficking; cellular response to stress; and Ca2+ storage (25, 33). Perturbation of Ca2+ homeostasis and the accumulation of unfolded proteins in the ER leads to ER stress and dysfunction, which triggers apoptosis, a series of events implicated in the pathogenesis of neurodegenerative disorders, including Alzheimer's and Parkinson's disease as well as stroke (25, 33). Ectopic overexpression of eIF4E rescues cells from apoptosis in response to death receptor ligation, growth factor withdrawal, and metabolic stress (21, 23, 24). Here we examine whether overexpression of eIF4E rescues cells from ER stress-mediated apoptosis and dissect the critical steps of rescue. We found that overexpressed eIF4E prevents apoptosis induced by ER stressors, perturbing protein glycosylation, active calcium transport across the ER membrane, and calcium flux across the plasma membrane. In detailed studies of cellular responses to the calcium ionophore A23187 [GenBank] , the anti-apoptotic effect of eIF4E was associated with the preservation of ER calcium homeostasis and failure of caspase-12 to translocate from the ER to the cytoplasm. The preservation of viability by eIF4E could not be accounted for by differences in calcium influx from the extracellular environment in response to A23187 [GenBank] or by interruption of cross-talk among apoptotic pathways. We therefore conclude that the mechanism of eIF4E rescue from ER stressors involves critical steps at or upstream of caspase-12 activation.

To date, three major apoptotic pathways have been identified. Each is orchestrated by a distinct cellular locus or organelle. The extrinsic apoptotic pathway is mediated by ligand-induced oligomerization of specific cell surface death receptors, resulting in caspase-8 activation (25, 27). One intrinsic apoptotic pathway is mediated by diverse metabolic stresses that converge on the mitochondrion, triggering cytochrome c release and activation of caspase-9 (7), processes antagonized by Bcl-2 (34, 35) and facilitated by the multidomain proapoptotic Bcl-2 family members Bax and Bak (4, 19). The other intrinsic apoptotic pathway is triggered by ER stressors, resulting in Ca2+ efflux from the ER and caspase-12 activation (13, 17, 18, 34, 35). Activation of caspase-8, caspase-9, or caspase-12 causes activation of downstream effector caspases, including caspase-3, -6, and -7, ultimately leading to apoptosis (26). Despite the diversity of mechanisms operative in these three pathways, our prior results (21, 36), along with data from the present study, indicate that ectopic expression of eIF4E blocks all three pathways.

One possible explanation for this robustness of eIF4E in preserving cell viability is that the three pathways are not actually independent. Indeed, in some cell types, cross-talk exists among the three apoptotic pathways. For example, in cells designated as Fas type II cells, a mitochondrion-mediated amplification loop is required for death ligand-induced apoptosis, which is initiated by activated caspase-8 (30, 31). Recent findings suggest that alterations in homeostatic concentrations of Ca2+ induced by ER stressors stimulate the release of cytochrome c from mitochondria in both a caspase-8- and Bid-dependent (37) and -independent manner (4). Caspase-7 functions as an important effector caspase for the mitochondrial death pathway, but in response to ER stress, is translocated from the cytosol to the ER surface where it cleaves and activates caspase-12 (18). Pro- and anti-apoptotic members of the Bcl-2 family also regulate apoptotic events in more than one cellular compartment. The "multidomain" Bax and Bak proteins, for example, can operate in both mitochondria and ER (4), whereas cytochrome c release can be blocked by Bcl-2 variants genetically engineered to reside either in mitochondrial or ER membranes or in both organelles (22, 29, 38). Thus, there is clear coordination among pathways and compartments in the apoptotic cascade.

What is the mechanism linking the cap-dependent translation machinery to the ER-mediated apoptotic pathway? Our previous data underscore the pleiotropic nature of eIF4E anti-apoptotic signaling, which includes pretranslational and translational activation of Bcl-XL (36). Bcl-2 and Bcl-XL can regulate ER-dependent Ca2+ homeostasis through several mechanisms. These include changes in the rate of Ca2+ efflux and reuptake by directly targeting store-operating Ca2+ channels and pumps, alterations in luminal Ca2+-binding chaperones, and modulation of upstream inositol 1,4,5-trisphosphate-signaling pathways (39). On the basis of these findings, it is reasonable to suggest that the activated cap-dependent translation apparatus can suppress ER-mediated apoptosis directly or by interdiction of other apoptotic pathways, function of which is essential for ER-mediated cell death to occur. In our cell system, neither caspase-8 nor -9 is activated by calcium ionophore, indicating that the apoptosis observed was independent of the extrinsic and mitochondrial pathways. We therefore conclude that the rescue observed in response to ectopic eIF4E resulted from the direct blockade of the ER stressor pathway. Thus, together with findings that overexpressed eIF4E protects cells against death receptor ligation as well as against mitochondrion-mediated apoptosis (36), we interpret the present results to indicate that critical apoptotic checkpoints at the cell surface and within key organelles are integrated and coordinated by translational control.

Our findings that ER-dependent apoptosis is regulated by components of the translational machinery are in accord with recent data documenting that elongation factor EF-1{alpha}, which is essential for both cap-dependent and cap-independent translation, rescues cells from growth factor withdrawal and ER stress. In this regard, when overexpressed in interleukin-3-dependent FL5.12 cells, EF-1{alpha} preserves cell viability in the context of interleukin-3 withdrawal and inhibits apoptosis in response to the ER stressors brefeldin A and thapsigargin (32). Together with our findings, these results support the notion that translational control cooperates with well established transcriptional and post-translational mechanisms to oversee the basic apoptotic machinery. Thus, by modulating signals from the plasma membrane, cytosol, mitochondrion, and ER, eIF4E is positioned to orchestrate the integration of key intracellular apoptotic checkpoints.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant IU01-CA91220 (to V. A. P.), National Institutes of Health Grants 2P50-HL50152 and IR01-HL073719 (to P. B. B.) and HL 07741-07 (to S. L.), and American Lung Association Grant RT-019-N (to D. M. P.). 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

{ddagger} To whom correspondence should be addressed. Tel.: 612-624-0999; Fax: 612-625-2174; E-mail: bitte001{at}umn.edu.

1 The abbreviations used are: ER, endoplasmic reticulum; eIF4E, eukaryotic translation initiation factor 4E; PBS, phosphate-buffered saline; DAPI, 4',6-diamidino-2-phenylindole; AM, acetoxymethyl ester; AMC, amino-methylcoumarin; AFC, amino-trifluoromethyl coumarin. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Janet Dubinsky (Department of Neuroscience, University of Minnesota) for assistance in measuring calcium flux using fura-2. We also thank Janet Peller and Gregory Veltri (Flow Cytometry Core Facility, University of Minnesota Cancer Center) for assistance in quantifying intracellular calcium. Special thanks go to Rachel McMullen, Hong-Yiou Lin, and Karen Smith (Medical School, University of Minnesota) for their technical support and to Pat Jung (Medical School, University of Minnesota) and Jerry Sedgewick (Biomedical Image Processing Laboratory, University of Minnesota) for assistance with photomicroscopy.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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