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J Biol Chem, Vol. 275, Issue 13, 9628-9635, March 31, 2000


Cross-talk between Phosphatidylinositol 3-Kinase and Sphingomyelinase Pathways as a Mechanism for Cell Survival/Death Decisions*

Matthew E. Burowabcd, Christopher B. Weldonbce, Bridgette M. Collins-Burowad, Nijm Ramseycd, Amy McKeec, Anke Klippelf, John A. McLachlanabd, Sanda Clejanacdg, and Barbara S. Beckmanabcdh

From the a Molecular and Cellular Biology Program, b Department of Pharmacology, c Tulane Cancer Center, e Department of Surgery, g Department of Pathology and Laboratory Medicine, d Tulane Center for Bioenvironmental Research, Tulane University School of Medicine, New Orleans, Louisiana 70112 and f Chiron Corporation, Emeryville, California 94608

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Peptide hormones act to regulate apoptosis through activation of multiple pro- and anti-apoptotic signaling cascades of which lipid signaling events represent an important facet of the cellular rheostat that determines survival and death decisions. Activation of sphingomyelinase, which generates ceramide, is an intermediate in cellular stress responses and induction of apoptosis in many systems. Conversely, phosphatidylinositol 3-kinase (PI3K) is a critical signaling molecule involved in regulating cell survival and proliferation pathways. In the present study, we investigate cross-talk between the PI3K and sphingomyelinase pathways as a mechanism for regulation of cell survival/death decisions. We show that phorbol ester, insulin-like growth factor 1, and a constitutively active PI3K suppress both tumor necrosis factor-induced apoptosis and ceramide generation. Conversely, inhibition of the PI3K pathway with expression of a kinase-dead PI3K both prevented survival signaling and enhanced tumor necrosis factor-induced ceramide generation. The ability of exogenous sphingomyelinase to induce ceramide generation was partially suppressed by expression of constitutively active PI3K and enhanced by inhibition of PI3K suggesting that cross-talk between PI3K and ceramide generation within cells is regulated subsequent to activation of sphingomyelinase.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Apoptotic signaling by TNF1 involves recruitment of specific proteins to the "death domain" of the p55 TNF receptor (TNFR1) including TRADD, TNF receptor-associated factors, receptor-interacting protein, receptor-interacting protein-associated Ich-1/CED-3 homologous protein with a death domain, and Fas-associated death domain (1-3). The formation of this TNFR1 complex leads to the activation of a number of signaling intermediates including c-Jun N-terminal kinases (JNK), phospholipase A2, sphingomyelinase, NF-kappa B, and caspases. Reports have implicated these pathways in both promotion and suppression of apoptosis by TNF as well as by other agents including chemotherapeutic drugs, radiation, and Fas (1-7). The magnitude and types of signaling events activated as well as their coordinate interaction with other pro/anti-apoptotic pathways determine the response of a cell to TNF and other apoptotic inducers. Therefore, the sum of these signals and their cross-regulation to each other determine apoptotic sensitivity and cell fate.

A diverse number of peptide hormones including insulin-like growth factor I (IGF-1), epidermal growth factor, nerve growth factor (NGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGF), insulin, granulocyte macrophage-colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), as well as the PKC-activating tumor promoter phorbol ester (PMA) have been shown to act as both proliferative and cell survival signals in a cell type-specific manner (8-20). In MCF-7 cells, IGF-I and insulin have been shown to enhance proliferation, whereas basic FGF and the protein kinase C (PKC)-activating phorbol ester (PMA) act to suppress proliferation (21-24). The ability of growth factors to mediate cell survival effects may be mediated through their ability to activate rapid signaling events within cells that then target and subvert the apoptotic cascade. Peptide hormones and phorbol esters have been shown to suppress apoptosis induced by a number of agents including growth factor withdrawal, ionizing radiation, chemotherapeutic drugs, cellular stress, as well as TNF and Fas. These diverse survival factors activate numerous cellular signaling pathways including PKC, mitogen-activated protein kinase, and PI3K (25-32). Therefore, it is of interest to determine if common signaling pathways exist among the survival factors that regulate apoptosis.

PI3K has emerged as a critical signaling molecule that regulates multiple cellular processes including survival and proliferation in numerous systems. PI3K is composed of a regulatory p85 subunit and a catalytic p110 subunit that as an active complex phosphorylate the 3-ring position of PI-4,5-bisphosphate to generate PI-3,4,5-triphosphate (PIP3) (33-36). Downstream targets activated subsequent to PIP3 include PKC isoforms, JNK, Ras, p70 S6 kinase, Rac, and PKB/Akt of which PKB/Akt has been implicated as an intermediate in PI3K-generated survival signals (31-43). Known activators of PI3K, including the peptide hormones PDGF, NGF, and IGF-1 as well as PMA act as survival factors suppressing apoptosis induced by a number of agents (6, 32-44). Additionally, transfection of cells with constitutively active PI3K or Akt results in inhibition of apoptosis induced by c-Myc, UV radiation, TGF-beta as well as Fas (31, 32, 37-41). PI3K activation of Akt/PKB and subsequent phosphorylation of Bad suggests one mechanism by which PI3K signaling acts to suppress apoptosis (45, 46). However, others (47) have demonstrated PKB/Akt survival signaling can occur independently of Bad phosphorylation, suggesting that the PI3K survival signal may target multiple components of the apoptotic cascade. Recently, ceramide has been shown to suppress Akt/PKB and its survival signaling suggesting cross-talk between these two pathways (33, 48-50). Based upon this information, we wished to examine both the ability of a PI3K signal to suppress apoptosis as well as the coordinate cross-regulation between the PI3K and ceramide pathways.

The lipid second messenger ceramide is an important component of the cell stress response pathway and has been shown to regulate cell proliferation, differentiation, and apoptosis. The effects of ceramide on numerous cellular processes is thought to be mediated through activation of a number of signaling molecules including, NF-kappa B, JNK, ceramide-activated protein kinase, and ceramide-activated protein phosphatase (4-6, 51, 52). In addition to TNF, a variety of extracellular stimuli and agents of injury or stress including chemotherapeutic drugs, heat shock, oxidative stress, UV and ionizing radiation, as well as certain cytokines and hormones activate sphingomyelinase resulting in the generation of ceramide (4-7). TNF receptor-induced ceramide generation occurs primarily through the activation of neutral and acidic sphingomyelinases and, in the case of other apoptotic stimuli such as chemotherapeutic drugs, through de novo synthesis via ceramide synthase (4-7). Ceramide levels within cells are regulated by a balance between its generation and metabolism through multiple enzymes including sphingomyelinases, ceramide synthase, ceramidases, ceramide kinase, sphingomyelin synthase, and glucosylceramide synthase (4-7, 53). Additionally, recent reports have shown that phorbol ester-mediated inhibition of TNF-induced apoptosis and suppression of ceramide generation are partially mediated through activation of ceramidase (54). Therefore, the effects of ceramide generation within cells may depend on the specific sphingomyelinase activated, the magnitude and duration of signaling, as well as the existence of other pathways that may regulate the cell stress response.

Here we demonstrate that manipulation of PI3K can determine the outcome of ceramide and TNF-induced cell death pathways in MCF-7 cells. Given the convincing role of ceramide in numerous cellular processes as a stress signal mediator and the role of PI3K in cell survival and proliferation decisions, we further investigated the role of PI3K in the regulation of ceramide levels. We show that PI3K is capable of regulating cellular ceramide levels induced by both TNF and exogenous SMase, demonstrating the existence of cross-talk between the PI3K and SMase pathways.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials and Cell Culture-- Hoechst 33258, insulin, phorbol ester, and Bacillus cereus sphingomyelinase were obtained from Sigma. Human recombinant TNF and IGF-I were obtained from R & D Systems. X-Gal was obtained from Fisher. LY 294002 and C8-ceramide were obtained from Biomol. Cell culture reagents and LipofectAMINE were obtained from Life Technologies, Inc. MCF-7 human breast epithelial cells were maintained in 10% DMEM (high glucose containing Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.)), 10-10 M insulin (Sigma), basal minimum essential amino acids, minimum essential medium, amino acids, penicillin, streptomycin, and sodium pyruvate (Life Technologies, Inc.) as described previously (55).

Expression Constructs and Transfections-- The constructs used were the constitutively activated PI3K (pCG-p110* (N-terminal myristoylation signal, Myc-tagged)) and the kinase-dead PI3K (pCG-p110*Delta kin (N-terminally myristoylation signal, Myc-tagged)) which have been previously described (16, 56, 57). The constitutively active Akt (CA-Akt) (AKT-RC25 (N-terminally myristoylated, HA-tagged)) has also been previously described (16, 38, 58). MCF-7 cells were transfected with 3 µg of plasmid DNA per 1 × 106 cells using the LipofectAMINE method according to manufacturer's protocol (Life Technologies, Inc.) in Opti-MEM (Life Technologies, Inc.) for 6 h at which point the media were replaced with 10% DMEM for 48 h prior to experiments. Prior to experimentation the media were replaced with 0% DMEM, and the cells were treated accordingly.

Apoptosis Assays-- For fluorescent microscopy, cells were harvested and fixed in formalin, stained with bisbenzimide (Hoechst 33258) (50 µg/ml), and visualized using an Olympus axioscope microscope using appropriate filters. For DNA fragmentation analysis, cells were harvested, and genomic DNA was isolated and run using gel electrophoresis as described previously (55). Viability was determined as the percentage of viable cells per 500 cell counts as measured by trypan blue dye exclusion. For the crystal violet viability assay, MCF-7 cells were plated in 96-well plates at 1 × 104 cells per well in 10% DMEM. The cells were allowed to adhere for 24 h at which point the media were removed, cells were washed in PBS, the media were replaced with 0% DMEM, and the cells were treated appropriately. Twenty four hours later the media were removed, and cells were stained with a 0.5% crystal violet solution for 10 min, washed with PBS to remove excess crystal violet, and lysed in a 0.1% SDS solution. Absorbance as 540 nM was measured using a 96-well microplate reader. Percent viability was determined based on 100% as representing untreated control cells. Decreased viability represents a decrease in percentage of staining as compared with 100% control values. For the beta -galactosidase apoptosis assay (59), MCF-7 cells were transfected with 2 µg of either empty vector, p110*, or CA-Akt along with 1 µg of pCMV-beta -galactosidase for 6 h using the LipofectAMINE method. Following this, media was replaced with 10% DMEM overnight. The next day the media were changed to 0% DMEM, and cells were treated with or without TNF (10 ng/ml). 24 h later cells were fixed with 0.05% glutaraldehyde in the treated medium for 15 min. Cells were then washed 3 times with PBS and incubated in beta -galactosidase staining solution (20 mM K3Fe(CN)6, 20 mM K4Fe(CN)6, 1 mM MgCl2, 1 mg/ml X-gal) overnight at 37 °C. The staining solution was removed, and the cells were visualized using a phase contrast microscope with percent cell death determined by counting the number of apoptotic (rounded blue) versus total transfected cells from three independent experiments. The beta -galactosidase assay was also used to confirm transfection efficiency into MCF-7 cells.

Ceramide Analysis-- Ceramide was quantified by the diacylglycerol (DAG) kinase assay as 32P-incorporated upon phosphorylation of ceramide to ceramide 1-phosphate by DAG kinase as described previously (55, 60, 61). Briefly, MCF-7 cells were treated with or without TNF-alpha (10 ng/ml) for the times indicated, washed in PBS, and fixed in ice-cold methanol. After extraction of the lipid, ceramide contained within the organic phase extract was resuspended in 20 µl of 7.5% alpha -octyl-beta -glucopyranoside, 5 mM cardiolipin, 1 mM diethylenetriaminepentaacetic acid (Sigma). Thereafter, 40 µl of purified DAG kinase in enzyme buffer (20 mM Tris-HCl, 10 mM dithiothreitol, 1.5 M NaCl, 250 mM sucrose, and 15% glycerol, pH 7.4) was added to the organic phase extract. [gamma -32P]ATP (20 µl 10 mM; 1000 dpm/pmol), in buffer, was added to start the reaction. After 30 min at 22 °C, the reaction was stopped by extraction of lipids with 1 ml of chloroform:methanol:hydrochloric acid (100:100:1, v/v). Buffered saline solution (170 µl; 135 mM NaCl, 1.5 mM CaCl2, 0.5 mM MgCl2, 5.6 mM glucose, and 10 mM Hepes, pH 7.2) and 30 µl of 100 mM EDTA were added. The lower organic phase was dried under N2. Ceramide 1-phosphate was resolved by TLC using CHCl3:CH3OH:acetic acid (65:15:5, v/v) as solvent and detected by autoradiography, and the incorporated 32P was quantified by phosphorimaging (Fugi BAS1000, Fugi Medical Systems, USA). The level of ceramide was determined by comparison to a concomitantly run standard curve composed of known amounts of ceramide. During initial examination of ceramide generation by TNF treatment in MCF-7 cells, our laboratory measured ceramide levels by both DAG kinase assay and high pressure liquid chromatographic analysis. These studies were in agreement with the recent publication of Garzotto et al. (61) which demonstrated a linear correlation between ceramide generation measured by high pressure liquid chromatography and DAG kinase assay.

Western Blot Analysis-- Analysis of Myc tagged-PI3K expression and poly(A)DP-ribose polymerase (PARP) cleavage were assessed using Western blot analysis as described previously (55). Briefly, 50 µg of cell lysate was run on 8% SDS-PAGE gels, transferred, and probed with anti-PARP-specific monoclonal antibodies (Santa Cruz Biotechnology) (1:5000) or anti-Myc-specific monoclonal antibodies (Invitrogen) (1:2500), followed by goat anti-mouse-specific secondary antibodies (1:5000) and developed using ECL chemiluminescent detection methods (Amersham Pharmacia Biotech).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Many peptide hormones including IGF-I, NGF, FGF, epidermal growth factor, IL-3, and GM-CSF act to support or promote survival in numerous cell types. Additionally, PMA, which binds to and activates PKC, also promotes survival in a cell type-specific manner. Significant research has focused on identification of signaling molecules that mediate the shared survival effects of these hormones. Phosphatidylinositol 3-kinase (PI3K) has emerged as a critical signaling molecule that regulates multiple cellular processes including survival and proliferation in numerous systems. In MCF-7 cells IGF-1 has been previously demonstrated to induce proliferation in a PI3K-dependent manner, whereas PMA treatment suppresses proliferation (21-24). We examined the ability of these two agents to promote cell survival of MCF-7 cells and to determine if PI3K represents a common intermediate for the survival pathways of both agents. Here we show that PMA and IGF-1 promote cell survival in MCF-7 cells treated with TNF (Fig. 1a). TNF (0.1-10 ng/ml) induced a dose-dependent decrease in cell viability that was inhibited by pretreatment with PMA (20 ng/ml) or IGF (100 ng/ml). This survival effect was blocked by the addition of LY 294002 (30 µM), a specific inhibitor of PI3K (62, 63). By using DNA fragmentation analysis to measure apoptosis, TNF-induced ladder formation was suppressed by the addition of PMA and IGF (Fig. 1b). The addition of LY 294002 blocked the PMA and IGF suppression of apoptosis, restoring DNA ladder formation. This finding demonstrated that both PMA and IGF-I are acting as survival factors in MCF-7 cells, suppressing the induction of apoptosis. We have previously demonstrated that in MCF-7 cells TNF (10 ng/ml) induces a time-dependent cleavage of PARP (55). The ability of PMA to suppress TNF-induced apoptosis correlates with the ability of this agent to suppress partially PARP cleavage (data not shown). The restoration of TNF-induced PARP cleavage by pretreatment with LY 294002 confirms a role for PI3K in survival signaling by PMA. Exposure of MCF-7 human breast carcinoma cells to TNF resulted in a dose-dependent induction of apoptosis as determined by DNA fragmentation analysis and the appearance of key morphological features of apoptosis as measured by fluorescence microscopy (Fig. 2, a and b) (55, 65). To investigate the ability of PI3K to suppress TNF-induced apoptosis, we transfected MCF-7 cells with vectors containing a PI3K construct that was mutated to be either constitutively active (p110*) or kinase-dead (p110*Delta kin) (36, 56, 57). Treatment with TNF revealed a dose-dependent decrease in cell viability in vector control cells (Vec), whereas cells expressing p110* were more resistant to TNF-mediated cell death (Fig. 2c). In comparison, the p110*Delta kin-expressing cells showed an enhanced cytotoxic effect in response to TNF. DNA fragmentation analysis confirmed that p110*-transfected cells were more resistant to TNF-induced apoptosis (Fig. 2d). The role of PI3K and its downstream signaling target Akt in regulating TNF-induced apoptosis were examined using the beta -galactosidase apoptosis assay (59). TNF (10 ng/ml) exposure reduced the viability of vector-transfected cells to 45.4 ± 9.7% viability as compared with control. The expression of either p110* or CA-AKT, prior to TNF exposure, enhanced viability to 84.5 ± 3.6 and 88.4 ± 4.0%, respectively, as compared with vector-transfected cells. This demonstrated that the presence of a constitutively active PI3K signal is capable of subverting the TNF apoptotic signal.


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  		Fig. 1.   IGF-I and PMA suppression of TNF-induced apoptosis is dependent on PI3K. a, MCF-7 cells grown in 96-well plates were pretreated with or without LY 294002 (30 µM) for 30 min followed by PMA (20 ng/ml) or IGF-I (100 ng/ml) prior to treatment with TNF (10 ng/ml). Cells were harvested 24 h later for crystal violet viability assay. Error bars represent the standard error of the mean for three experiments, each with four replicates. diamond , TNF; , TNF and PMA; black-square, TNF and PMA and LY 294002; open circle , TNF and IGF; , TNF and IGF and LY 294002. b, IGF-I and PMA suppression of TNF-induced DNA fragmentation is dependent on PI3K. MCF-7 cells were pretreated with or without LY 294002 (Ly) (30 µM) for 30 min followed by PMA (20 ng/ml) or IGF-I (100 ng/ml) prior to treatment with TNF (10 ng/ml). Cells were harvested 48 h later for DNA fragmentation analysis. Con, control.


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  		Fig. 2.   PI3K-mediated regulation of TNF-induced apoptosis in MCF-7 cells. a, MCF-7 cells were treated with recombinant human TNF (10 ng/ml) and harvested after 48 h for assessment of apoptotic nuclear morphology by fluorescence microscopy. b, MCF-7 cells were treated with recombinant human TNF (0.01-100 ng/ml) and harvested after and 72 h for DNA fragmentation analysis. c and d, MCF-7 cells were transfected with a constitutively active PI3K construct (CA-PI3K), a kinase-dead PI3K construct (DN-PI3K), or empty vector (Vec). Transfected MCF-7 cells were serum-starved for 1 h prior to treatment with TNF at the concentrations indicated. Cells were harvested at 24 h, and viability was determined using trypan blue exclusion. Error bars represent standard error of the mean of three experiments with the absence of error bars indicating less that 2.5% S.E. Cells treated with TNF (0.01-10 ng/ml) were harvested after 72 h for DNA fragmentation analysis.

The ability of PMA, FGF, and PDGF to inhibit ceramide generation is correlated with the ability of these survival factors to suppress apoptosis (5-7, 54) suggesting sphingomyelinase activation represents one level of apoptotic regulation by survival factors. Consistent with previous studies, TNF induced a rapid and transient increase in ceramide over control with a 3.5-fold maximum occurring at 15 min (Fig. 3) (55, 62). To determine if the effects of PI3K manipulation on TNF-induced apoptosis are mediated through alteration in ceramide generation, we used both the MCF-7/p110*Delta kin and MCF-7/p110* cells. In cells expressing p110*, TNF-induced ceramide generation was completely suppressed, whereas transfection of p110*Delta kin resulted in an increase in both the magnitude and duration of ceramide generation (Fig. 3). We next evaluated the effect of DN-PI3K on the ability of PMA and IGF to suppress TNF-induced ceramide generation (Fig. 4, a and b). Transfection of DN-PI3K into MCF-7 cells completely reversed PMA's suppression of ceramide generation. DN-PI3K also affected IGF-induced suppression but not as strikingly.


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  		Fig. 3.   PI3K-mediated regulation of TNF-induced ceramide generation in MCF-7 cells. MCF-7 cells expressing either CA-PI3K, DN-PI3K, or empty vector were treated with TNF (10 ng/ml) for times shown and harvested for ceramide analysis. Error bars represent standard error of the mean of five experiments with the absence of error bars indicating less than 2.5% S.E.


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  		Fig. 4.   PMA- and IGF-mediated suppression of ceramide generation is dependent on PI3K. a, MCF-7 cells expressing either DN-PI3K (diamonds) or empty vector (Vec) (squares) were pretreated with PMA (20 ng/ml) (filled symbols) or without (open symbols) for 15 min followed by treatment with TNF (10 ng/ml) for the times indicated. Error bars represent standard error of the mean of three experiments with the absence of error bars indicating less that 0.15-fold S.E. b, MCF-7 cells expressing either DN-PI3K (diamonds) or empty vector (squares) were pretreated with IGF-I (100 ng/ml) (filled symbols) or without (open symbols) for 15 min followed by treatment with TNF (10 ng/ml) for the times indicated. Error bars represent standard error of the mean of three experiments with the absence of error bars indicating less than 0.15-fold S.E.

To evaluate further the ability of PI3K manipulation to affect the apoptotic response, MCF-7 cells were exposed to the water-soluble ceramide analogue, D-erythro-octanoylsphingosine (C8-ceramide). Treatment of MCF-7/Vec and MCF-7/p110* cells with the C8-ceramide analogue induced a dose-dependent loss of viability and apoptosis (Fig. 5, a and b). However MCF-7/P110*Delta kin cells underwent a more profound cell death and apoptosis with ceramide exposure at a lower concentration. This suggests that the ability of a constitutively active PI3K acts to suppress TNF and ceramide-induced apoptosis subsequent to or at the level of ceramide within the cell death cascade.


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  		Fig. 5.   PI3K-mediated regulation of ceramide-induced apoptosis in MCF-7 cells. MCF-7 cells were transfected with a constitutively active PI3K construct (CA-PI3K), a kinase-dead PI3K construct (DN-PI3K) or empty vector (Vec) for 48 h. Transfected MCF-7 cells were serum-starved for 1 h prior to treatment with D-erythro-N-octanoylsphingosine (C8-ceramide) at the concentrations indicated. a, cells were harvested at 24 h and viability determined using trypan blue exclusion. Error bars represent standard error of the mean of three experiments with the absence of error bars indicating less that 2.5% S.E. b, cells treated with C8-ceramide (20-80 µM) were harvested after 72 h for DNA fragmentation analysis.

We next investigated the possibility that the increased magnitude and duration of TNF-induced ceramide generation in p110*Delta kin cells and the suppression of ceramide generation in p110* cells may be due to the ability of PI3K to regulate cellular ceramide levels subsequent to the activation of sphingomyelinase. To examine this we used exogenous bacterial sphingomyelinase that has previously been shown to result in increased cellular ceramide levels (51, 66). The addition of B. cereus sphingomyelinase to MCF-7 cells resulted in a maximal 4.2-fold increase in ceramide levels at 15 min post-treatment which dropped and remained at a constant level (1.8-2.1-fold) out to 4 h (Fig. 6). However, in MCF-7/p110* cells treated with sphingomyelinase the early 15-min peak in ceramide was suppressed, and a maximal 2.4-fold level was reached at 120 min (Fig. 7a). To evaluate the effects of PI3K inhibition on sphingomyelinase-induced ceramide levels, MCF-7 cells were treated with the specific PI3K antagonist LY 294002 (30 µM) prior to sphingomyelinase exposure. The effects of PI3K inhibition enhanced sphingomyelinase-induced ceramide generation 2- and 1.72-fold over sphingomyelinase treatment alone at 15 min and 24 h, respectively (Fig. 7b).


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  		Fig. 6.   B. cereus sphingomyelinase induces generation of ceramide in MCF-7 cells. MCF-7 cells were treated with 300 milliunits/ml BcSMase for times indicated and harvested for ceramide analysis. Error bars represent standard error of the mean of two experiments with the absence of error bars indicating less that 2.5% S.E.


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  		Fig. 7.   PI3K-mediated regulation of sphingomyelinase-induced ceramide generation in MCF-7 cells. a, MCF-7 cells expressing either CA-PI3K or empty vector (Vec) were treated with 300 milliunits/ml BcSMase for times indicated and harvested for ceramide analysis. b, MCF-7 cells were pretreated with or without LY 294002 (30 µM) followed by BcSMase (300 milliunits/ml) for either 15 min or 24 h and harvested for ceramide analysis. Error bars represent standard error of the mean of three experiments with the absence of error bars indicating less that 2.5% S.E.

Consistent with the literature, B. cereus sphingomyelinase treatment did not result in apoptosis (Fig. 8, a and b) (66). However pretreatment of MCF-7 cells with LY 294002 prior to sphingomyelinase treatment resulted in the induction of apoptosis. Caspase activation represents a critical step in apoptotic signaling, and previous reports have indicated both ceramide analogues and TNF-induced cleavage of the caspase target PARP in MCF-7 cells (44, 52, 55). Treatment with sphingomyelinase resulted in PARP cleavage in combination with LY 294002 but not in its absence (Fig. 8c). Additionally, PARP cleavage in TNF-treated cells was enhanced by the addition of LY 294002 (data not shown). These data suggest that the blockade of the PI3K pathways in the presence of sphingomyelinase activation favors induction of apoptosis.


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  		Fig. 8.   Potentiation of sphingomyelinase-mediated apoptosis and PARP cleavage by inhibition of PI3K in MCF-7 cells. a, MCF-7 cells were pretreated with LY 294002 (30 or 60 µM) followed by either TNF (10 ng/ml) or BcSMase (300 milliunits/ml) and harvested after 24 h for trypan blue viability. Error bars represent standard error of the mean of three experiments with the absence of error bars indicating less that 2.5% S.E. b, MCF-7 cells were pretreated with LY 294002 followed by BcSMase (300 milliunits/ml) and harvested after 48 h for DNA fragmentation analysis. c, MCF-7 cells were pretreated with LY 294002 followed by BcSMase (300 milliunits/ml) and harvested after after 24 h for analysis of PARP cleavage.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phorbol esters as well as certain peptide hormones like IGF-1 act as survival factors suppressing apoptosis in a number of systems. In MCF-7 cells, IGF-I has been demonstrated to possess potent proliferative effects that are dependent on PI3K (21). Although in some systems PMA has been shown to activate PI3K (43), in MCF-7 cells it appears to inhibit proliferation through a prolonged activation of the ERK signaling cascade and subsequent up-regulation of P21/Waf1/Cip1 (22, 64). Despite their divergent effects on MCF-7 cell proliferation, both agents potently protected MCF-7 cells from TNF-induced cell death. This suggests that both compounds potentially activate common survival signaling intermediates. The addition of the PI3K inhibitor, LY 294002, blocked the cell survival effects of both agents and restored TNF-induced apoptosis, suggesting that an intact PI3K pathway is required for the full survival effects of both PMA and IGF. Other peptide hormones including NGF, PDGF, IL-3, GM-CSF, EPO, FGF, as well as IGF-1 and PMA have been shown to activate PI3K that has been proposed as a critical survival intermediate in a number of systems. These reports have, however, primarily studied peptide hormone and PI3K-mediated survival signaling in suppression of survival factor withdrawal-induced cell death. In these systems withdrawal or inhibition of survival factors results in apoptosis by default.

Here we demonstrate the ability of a PI3K-mediated pathway to suppress or delay the function of a direct inducer of cell death. The involvement of PI3K in suppression of TNF-induced apoptosis is confirmed by the expression of a constitutively active PI3K construct, which increased survival. Additionally, the transfection of a kinase-dead mutant PI3K construct resulted in an enhancement of TNF-induced apoptosis.

These studies established that cross-talk exists between TNF-activated cell death and PMA or IGF-mediated survival cascades. The importance of ceramide as an early intermediate for cell death signaling prompted us to investigate if regulation of ceramide levels by survival factors may represent a mechanism of action for these factors. PMA- and FGF-mediated suppression of ceramide generation by apoptotic inducers has been previously demonstrated in other cell types (5-7, 10, 14). In MCF-7 cells we showed an early generation of ceramide by TNF occurring maximally at 15 min and then returning to control levels by 60 min. To evaluate the possibility that PI3K regulated ceramide levels within MCF-7 cells, we again used transient transfection of either a CA-PI3K or a DN-PI3K. The transfection of a CA-PI3K resulted in a suppression of the TNF-induced ceramide peak to near control levels. However, in the DN-PI3K-transfected cells, both the magnitude and duration of the ceramide peak was increased, occurring maximally at 30 min and not returning to control levels even at 360 min. This suggested that molecular suppression of the PI3K cascade might occur through either direct regulation of the duration of SMase activation or at levels occurring subsequent to the generation of ceramide. We also demonstrated that transfection of a kinase-dead PI3K affected the ability of PMA and IGF to suppress ceramide levels. Whereas the early activation peak of ceramide was suppressed by PMA, the transfection of kinase-dead PI3K reversed this effect. The suppression of the early peak of ceramide generation by IGF was only partially reversed by DN-PI3K. The suppression of ceramide levels by exogenous agents such as PMA, IGF, and possibly other growth factors, appear to be dependent, in part, upon an intact PI3K pathway. However the differences observed between the temporal effects of IGF versus PMA on TNF-induced ceramide levels is suggestive of additional signaling pathways activated by these two factors. Both PMA and IGF appear to require an intact PI3K pathway to regulate ceramide levels but to differing degrees. These results suggest that the regulation of ceramide levels within cells may be dependent, both on activation by cell stress and apoptotic inducers, as well as through survival factors activating negative regulatory pathways that keep ceramide levels in check.

The intriguing cross-talk between the PI3K and SMase pathways prompted us to investigate further the mechanism of action by which this occurs. The addition of exogenous short chain water-soluble ceramide analogues has been previously shown to induce apoptosis in many systems including MCF-7 cells (65, 66). We demonstrated that transfection of CA-PI3K into MCF-7 cells resulted in a suppression of C8-ceramide and transfection of DN-PI3K- enhanced cell death by C8-ceramide. This suggests that the effects of PI3K in these cells are not exclusive to TNF, and given the role of ceramide in TNF-induced signaling, the PI3K survival pathway may mediate survival effects at the level of or subsequent to the generation of ceramide.

We next wished to investigate the mechanism by which PI3K activation or suppression affects ceramide levels and potentially mediated anti-apoptotic effects. One possibility is through direct effects on SMase activity. In this scenario a basal PI3K pathway may keep activation of SMase to a minimum and prevent full generation of ceramide after stimulation. Blockade of the PI3K-dependent suppression of SMase would then enhance TNF-induced signaling. However, this is unlikely because neither treatment with LY 294002 nor transfection of DN-PI3K alone affected ceramide levels in the absence of an inducer. The inability of either the CA-PI3K or the DN-PI3K to affect basal ceramide levels suggested that the role of PI3K in ceramide regulation occurred subsequent to its generation by SMase. To investigate this we used a novel approach. Previously identified and purified bacterial SMase from B. cereus (BcSMase) when added to cells in culture has been shown to generate ceramide levels through interaction and cleavage of sphingomyelin in the external membrane of cells (54, 65, 66). Although this does increase intracellular levels of ceramide, the ability of BcSMase to induce cell death is both system- and concentration-dependent. We have shown that similar to TNF, BcSMase induces an early peak of ceramide levels in MCF-7 cell occurring at 15 min. However, unlike TNF, BcSMase-induced ceramide levels do not return completely to control. The early peak in ceramide generation may be due to the BcSMase-induced sphingomyelin cleavage and subsequent metabolism to near control levels. However, the constant presence of activated BcSMase in the media provides a continuous cleavage of sphingomyelin in the external membrane. Transfection of a CA-PI3K into MCF-7 cells does suppress the early peak in ceramide generation, but the ceramide levels increase above control out to 2 h. Additionally, the suppression of PI3K by LY 294002 enhanced the levels of ceramide in MCF-7 cell treated with BcSMase at both the early 15-min time point and at 24 h.

The ability of PI3K to suppress the early activity of BcSMase-induced ceramide suggests this pathway is involved in affecting the ceramide levels in cells independent of the activation or presence of SMase and occurs through subsequent metabolism of ceramide within cells. It has been previously demonstrated that very high concentrations of BcSMase induced cell death in MCF-7 cells (65), but at lower concentrations, we did not observe apoptosis with BcSMase alone. Additionally, Zhang et al. (66) observed that the BcSMase treatment did not result in apoptosis that may be attributed to the differences in cellular localization of ceramide generation and/or accumulation. However, we observed that with the inhibition of PI3K, the BcSMase treatment resulted in apoptosis and in subsequent activation of the caspase cascade. We propose that ceramide acting as a cellular stress response intermediate alone is insufficient to induce apoptosis. This is consistent with the suggestion that TNF-induced apoptosis occurs primarily through a TNFR1-TRADD-Fas-associated death domain-caspase-dependent pathway with ceramide generation being dependent upon caspase activation (67, 68). However, with the suppression of PI3K, the prolonged and enhanced ceramide signal is capable of functioning to promote apoptosis. In contrast, overactivation of the PI3K pathway suppresses the ceramide signal thereby facilitating cell survival. It is therefore interesting to speculate that it is the relative balance between these two early lipid signaling pathways that in part determines the outcome of cell survival/death decisions and ultimately cell fate.

The plethora of recent studies on PI3K has strongly implicated this pathway as a critical intermediate in survival signaling by a number of agents. We along with others (25, 40, 69) have now demonstrated the ability of PI3K to suppress cell death induced by members of the death receptor family. Whereas a constitutively active PI3K suppresses both TNF and ceramide-induced apoptosis, a kinase-dead mutant of PI3K is capable of sensitization of MCF-7 cells to apoptosis induced by both agents. Although a link between PI3K/Akt and Bad phosphorylation has been proposed as a mechanism for cell survival, the ability of PI3K to determine cell fate may occur through the regulation of multiple pathways (45- 47, 69). Recently, ceramide has been shown to suppress PKB/Akt activity suggesting a potential cross-talk between the SMase and PI3K lipid signaling pathways (33, 48-50). Here we demonstrate that both TNF and exogenous SMase-induced ceramide levels within MCF-7 cells are regulated by manipulation of PI3K activity. Taken together these findings along with our results establish that cross-talk occurs between the PI3K and SMase pathways which may function as a mechanism for cell survival. Given the convincing role of ceramide in numerous cellular processes as a stress signal mediator and the role of PI3K in cell survival and proliferation decisions, we propose a more universal role for these two signals as a part of the cellular machinery which regulates early lipid signaling events, the output of which determines the fate of the cell.

    FOOTNOTES

* This work was supported by a predoctoral fellowship from the United States Department of Defense Breast Cancer Research Program DAMD17-97-1-7024 (to M. E. B.), National Institutes of Health Grant 1 T32 CA65436-01A3 (to C. B. W.), the Cancer Association of Greater New Orleans (to B. S. B. and A. M.), the Tulane Cancer Center (to B. S. B.), and the Tulane-Xavier Center for Bioenvironmental Research (to J. A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

h To whom correspondence should be addressed: Dept. of Pharmacology, Tulane University Medical Center, 1430 Tulane Ave. SL-83, New Orleans, LA 70112. Tel.: 504-584-2631; Fax: 504-588-5283; E-mail: bbeckman@tmcpop.tmc.tulane.edu.

    ABBREVIATIONS

The abbreviations used are: TNF, tumor necrosis factor-alpha ; TNFR1, TNF receptor 1; TRADD, TNF receptor 1-associated death domain; JNK, c-Jun N-terminal kinase; NF-kappa B, nuclear factor kappa B; PI3K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol-3,4,5-phosphate; PKC, protein kinase C; PKB, protein kinase B; PDGF, platelet-derived growth factor; NGF, nerve growth factor; IGF, insulin-like growth factor 1; PMA, phorbol ester; PARP, poly(ADP)ribose polymerase; SMase, sphingomyelinase; BcSMase, B. cereus sphingomyelinase; FGF, fibroblast growth factor; GM-CSF, granulocyte macrophage-colony-stimulating factor; IL-3, interleukin-3; X-gal, 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside; DMEM, Dulbecco's modified Eagle's medium; DAG, diacylglycerol; DN-PI3K, kinase-dead PI3K construct; CA-PI3K, constitutively active PI3K construct.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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