Ceramide-induced Translocation of Protein Kinase C-delta and -epsilon to the Cytosol
IMPLICATIONS IN APOPTOSIS*

(Received for publication, July 15, 1996, and in revised form, September 16, 1996)

Hirofumi Sawai Dagger , Toshiro Okazaki Dagger §, Yasushi Takeda Dagger , Masaro Tashima Dagger , Hiroyoshi Sawada Dagger , Minoru Okuma Dagger , Shuji Kishi par , Hisanori Umehara § and Naochika Domae §

From the Dagger  Department of Hematology and Oncology, Clinical Sciences for Pathological Organs, Graduate School of Medicine, Kyoto University, 54 Shogoin-kawaramachi, Sakyo-ku, Kyoto 606, Japan, the § Department of Medicine, Osaka Dental University, 1-5-31 Otemae, Chuo-ku, Osaka 540, Japan, and par  Pharmaceutical Basic Research Laboratories, Japan Tobacco Inc., 13-2 Fukuura, 1-chome Kanazawa-ku, Yokohama, Kanagawa 236, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Ceramide is now recognized as an intracellular lipid signal mediator, which induces various kinds of cell functions including apoptosis. Ceramide-induced apoptosis was reported to be blocked by 12-O-tetradecanoylphorbol 13-acetate, a protein kinase C (PKC) activator, but its mechanism remained unclear. Therefore, we investigated whether ceramide has any effects on PKC in the induction of apoptosis. We here report that N-acetylsphingosine (synthetic membrane-permeable ceramide) induced translocation of PKC-delta and -epsilon isozymes from the membrane to the cytosol within 5 min in human leukemia cell lines. Treatment with sphingomyelinase, tumor necrosis factor-alpha , or anti-Fas antibody, all of which can induce apoptosis by generating natural ceramide, similarly induced cytosolic translocation of PKC-delta and -epsilon . In Fas-resistant cells anti-Fas antibody did not induce cytosolic translocation of PKC-delta and -epsilon because of no generation of ceramide, whereas N-acetylsphingosine induced apoptosis with cytosolic translocation of PKC-delta and -epsilon . Furthermore, both 12-O-tetradecanoylphorbol 13-acetate and a nonspecific kinase inhibitor, staurosporine, prevented ceramide-induced apoptosis by inhibiting cytosolic translocation of PKC-delta and -epsilon . These data suggest that cytosolic translocation of PKC-delta and -epsilon plays an important role in ceramide-mediated apoptosis.


INTRODUCTION

Sphingolipids have recently emerged as intracellular signal mediators in a variety of cell functions (1). Hannun et al. (2, 3) reported that sphingosine, the backbone of sphingolipids, and lysosphingolipids inhibit PKC1 in vitro and in vivo. Thereafter, the "sphingomyelin cycle," transient hydrolysis of sphingomyelin and concomitant generation of ceramide, was discovered in the early phase of monocytic differentiation of human leukemia HL-60 cells induced by 1alpha ,25-dihydroxy-vitamin D3 (4, 5). Many reports have supported the idea that ceramide is an intracellular signal mediator transducing the effects of various extracellular stimulants including tumor necrosis factor-alpha (TNF-alpha ) (6, 7), gamma -interferon (6), interleukin-1 (8), and nerve growth factor (9). Recent studies have shown that ceramide plays an important role in apoptosis (10). Besides TNF-alpha , apoptosis-inducing stimuli such as cross-linking of Fas (11, 12), ionizing radiation (13), glucocorticoid (14), anti-immunoglobulin antibody (15), anti-cancer drugs (16, 17), and serum deprivation (18) have been reported to induce the sphingomyelin hydrolysis and/or generation of ceramide.

PKC is a family of serine/threonine kinases that takes part in various cellular responses (19). Molecular cloning and biochemical studies have revealed the presence of at least 10 PKC isozymes that can be classified into three subgroups. The classical PKC members (alpha , beta I, beta II, and gamma ) are activated by Ca2+, phosphatidylserine, and diacylglycerol (DAG) or phorbol esters. The novel PKC members (delta , epsilon , eta /L, and theta ), which lack the C2 region, are activated by phosphatidylserine and DAG or phorbol esters without Ca2+. The atypical PKC members (zeta  and lambda ), which have only one cysteine-rich region, are dependent on phosphatidylserine but are not affected by DAG, phorbol esters, or Ca2+. The role of different PKC isozymes in cellular functions remains unclear.

It has been reported that ceramide has no effect on PKC activity in vitro (2), but it remains unclear whether ceramide has any effect on PKC in vivo. It is known that activation of PKC by DAG or phorbol esters induces the translocation of PKC from the cytosol to the membrane fraction (20) and inhibits ceramide-induced apoptosis (10, 18, 21). Although it was recently reported that ceramide inhibited the membranous translocation of PKC induced by PKC-activating stimuli (22, 23) and inactivated PKC-alpha (24), the mechanisms by which PKC activators inhibit ceramide-induced apoptosis are still not known. We therefore investigated the change of the subcellular distribution of each PKC isozyme in the apoptosis-inducing process by ceramide and ceramide-generating signals. We here show that N-acetylsphingosine (C2-ceramide), membrane-permeable synthetic ceramide, induced the translocation of PKC-delta and -epsilon from the membrane to the cytosol fraction in three human leukemia cells (HL-60, U-937, and HPB-ALL cells). Induction of apoptosis by neutral bacterial sphingomyelinase, TNF-alpha , or anti-Fas antibody, all of which generate natural ceramide, induced cytosolic translocation of PKC-delta and -epsilon as well. Furthermore, we show the following lines of evidence demonstrating how ceramide-induced cytosolic translocation of PKC-delta and -epsilon is indispensable in leukemic cell apoptosis: 1) HPB-alpha FR cells are resistant to anti-Fas antibody-inducing apoptosis because of no generation of ceramide by anti-Fas antibody and the consequent failure of cytosolic translocation of PKC-delta and -epsilon ; 2) ceramide can induce both apoptosis and cytosolic translocation of PKC-delta and -epsilon in HPB-alpha FR cells; 3) translocation of PKC-delta and -epsilon to the membrane by 12-O-tetradecanoylphorbol 13-acetate (TPA) or staurosporine inhibits ceramide-induced apoptosis as a consequence of prevention of cytosolic translocation of PKC-delta and -epsilon . These data suggest that ceramide-induced cytosolic translocation of PKC-delta and -epsilon plays an important role in the signaling pathway leading to apoptosis. We also discuss the topological meanings of PKC translocation in apoptotic signals.


EXPERIMENTAL PROCEDURES

Materials

D-erythro-C2-ceramide was purchased from Matreya, Inc. D-erythro-N-Acetyldihydrosphingosine (D-erythro-C2-dihydroceramide) was kindly provided by Dr. Y. A. Hannun (Duke University). p-Amidinophenyl methanesulfonyl fluoride hydrochloride was purchased from Wako (Osaka, Japan). Recombinant neutral bacterial sphingomyelinase was purchased from Higeta-Shoyu (Choshi, Japan). Other chemicals were obtained from Sigma.

Cell Culture

Human myelogenous leukemia HL-60 cells, human monoblastic leukemia U-937 cells, and human T-lymphoblastic leukemia HPB-ALL cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum at 37 °C in a 5% CO2 incubator. HL-60 and U-937 cells in exponentially growing phase were washed once in RPMI 1640 medium containing 5 µg/ml transferrin and 5 µg/ml insulin instead of serum, resuspended in the serum-free medium at a concentration of 5 × 105 cells/ml overnight, and then used for experiments. HPB-ALL cells were treated in serum-containing medium. The HPB-alpha FR subline, resistant to anti-Fas antibody, was obtained by continuous culture of HPB-ALL cells with anti-Fas antibody.

Preparation of Cell Extracts

Subcellular fractionation was performed as described (25, 26) with modifications. The cells were washed once with ice-cold phosphate-buffered saline and lysed in buffer A (20 mM Tris/HCl, pH 7.4, 10 mM EDTA, 5 mM EGTA, 0.1% 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 1 mM p-amidinophenyl methanesulfonyl fluoride hydrochloride, 100 µg/ml leupeptin, 0.15 unit/ml aprotinin) by passing through a 27-gauge needle. Complete cell lysis was confirmed by microscopy. The lysate was centrifuged at 800 × g for 5 min at 4 °C in order to remove nuclei, and the supernatant was centrifuged at 100,000 × g for 20 min at 4 °C in a Beckman TL-100s ultracentrifuge. The supernatant was collected and used as the cytosol fraction. The membrane pellet was solubilized in buffer A containing 1% Triton X-100 by bath sonication and centrifuged at 12,000 × g for 10 min at 4 °C in a microcentrifuge, and the supernatant was used as the membrane fraction. Protein concentration was determined by using the Protein Assay kit (Bio-Rad).

Western Blot Analysis

The samples (50 µg) were denatured by boiling in Laemmli sample buffer for 5 min, subjected to SDS-polyacrylamide gel electrophoresis using a 7.5% running gel, and electroblotted to Immobilon-P transfer membrane (Millipore Corp.) as described (27). Nonspecific binding was blocked by incubation of the membrane with PBS containing 5% skim milk and 0.1% Tween 20 for more than 1 h. Then the membrane was washed in PBS containing 0.1% Tween 20 (PBS-T) for 15 and 5 min and incubated with a 1:200 dilution of rabbit anti-PKC isozymes (Santa Cruz Biotechnology) in PBS-T for 1 h. The membrane was washed in PBS-T for 15 and 5 min and incubated with a 1:4000 dilution of goat anti-rabbit immunoglobulin peroxidase conjugate (Tago) in PBS-T for 1 h. After washing the membrane for 3 × 5 min in PBS-T, detection of PKC isozymes was performed using ECL Western blotting detection reagents (Amersham Corp.) according to the manufacturer's protocol. Recombinant human PKC isozymes were kindly provided by Dr. D. J. Burns and used as positive controls.

Analysis of DNA Fragmentation

DNA extraction was performed using the G NOME DNA isolation kit (BIO 101) according to the manufacturer's protocol with modification. Briefly, the cells (1 × 106) were washed once in PBS and resuspended in 185 µl of cell suspension solution. After the addition of 5 µl of RNase Mixx and 10 µl of cell lysis/denaturing solution, the cell lysate was incubated at 55 °C for 15 min. Then 3 µl of Protease Mixx was added to the lysate, and the mixture was further incubated for 2 h. After the addition of 50 µl of Salt Out mixture, the mixture was cooled on ice for 10 min and centrifuged at 100,000 × g for 10 min at 4 °C in a Beckman TL-100s ultracentrifuge. Then 1 ml of 80% ethanol diluted with TE buffer (10 mM Tris/HCl, pH 8.0, 1 mM EDTA) was added to the supernatant, stored at -20 °C for 2 h, and centrifuged at 12,000 × g for 15 min at 4 °C in a microcentrifuge. The DNA pellet was dissolved in TE buffer (pH 8.0). The concentration of DNA was calculated by determining the absorbance at 260 nm. Electrophoresis was carried out through 3% NuSieve-agarose (FMC BioProducts) minigel in 1 × TAE buffer (0.04 M Tris acetate, 0.001 M EDTA) at 50 V for 1.5 h. DNA was visualized under UV light after staining with ethidium bromide.

Ceramide Measurement

Extraction of cellular lipids and ceramide measurement using DAG kinase were performed as described (4). The solvent system to separate ceramide phosphate and phosphatidic acid was chloroform/acetone/methanol/acetic acid/H2O (10:4:3:2:1).


RESULTS

Translocation of PKC-delta and -epsilon from Membrane to Cytosolic Fraction by Treatment with C2-ceramide

Because treatment with 10 µM C2-ceramide induced apoptosis (internucleosomal DNA fragmentation) of human myelogenous leukemia HL-60 cells within 2 h (data not shown), we examined the effect of 10 µM C2-ceramide on the cellular distribution of PKC isozymes within 1 h before the cells showed apoptotic characteristics. As shown in Fig. 1, A and B, PKC-delta and -epsilon , which initially existed more abundantly in the membrane than in the cytosol fraction, showed translocation from the membrane to the cytosol fraction, reaching a maximum within 2-5 min by treatment with C2-ceramide. The increased levels of PKC-delta and -epsilon in the cytosol fraction subsequently decreased near the control levels after 30 min, whereas the levels of PKC-delta and -epsilon in the membrane fraction once recovered after 15 min but decreased after 30 min (Fig. 1B). The reason for the transient nature of cytosolic translocation of PKC-delta and -epsilon may not be due to the metabolism of ceramide to other inactive compounds because the metabolism of C2-ceramide is not so rapid in HL-60 cells as we reported before (5), whereas another report showed the extensive metabolism of C2-ceramide in Chinese hamster ovary cells (28). Although it remains to be elucidated why PKC-delta and -epsilon disappeared from the cytosol, one possibility is that PKC-delta and -epsilon may be degraded after translocation to the cytosol by some proteases as mentioned in a recent report (29). In terms of the brief reappearance of PKC-delta and -epsilon in the membrane, the increase of DAG by treatment with ceramide might be the cause, but it is at present unclear. The classical PKC (alpha , beta II, and gamma ) and the atypical PKC-zeta did not show significant changes in the subcellular distribution by treatment with C2-ceramide (Fig. 1A). Further experiments were performed to determine whether ceramide translocated PKC-delta and -epsilon to the nucleus, but no significant changes of PKC-delta and -epsilon were detected in the nuclear fraction (data not shown).


Fig. 1. Cytosolic translocation of PKC-delta and -epsilon induced by ceramide. Subcellular fractionation and Western blot analysis were performed as described under "Experimental Procedures." The results are representative of at least three different experiments. A, effects of C2-ceramide on the subcellular localization of PKC-alpha , -beta II, -gamma , -delta , -epsilon , and -zeta isozymes in HL-60 cells. The cells were treated with 10 µM C2-ceramide for 0, 5, 15, and 30 min. A single band was detected at the position corresponding to each recombinant PKC isozyme except PKC-gamma , detected as double bands. The position of PKC-zeta is indicated by the arrow. B, time course of the ceramide-induced cytosolic translocation of PKC-delta and -epsilon . HL-60 cells were treated with 10 µM C2-ceramide for the indicated times. The densities were assessed using NIH Image version 1.47, and -fold induction was calculated by comparing each density with that of the control in the cytosol (left) or in the membrane (right) fraction. Squares, PKC-delta ; circles, PKC-epsilon . C, dose dependence of the ceramide-induced cytosolic translocation of PKC-delta and -epsilon . HL-60 cells were treated with the indicated concentrations of C2-ceramide for 5 min. D and E, effects of C2-ceramide on the subcellular localization of PKC-delta and -epsilon in U-937 cells (D) or in HPB-ALL or HPB-alpha FR cells (E). The cells were treated with the indicated concentrations of C2-ceramide for 5 min.
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C2-ceramide induced the cytosolic translocation of PKC-delta and -epsilon in a dose-dependent manner (Fig. 1C). The increase of PKC-delta and -epsilon in the cytosol fraction was detected at the concentration as low as 1 µM C2-ceramide, which began to inhibit HL-60 cell growth (5). Because ceramide induced apoptosis in monoblastic leukemia U-937 cells and T-lymphoblastic leukemia HPB-ALL cells as well (data not shown), we investigated whether ceramide could induce translocation of PKC-delta and -epsilon to the cytosol fraction in these cell lines. Translocation of PKC-delta and -epsilon from the membrane to the cytosol was also detected in these cell lines (Fig. 1, D and E), suggesting that this translocation of PKC-delta and -epsilon might be a general phenomenon in ceramide-induced leukemic cell apoptosis.

Specificity of Cytosolic Translocation of PKC-delta and -epsilon Induced by Ceramide

C2-dihydroceramide, which has almost the same chemical structure as C2-ceramide except for lacking the C4-C5 double bond of sphingoid backbone, did not induce apoptosis in HL-60 cells in contrast to C2-ceramide (30). C2-dihydroceramide did not induce significant translocation of PKC-delta and -epsilon (Fig. 2A), demonstrating the biological specificity of ceramide effects on the cytosolic translocation of PKC-delta and -epsilon . The effects of natural ceramide on translocation of PKC-delta and -epsilon to the cytosol were investigated by treating HL-60 cells with exogenous neutral bacterial sphingomyelinase (SMase). Because SMase purified from bacteria may contain some other phospholipases, we used recombinant neutral SMase (Higeta-Shoyu, Japan) (31, 32). The cytosolic translocation of PKC-delta and -epsilon was detected 60 min after treatment with 100 milliunits/ml SMase (Fig. 2B), which induced DNA fragmentation within 3 h in HL-60 cells (data not shown). SMase-induced translocation occurred later and more weakly than C2-ceramide-induced translocation, presumably due to insufficient action of ceramide generated in the outer membrane by exogenous SMase. In order to examine the specificity of SMase action, we checked the effects of other phospholipases on the subcellular localization of PKC-delta and -epsilon (Fig. 2B). Treatment with the phospholipases other than SMase did not induce apoptosis within at least 6 h in HL-60 cells, whereas 30-50% of the cells showed apoptotic changes by treatment with SMase in 6 h. PKC-delta in the cytosol fraction was decreased within 5 min of treatment with phosphatidylinositol-specific phospholipase C, which generates DAG and inositol trisphosphate from phosphatidylinositol, and remained under the control until 60 min after treatment. It was likely that translocation of PKC from the cytosol to the membrane was induced by phosphatidylinositol-specific phospholipase C-generated DAG. Neither phospholipase D, which generates phosphatidic acid mainly from phosphatidylcholine, nor phospholipase A2, which generates arachidonic acid and lysophosphatidylcholine, induced significant changes in the subcellular localization of PKC-delta . No significant changes of translocation of PKC-epsilon were detected by phosphatidylinositol-specific phospholipase C, phospholipase D, or phospholipase A2. These results suggest that natural ceramide generated by SMase was specifically involved in translocation of PKC-delta and -epsilon to the cytosol in contrast to other lipid second messengers including DAG, phosphoinositide, phosphatidic acid, and arachidonic acid.


Fig. 2. Specificity of the effect of ceramide on the cytosolic translocation of PKC-delta and -epsilon . Subcellular fractionation and Western blot analysis were performed as described under "Experimental Procedures." The results are representative of three different experiments. A, effects of dihydroceramide on the subcellular distribution of PKC-delta and -epsilon . HL-60 cells were treated with 0.1% ethanol vehicle (-), 10 µM C2-ceramide (C2), or 10 µM C2-dihydroceramide (DH) for 5 min. B, effects of recombinant Bacillus cereus neutral SMase (SMase), phosphatidylinositol-specific phospholipase C from B. cereus (PLC), phospholipase D from Streptomyces chromofuscus (PLD), or phospholipase A2 from Streptomyces violaceoruber (PLA2) on the subcellular distribution of PKC-delta and -epsilon in HL-60 cells. The cells were either untreated (-) or treated (+) with 100 milliunits/ml SMase or other phospholipases for 60 min.
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TNF-alpha and Anti-Fas Antibody Induced Cytosolic Translocation of PKC-delta and -epsilon

Generation of ceramide has been reported to be induced by various stimuli including TNF-alpha (6, 7) and anti-Fas antibody (11, 12). We investigated whether these biological stimuli translocated PKC-delta and -epsilon to the cytosol in the process of apoptosis. As shown in Fig. 3, TNF-alpha in U-937 cells induced cytosolic translocation of PKC-delta and -epsilon more evidently than in HL-60 cells, probably because U-937 cells were more susceptible to the apoptosis-inducing effect of TNF-alpha than HL-60 cells (data not shown). Cytosolic translocation of PKC-delta and -epsilon was observed within 5-15 min after treatment with TNF-alpha in U-937 cells. Because HL-60 and U-937 cells were not very sensitive to anti-Fas antibody (data not shown), we used HPB-ALL cells, highly sensitive to anti-Fas antibody, to examine its effect on PKC translocation. Treatment with anti-Fas antibody induced cytosolic translocation of PKC-delta and -epsilon within 15-60 min (Fig. 4A). In order to confirm the role of translocation to the cytosol in induction of apoptosis, we used a Fas-resistant HPB-ALL subline (HPB-alpha FR) in which anti-Fas antibody hardly induced apoptosis (Fig. 4B) and did not generate ceramide at least within 3 h, whereas in HPB-ALL cells anti-Fas antibody induced ceramide generation (Fig. 4C). In HPB-alpha FR cells, treatment with anti-Fas antibody did not induce cytosolic translocation of PKC-delta and -epsilon (Fig. 4A), whereas treatment with C2-ceramide translocated PKC-delta and -epsilon from the membrane to the cytosol and subsequently induced apoptosis (Figs. 1E and 4B). These results suggest that physiological apoptotic stimuli including TNF-alpha and anti-Fas antibody may require ceramide-mediated cytosolic translocation of PKC-delta and -epsilon .


Fig. 3. Effects of TNF-alpha on cytosolic translocation of PKC-delta and -epsilon in HL-60 cells and U-937 cells. The cells were either untreated or treated with TNF-alpha (100 ng/ml) for 5 min. Subcellular fractionation and Western blot analysis were performed as described under "Experimental Procedures." The results are representative of three different experiments.
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Fig. 4. Effects of anti-Fas antibody or C2-ceramide on cytosolic translocation of PKC-delta and -epsilon in Fas-sensitive HPB-ALL cells and Fas-resistant HPB-alpha FR cells. A, the cells were either untreated (-) or treated with anti-Fas antibody (100 ng/ml) for 15 min (+). Subcellular fractionation and Western blot analysis were performed as described under "Experimental Procedures." The results are representative of three different experiments. B, effects of anti-Fas antibody or C2-ceramide on the growth of HPB-ALL cells and HPB-alpha FR cells. The cells were treated with the indicated concentrations of anti-Fas antibody or C2-ceramide for 24 h, and the viable cell number was counted by the trypan blue dye exclusion test. C, effects of anti-Fas antibody on the levels of ceramide in HPB-ALL or HPB-alpha FR cells. The cells were treated with 100 ng/ml anti-Fas antibody for 5-180 min, and ceramide measurement was performed as described under "Experimental Procedures." The results were obtained from three different experiments. The bars indicate one S.D.
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Inhibition of Both Ceramide-induced DNA Fragmentation and Cytosolic Translocation of PKC-delta and -epsilon by Treatment with TPA and Staurosporine

Previous reports have shown that phorbol esters inhibited ceramide-induced apoptosis (10, 18). We confirmed that TPA inhibited DNA fragmentation induced by C2-ceramide in a dose-dependent manner in HL-60 cells (Fig. 5A). The cytosolic translocation of PKC induced by C2-ceramide was also inhibited by treatment with TPA at the concentrations that blocked ceramide-induced apoptosis (Fig. 5B). Because higher concentrations of TPA induced rapid down-regulation of PKC-delta , the amount of PKC-delta in the membrane fraction was decreased within 5 min by treatment with C2-ceramide and 10 nM TPA and remained down-regulated at least after 30 min. PKC-epsilon was also down-regulated within 15 min of treatment with TPA (data not shown). Conversely TPA-induced translocation of PKC-delta and -epsilon from the cytosol to the membrane was competed by C2-ceramide dose-dependently (Fig. 5C), and higher concentrations of C2-ceramide induced apoptosis against treatment with TPA (Fig. 5D). Furthermore, we investigated the effects of a synthetic DAG analogue, 1,2-dioctanoyl-sn-glycerol (diC8) on the ceramide-induced translocation of PKC-delta and -epsilon to the cytosol, because DAG is a critical physiological activator of PKC. We found that diC8 inhibited the translocation of PKC-delta and -epsilon to the cytosol by C2-ceramide as well as TPA did (Fig. 5E). Although it was reported that diC8 inhibited ceramide-induced apoptosis in HL-60 cells (18, 21), in our hands it did not inhibit ceramide-induced DNA fragmentation, presumably because of the short duration of action due to its rapid metabolism compared with TPA and because a higher concentration (20 µM) of diC8 induced DNA fragmentation itself (data not shown).


Fig. 5. Effects of TPA on ceramide-induced apoptosis and translocation of PKC-delta and -epsilon . A, inhibition of ceramide-induced DNA fragmentation by TPA in HL-60 cells. The cells were treated with the indicated concentrations of TPA in the presence (+) or absence (-) of 5 µM C2-ceramide (C2) for 6 h. Analysis of DNA fragmentation was performed as described under "Experimental Procedures." The results are representative of three different experiments. B, inhibition of ceramide-induced cytosolic translocation of PKC-delta and -epsilon by TPA. HL-60 cells were treated with the indicated concentrations of TPA in the presence (+) or absence (-) of 10 µM C2-ceramide (C2) for 5 min. Subcellular fractionation and Western blot analysis were performed as described under "Experimental Procedures." The results are representative of three different experiments. C, inhibition of TPA-induced translocation of PKC-delta and -epsilon to the membrane by ceramide. HL-60 cells were treated with the indicated concentrations of C2-ceramide in the presence (+) or absence (-) of 1 nM TPA for 5 min. Subcellular fractionation and Western blot analysis were performed as described under "Experimental Procedures." The results are representative of two different experiments. D, induction of apoptosis by increasing doses of ceramide against TPA. HL-60 cells were treated with the indicated concentrations of C2-ceramide in the presence (+) or absence (-) of 1 nM TPA for 3 h. Analysis of DNA fragmentation was performed as described under "Experimental Procedures." The results are representative of two different experiments. E, competing effects of DAG on ceramide-induced cytosolic translocation of PKC-delta and -epsilon . HL-60 cells were treated with the indicated concentrations of diC8 in the presence (+) or absence (-) of 5 µM C2-ceramide (C2) for 5 min. Subcellular fractionation and Western blot analysis were performed as described under "Experimental Procedures." The results are representative of three different experiments. bp, base pairs.
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Because staurosporine, a nonspecific kinase inhibitor, was reported to induce the translocation of PKC-epsilon from the cytosol to the membrane in SH-SY5Y human neuroblastoma cells (33), we examined its effect on the ceramide-induced cytosolic translocation of PKC-delta and -epsilon . We found that staurosporine translocated PKC-delta as well as PKC-epsilon in HL-60 cells and inhibited the cytosolic translocation of PKC-delta and -epsilon induced by C2-ceramide (Fig. 6A). Furthermore, we investigated the effect of staurosporine on ceramide-induced apoptosis and found that ceramide-induced DNA fragmentation was suppressed by staurosporine (Fig. 6B). These results demonstrated that ceramide-induced apoptosis and cytosolic translocation of PKC-delta and -epsilon was inhibited by both PKC activator and non-PKC activator, which translocated PKC-delta and -epsilon from the cytosol to the membrane, and therefore membranous translocation (rather than activation) of PKC-delta and -epsilon might be necessary to inhibit ceramide-induced apoptosis. In other words, it is suggested that cytosolic translocation of PKC-delta and -epsilon plays an important role in ceramide-mediated apoptosis.


Fig. 6. Effects of staurosporine on ceramide-induced apoptosis and cytosolic translocation of PKC-delta and -epsilon . A, inhibition of ceramide-induced cytosolic translocation by staurosporine. HL-60 cells were treated with 10 nM staurosporine (St), 10 µM C2-ceramide (C2), or both. Subcellular fractionation and Western blot analysis were performed as described under "Experimental Procedures." The results are representative of three different experiments. B, inhibition of ceramide-induced apoptosis by staurosporine. HL-60 cells were treated for 3 h with ethanol vehicle (C), 10 µM C2-ceramide (C2), or both 3 nM staurosporine and 10 µM C2-ceramide (C2 + St). Analysis of DNA fragmentation was performed as described under "Experimental Procedures." The results are representative of three different experiments. bp, base pairs.
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DISCUSSION

Although it is known that sphingosine or lysosphingolipids inhibit PKC activity (2, 3), ceramide has been considered to have no direct effect on PKC. Recent studies have shown that treatment with C2-ceramide (or sphingomyelinase) inhibits membranous translocation of PKC by PKC-activating stimuli (22, 23) and inactivates PKC-alpha (24), but there have been no reports that demonstrate the biological effect of ceramide on PKC in induction of apoptosis. We here showed that treatment with C2-ceramide translocated PKC-delta and -epsilon from the membrane to the cytosol fraction in three different human leukemia cells (promyelocytic HL-60, monoblastic U-937, and T cell HPB-ALL cells). Neither treatment with dihydroceramide lacking the C4-C5 double bond of ceramide structure nor exogenous phospholipases (PLC, phospholipase D, and phospholipase A2 except SMase) induced cytosolic translocation of PKC-delta and -epsilon , demonstrating the specificity of ceramide effect on the cytosolic translocation of PKC-delta and -epsilon . Furthermore, treatment with TNF-alpha or anti-Fas antibody, which induced apoptosis in the consequence of generating ceramide by hydrolysis of sphingomyelin (6, 7, 11, 12), translocated PKC-delta and -epsilon to the cytosol as well as ceramide. These data suggest that cytosolic translocation of PKC-delta and -epsilon may be closely related to the induction of apoptosis by ceramide and ceramide-generating stimuli including TNF-alpha and anti-Fas antibody.

In order to confirm that ceramide and ceramide-generating stimuli required the translocation of PKC-delta and -epsilon to the cytosol for completing apoptotic signals, we investigated 1) whether cytosolic translocation of PKC-delta and -epsilon was blocked by the failure of ceramide generation and 2) whether apoptosis was affected by the inhibition of ceramide-induced cytosolic translocation of PKC-delta and -epsilon . The results showed that in HPB-alpha FR cells neither cytosolic translocation of PKC-delta and -epsilon nor apoptosis was induced because of no generation of ceramide by the anti-Fas antibody and that the resistance to apoptosis in HPB-alpha FR cells was overcome by ceramide treatment accompanied by cytosolic translocation of PKC-delta and -epsilon . We also showed that ceramide-induced apoptosis was suppressed by competitive inhibition of cytosolic translocation of PKC-delta and -epsilon with TPA or a nonspecific kinase inhibitor staurosporine, both of which induced translocation to the membrane of PKC-delta and -epsilon . Taken together, cytosolic translocation of PKC-delta and -epsilon seemed to be indispensable to ceramide-mediated apoptosis in leukemia cells.

It has been reported that phorbol esters and DAG analogues, both activators of PKC, inhibited apoptosis induced by ceramide or ceramide-generating stimuli (10, 18, 21). As shown in Fig. 5, ceramide-induced apoptosis and cytosolic translocation of PKC-delta and -epsilon were overcome by TPA, and increasing doses of ceramide induced apoptosis and cytosolic translocation of PKC-delta and -epsilon against TPA. These data suggested that TPA and ceramide performed competitively in terms of the translocation of PKC-delta and -epsilon between the cytosol and the membrane. Although diC8 inhibited ceramide-induced cytosolic translocation of PKC-delta and -epsilon , apoptosis was not suppressed by diC8, probably due to its rapid metabolism and because higher concentrations of diC8 induced apoptosis with DNA fragmentation. On the other hand, we here investigated the effects of staurosporine on the ceramide-induced cytosolic translocation of PKC-delta and -epsilon because Jalava et al. (33) showed translocation to the membrane of PKC-epsilon by staurosporine. Surprisingly, staurosporine suppressed both ceramide-induced cytosolic translocation of PKC-delta and -epsilon and DNA fragmentation. Whereas higher concentrations of staurosporine (100 nM or more) were reported to induce apoptosis, conceivably due to nonspecific inhibition of various kinases (34, 35), at the concentration (3 nM) used in our experiments staurosporine might inhibit PKC more effectively than cyclic AMP-dependent kinase or tyrosine kinase, judging from IC50 values (2.7, 8.2, and 6.4 nM, respectively). These results suggest that translocation of PKC-delta and -epsilon to the membrane may be more strongly related to the inhibition of ceramide-induced apoptosis than to the activation of PKC. More complex modulation of PKC activity related to its topological changes may be critical to dissect the signal transduction between apoptosis and proliferation. In other words, the increase (possibly activation) of cytosolic PKC-delta and -epsilon may be the decisive signal to ceramide-mediated apoptosis as discussed below.

It therefore remains to be elucidated how PKC-delta and -epsilon translocated to the cytosol are involved in the signal transduction pathway leading to apoptosis. It was reported that overexpression of PKC-delta induced morphological change and growth inhibition by treatment with TPA in Chinese hamster ovary cells and NIH 3T3 cells, whereas overexpression of PKC-epsilon increased the growth rate in NIH 3T3 cells and induced malignant transformation in Rat 6 fibroblasts (36-38). Although these data suggest that activation of PKC-delta may inhibit cell growth or cell cycle progression, whereas that of PKC-epsilon may have the opposite effects, neither the mechanism of topological changes of PKC between the cytosol and the membrane nor the relation between the activity of each PKC isozyme and the induction of apoptosis is known. Recently, proteolytic activation of PKC-delta to a cytosolic 40-kDa fragment in apoptotic cells treated with radiation, TNF-alpha , or anti-Fas antibody was reported (29). These findings may be a clue to clarifying the biological meanings of ceramide-induced cytosolic translocation of PKC-delta and -epsilon in induction of apoptosis, because we found that treatment with TNF-alpha or anti-Fas antibody degraded not only PKC-delta but also PKC-epsilon in the cytosol fraction.2 These suggest that ceramide-induced cytosolic translocation of PKC-delta and -epsilon may be a prerequisite for their proteolytic activation in TNF-alpha or anti-Fas antibody-induced apoptosis. The more precise implications of ceramide-induced cytosolic translocation of PKC-delta and -epsilon in apoptosis will be defined by the further biochemical and biological investigations on the relation between cytosolic translocation and the activation of PKC-delta and -epsilon consequent to their degradation in the cytosol.


FOOTNOTES

*   This work was supported by Grant-in-Aid for Scientific Research on Priority Areas 05274108 (to T. O.) and 08671226 (to M. T.) from the Japanese Ministry of Education, Science and Culture and grants from the Otsuka Medical Research Fund (to T. O.), the Science Research Promotion Fund of Japan Private School Promotion Foundation, and the Osaka Dental University Research Foundation (to T. O., H. U., and N. D.). 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.
   To whom correspondence should be addressed: Dept. of Medicine, Osaka Dental University, 1-5-31 Otemae, Chuo-ku, Osaka 540, Japan. Tel.: +81-6-943-6521, Fax: +81-6-943-8051, E-mail: toshiroo{at}kuhp.kyoto-u.ac.jp.
1    The abbreviations and trivial names used are: PKC, protein kinase C; TNF-alpha , tumor necrosis factor-alpha ; DAG, diacylglycerol; C2-ceramide, N-acetylsphingosine; TPA, 12-O-tetradecanoylphorbol 13-acetate; C2-dihydroceramide, N-acetyldihydrosphingosine; SMase, sphingomyelinase; diC8, 1,2-dioctanoyl-sn-glycerol; PBS, phosphate-buffered saline.
2    H. Sawai, T. Okazaki, and M. Tashima , unpublished observations.

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