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J Biol Chem, Vol. 275, Issue 11, 7668-7676, March 17, 2000


Role of c-jun Expression Increased by Heat Shock- and Ceramide-activated Caspase-3 in HL-60 Cell Apoptosis
POSSIBLE INVOLVEMENT OF CERAMIDE IN HEAT SHOCK-INDUCED APOPTOSIS*

Tadakazu KondoDagger §, Tomoko MatsudaDagger §, Toshiyuki KitanoDagger , Atsushi TakahashiDagger , Masaro TashimaDagger , Hiroto Ishikura, Hisanori Umehara||, Naochika Domae||, Takashi UchiyamaDagger , and Toshiro OkazakiDagger **

From the Dagger  Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawaramachi, Sakyo-ku, Kyoto 606-8507, the  Transfusion Division, Shimane Medical University, Izumo 693-0027, and the || Department of Medicine, Osaka Dental University, 1-5-17 Otemae, Cyuo-ku, Osaka 540-0008, Japan

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ceramide has emerged as a lipid mediator in apoptosis induced by a variety of stresses. As we previously showed that the activation of AP-1, a nuclear transcription factor was indispensable to ceramide-induced apoptosis in human leukemia HL-60 cells (Sawai, H., Okazaki, T., Yamamoto, H., Okano, H., Takeda, Y., Tashima, M., Sawada, H., Okuma, M., Ishikura, H., Umehara, H., and Domae, N. (1995) J. Biol. Chem. 270, 27326-27331), the role and mechanism of heat shock (HS)-increased c-jun expression in apoptosis was here investigated. HS increased morphological changes compatible with apoptosis in human leukemia HL-60 cells, and induced ceramide generation and sphingomyelin hydrolysis with an increase of neutral magnesium-dependent sphingomyelinase activity. When HS failed to induce apoptosis in HS-resistant HL-60 cells, ceramide generation was not detected, suggesting that ceramide was involved in downstream signals required for HS-induced apoptosis. Both HS and N-acetylsphingosine (C2-ceramide) increased the expression of c-jun/c-fos mRNAs with the peak 2 h after treatment. When we examined whether the inhibition of c-jun expression by its antisense oligodeoxynucleotides (AS) blocked HS- or C2-ceramide-induced apoptosis, AS of c-jun gene inhibited apoptotic morphological changes and DNA fragmentation whereas did not sense oligodeoxynucleotides. Moreover, a synthetic tetrapeptide, acetyl-Asp-Met-Gln-Asp-aldehyde (DMQD-CHO), which inhibited the formation of active form of caspase-3 more efficiently than those of caspase-4, -6, -7, and -8, blocked both caspase-3 like activity, c-jun expression and apoptosis induced by HS or C2-ceramide, although DMQD-CHO did not affect HS-induced ceramide generation. These results suggested that the ceramide was generated through sphingomyelin hydrolysis by HS-activated neutral, magnesium-dependent sphingomyelinase and that subsequent c-jun expression through activation of caspase-3 played a role in HS-induced HL-60 cell apoptosis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Heat shock (HS)1 treatment is a clinically effective procedure for cancer therapy (1), and the execution of apoptosis is proposed as a mechanism by which HS induces cell death (2, 3). HS induces many kinds of proteins named HS proteins (HSPs), which are known to play a protective role for cell apoptosis owing to its function as a chaperon (4). For example, the overexpression of genes for HSP-70 and -27 protected the cells from tumor necrosis factor (TNF)-alpha and Fas-induced apoptosis, respectively (5, 6), and HSP-70 was reported to prevent HS-, oxidative stress- and ultraviolet-induced apoptosis by inhibiting pro-apoptotic signalings via p38 mitogen-activated protein kinase and c-Jun N-terminal kinase (JNK) (7). Thus, HSPs seem to function as an anti-apoptotic system to protect the cells from HS-induced apoptosis. It remains, however, to be clarified what the intracellular signaling molecules to mediate pro-apoptotic signals in HS-induced apoptosis are.

Since we reported that ceramide was generated by a hydrolysis of sphingomyelin (SM) through the pathway named "SM cycle" in 1alpha ,25-dihydroxyvitamin D3-induced human leukemia HL-60 cell differentiation (8), ceramide has been recognized as an important lipid mediator of various cell functions including differentiation, secretion, adhesion, senescence, and apoptosis (9-12). A diverse array of inducers such as TNF-alpha , Fas cross-linkage, irradiation, and 1-beta -D-arabinofuranosylcytosine as well as HS were reported to increase the intracellular ceramide levels in induction of apoptosis (13-16). Although several pathways related to serine/threonine kinase and phosphatase, Raf-1 kinase, mitogen-activated protein kinase, and NF-kappa B are proposed as downstream signals of ceramide (17), it is controversial what the indispensable mechanisms for ceramide-mediated apoptosis are. We previously showed that a nuclear transcription factor, AP-1, plays a crucial role in ceramide-induced apoptosis (9). Ceramide increased mRNA levels of c-jun and DNA binding activity of AP-1, and antisense deoxyoligonucleotides (AS) of c-jun inhibited ceramide-induced apoptotic cell death (9). Although HS was reported to increase c-jun mRNA, its mechanism and implications in the induction of HL-60 cell apoptosis are unknown.

Caspases are a family of cysteine protein proteases, which consist of more than 10 enzymes and cleave aspartate residue of apoptosis-related proteins including interleukin-1beta -converting enzyme, poly(ADP-ribose) polymerase, lamin B, and protein kinase C delta  (18-21). They are grouped into three categories, based on their cleavage sites and plausible functions (22). Group I containing caspase-1, -4, and -5 prefers the tetrapeptide sequence WEHD for autocleavage and activation, and is believed to play a role in cytokine secretion and inflammation. Groups II (caspase-2, -3, and -7) and III (caspase-6, -8, and -9) recognize DEXD and EXD as an optimal cleaving sequence, respectively, and were reported to be involved in initiation and execution of apoptosis. Among those caspase-3 seems to be of the most importance for the process of apoptosis because only caspase-3 knock out mice showed disregulation of brain apoptosis (23). Even though recently caspase-9 mutant mice also showed the similar neurological abnormality, it seems to be owing to the subsequent inhibition of caspase-3 (24, 25). In hematopoietic cells, the activated caspase-3 by cleaving pro-caspase-3 was required for a downstream signaling of ceramide-induced apoptosis (26, 27), but the role of caspase-3 in HS-induced apoptosis has not been examined.

In summary, ceramide-induced c-jun expression and caspase-3 activation seem to be the candidates for pro-apoptotic downstream signals in HS-induced apoptosis, but there were no data showing HS increased caspase-3 so far as we know. Moreover, it remains to be clarified how ceramide-induced c-jun expression and caspase-3 are involved in HS-induced apoptosis. We, therefore, investigated the mechanisms of ceramide generation by HS and examined whether HS- and ceramide-increased c-jun expression via caspase-3 activation is required to the induction of apoptosis by using a specific tetrapeptide caspase-3 inhibitor, acetyl-Asp-Met-Gln-Asp-aldehyde (DMQD-CHO) and antisense (AS) oligodeoxynucleotides of c-jun.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Human leukemia HL-60 cells were a kind gift from Dr. M. Saito (Hokkaido University, Hokkaido, Japan). C2-Ceramide was obtained from Matreya (Pleasant Gap, PA). DMQD-CHO was purchased from the Peptide Institute (Osaka, Japan). Murine monoclonal antibody VJ-41 (IgM) was kindly provided by Dr. M. Iwamori (Tokyo University, Tokyo, Japan). Other chemicals were purchased from Sigma.

Cell Culture-- Human leukemia HL-60 cells and a subline named HL-60/HS, which were resistant to HS-induced apoptosis, were grown in RPMI 1640 (Nissui, Tokyo, Japan) supplemented with 10% fetal calf serum (FCS, JRH Biosciences) and kanamycin sulfate (80 ng/ml) at 37 °C in a humidified atmosphere containing 5% CO2. The cell numbers were counted by a hemocytometer, and the viability was always greater than 95% in all experiments as assayed by 0.025% trypan blue dye exclusion method. Before the experiments, the cells were washed with phosphate-buffered saline (PBS) and incubated overnight in the RPMI 1640 supplemented with 2% FCS if not described particularly.

HS Treatment-- HL-60 cells were resuspended into the culture tubes at 5 × 105 cells/ml with a preheated medium, immersed in the water bath (Thermominder Mini-80; Aitec, Saitama, Japan) at various temperatures for the indicated durations, and then returned to incubate at 37 °C in 5% CO2. After HS treatment, the viable cells were counted by trypan blue exclusion method.

Measurement of Morphologically Apoptotic Cells and DNA Fragmentation-- The cells were treated with HS at the indicated temperatures for the different durations or with 5 µM C2-ceramide, harvested, and then stained with May-Giemsa or 4',6-diamidino-2-phenylindole (DAPI) staining method. At least 200 cells were counted under light microscope, and the cells with nuclear condensation and fragmentation were judged as apoptotic cells. For DNA fragmentation assay, the extraction of DNA was performed using G NOME DNA isolation kit (BIO 101) according to the manufacturer's protocol with a little modification. Briefly, the cells (1 × 106) were washed once in PBS and resuspended in 185 µl of Cell Suspension Solution. After 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 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 (pH 8.0) 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 optical density at 260 nm. Electrophoresis was carried out through 3% NuSieve agarose (FMC BioProducts) mini-gel in 1× TAE buffer at 50 V for 1.5 h. DNA was visualized under UV light after staining with ethidium bromide.

Metabolic Labeling of Sphingomyelin with [14C]Choline-- The cells were washed with PBS, seeded at 5 × 105 cells/ml in 2% FCS RPMI medium with [14C]choline chloride (0.1 µCi/ml) and incubated at 37 °C in 5% CO2 for 36 h. The labeled cells were treated with HS as described above and incubated at 37 °C in 5% CO2 for 2 h. After harvesting the cells, the lipids were extracted by the method of Bligh and Dyer (10). The samples were dried down by N2 gas and dissolved in 100 µl of chloroform. Twenty µl was applied on a Silica Gel 60 TLC plate (Merck), and 40 µl were used to measure phospholipid phosphate. TLC plate was developed in the solvent containing chloroform/methanol/2 N NH4OH (60: 35: 5), and then spots corresponding to SM were scraped and counted by using a scintillation counter. Radioactivity corresponding to SM was corrected by the amount of phospholipids in each sample.

Sphingomyelin Quantitation-- After HS treatment, the cells (1 × 107) were harvested and lipids were extracted from the cells by the method of Bligh and Dyer (10), and developed with a solvent system of chloroform/methanol/water (60: 35: 8, by vol) on a plastic TLC plate (catalog no. 805013, Sigma) with standard sphingomyelin. For TLC immunostaining, the plastic TLC plate was incubated with the blocking buffer containing 1% polyvinylpyrrolidone, 1% ovalbumin, and 0.02% NaN3 in PBS at 4 °C overnight, and then was reacted with 1 µg/ml murine monoclonal antibody VJ-41 (IgM), which preferentially reacted with SM (28), in 3% polyvinylpyrrolidone in PBS at 37 °C for 1 h. After washing the plate five times with 0.1% Tween 20 in PBS, the plate was incubated with peroxidase-conjugated goat anti-mouse IgM antibody (Cappel Laboratories, Cochraville, PA), diluted 1:1000, at 37 °C for 1 h, and washed five times with 0.1% Tween 20 in PBS. The antibody bound on the TLC plate was detected using ECL Western blotting reagents (Amersham Pharmacia Biotech) according to the manufacturer's protocol. The spots corresponding to SM were scraped, lipids were extracted by the Bligh and Dyer method, and then inorganic phosphate in extract was measured by ammonium molybdate/ascorbic acid method (10) to calculate SM content.

Metabolic Labeling of Ceramide with [14C]Serine-- The cells were washed with PBS, seeded at 5 × 105 cells/ml in serum-free medium containing 10 µCi/ml L-[U-14C]serine (Amersham Pharmacia Biotech), and incubated at 37 °C in 5% CO2 for 24 h. The labeled cells were treated with HS for 30 min at 42 °C, and then incubated at 37 °C in 5% CO2 for 2 h. After harvesting the cells, lipids extracted from the cells by the method of Bligh and Dyer were separated on TLC plates with a solvent of methyl acetate, propanol-1, chloroform, methanol, 0.25% KCl (25:25:25:10:9 by volume), the spots corresponding to ceramide were visualized, and the relative radioactivity was determined by using a BAS 2000 Image Analyzer (Fuji), corrected by the amount of phospholipids in each sample.

Ceramide Quantitation-- After extracting the lipids according to the Bligh and Dyer method, ceramide levels were enzymatically measured by using Escherichia coli diacylglycerol kinase, which converts ceramide to ceramide 1-phosphate, and corrected by phospholipid phosphate as described before (9). No changes of diacylglycerol kinase activity during the procedure were confirmed to add 40 nmol of C2-ceramide to the reaction mixture as an internal standard, and the amount of phospholipid phosphate paralleled viable cell numbers as described in the text.

Measurement of Sphingomyelinase-- After each treatment, HL60 cells (1 × 107) were harvested, washed twice with ice-cold PBS, and homogenized in 0.5 ml of lysis buffer containing 10 mM Tris-HCl (pH 7.5),1 mM EDTA, and 0.1% Triton X-100. The homogenate was centrifuged at 100,000 × g for 1 h at 4 °C. The supernatant was used as an enzyme source. The assay mixture for the measurement of magnesium-dependent neutral sphingomyelinase contained 0.1 M Tris-HCl (pH 7.5), 60 nmol of [methyl-14C]sphingomyelin (specific activity = 1.74 GBq/mmol; Amersham Pharmacia Biotech), 10 mM MgCl2, 0.2% Triton X-100, and 200 µg of enzyme in a final volume of 0.1 ml. For magnesium-dependent neutral sphingomyelinase, MgCl2 was removed from the reaction mixture. For acid sphingomyelinase, 0.1 M sodium acetate (pH 5.0) was used instead of Tris-HCl. Incubation was carried out at 37 °C for 1 h. The reaction was stopped by adding 1.5 ml of chloroform/methanol (2:1). Then 0.2 ml of double-distilled water was added to the tubes and vortexed. The tubes were centrifuged at 1000 × g for 5 min to separate the two phases. The clear upper phase (0.4 ml) was removed, placed in a glass scintillation vial, and counted with a scintillation counter (Packard Instrument Co.). Protein concentrations were determined using a Bio-Rad protein assay kit.

RNA Preparation and Northern Blotting-- Total RNA was prepared using Trizol reagent (Life Technologies, Inc.) according to the manufacturer's protocol, and 20 µg of total RNA was used for the Northern blotting analysis as described previously (10). Briefly, human c-jun and c-fos oligonucleotide probes (Oncogene Science) were labeled with [alpha -32P]dCTP using a multiprime labeling kit (Amersham Pharmacia Biotech). After electrophoresis and transferring RNA to Immobilon S membrane, hybridizations with labeled c-jun/c-fos probes were performed at 42 °C for 24 h and the membranes were washed in 2× SSC, 0.1% SDS (1× SSC containing 0.15 M NaCl and 15 mM sodium citrate) at room temperature for 30 min and at 50 °C for 20 min. The membranes were exposed to Fuji x-ray films with the intensifying screens at -80 °C and calculated by BAS imaging analyzer (Fuji Photo Film Co, Minaiasigara, Kanagawa, Japan). Equal loading of RNA was confirmed by methylene blue staining or the amount of beta -actin mRNA in each sample.

Oligodeoxynucleotides and Antisense Preparation-- The oligodeoxynucleotides of c-jun sense (5'-ACTGCAAAGATGGAAACG-3'), c-jun antisense (5'-CGTTTCCATCTTTGCAGT-3'), c-fos sense (5'-TTCTCGGGCTTCAACGCA-3'), and c-fos antisense (5'-TGCGTTGAAGCCCGAGAA-3') were synthesized. The nucleotide sequences were to the first 18 bases following the AUG sequences of human c-jun (29) and c-fos (30). The cells were incubated with c-fos/c-jun sense or antisense (50 µg/ml) at the concentration of 2.5 × 105 cells/ml in RPMI 1640 medium supplemented with 10% FCS for 1 day before heat shock treatment at 42 °C for 30 min or C2-ceramide addition.

Measurement of Caspase-3-like Activity-- The cells after each treatment were homogenized in lysis buffer containing 10 mM HEPES/KOH (pH 7.4), 2 mM EDTA, 0.1% CHAPS, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 100 µM pepstatin, 0.15 unit/ml aprotinin, and 50 µg/ml leupeptin, and centrifuged at 10,000 × g for 10 min. The supernatant was collected as an enzyme source and added to the reaction mixture (10% sucrose, 10 mM Hepes/KOH (pH 7.4), 5 mM dithiothreitol, 0.1% CHAPS, and 10 µM DEVD-MCA), which was followed by incubation at 25 °C for 60 min. Fluorescence was measured by a microplate reader (MTP-100F, Corona Electric, Katsuda, Japan) using 360 nm excitation and 450 nm emission filters. Concentrations of 7-amino-4-methylcoumarin liberated as a result of cleavage were calculated comparing with standard 7-amino-4-methylcoumarin solutions.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

HS-increased Cell Growth Inhibition and Apoptosis in HL-60 Cells-- When human leukemia HL-60 cells were treated with HS at 37, 40, 42, and 44 °C for 30 min, the viable cell numbers decreased in a temperature- and time-dependent manner (Fig. 1A). The morphologically apoptotic changes such as nuclear condensation and fragmentation without a membrane rupture were measured under light microscope after staining with May-Giemsa dye. The percents of apoptotic cells induced by 30 min of HS at 37, 40, 42, and 44 °C were 2 ± 2%, 20 ± 5%, 44 ± 10%, and 68 ± 4%, respectively, 4 h after cessation of treatment, while their viable cell numbers did not change significantly (Fig. 1, A and B). By HS treatment at more than 46 °C, the cells died of necrosis without showing any changes compatible with apoptosis (data not shown).


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  		Fig. 1.   Decrease of viable cell number and increase of apoptosis by HS treatment. The cells were treated with the indicated temperatures for 30 min at the initial concentration of 5.0 × 105 cells/ml, resuspended at 37 °C, and then harvested after the indicated times. The viable cell numbers were counted by a trypan blue dye exclusion method (A), and the percentages of apoptosis were measured under light microscope (B) 4 h after HS treatment as described under "Experimental Procedures." The results were obtained from three different experiments. The bars indicate 1 S.D.

HS-induced Sphingomyelin Hydrolysis and Ceramide Generation-- The biochemical mechanism of ceramide generation by HS treatment was not investigated in mammalian cells, even though the activation of ceramide synthase has recently been reported to involved in HS-induced ceramide generation in Saccharomyces cerevisiae (31). We first examined SM turnover and ceramide generation at the various temperatures after HS treatment in HL-60 cells. The levels of SM labeled with [14C]choline decreased to 84 ± 3% of the control level 2 h after 30 min treatment at 44 °C (Fig. 2A). In contrast, HS-induced ceramide generation measured by DGK assay increased in a temperature-dependent manner (Fig. 2B). Two hours after HS treatment at 44 °C increased ceramide levels to 172 ± 5% (8.6 ± 0.5 pmol/nmol phospholipid) as compared with the control levels (5.0 ± 0.2 pmol/nmol phospholipid). In addition, we examined the time course of ceramide generation after HS. In these experiments, we generalized DGK activity by adding known amount of C2-ceramide to each reaction mixture as an internal standard and measured phospholipids phosphate in the same numbers of living cells after HS treatment. As shown in Fig. 3, the levels of ceramide, which were measured by phosphorylating ceramide to ceramide 1-phosphate, significantly increased as compared with the levels of phosphorylated internal standard (40 nmol of C2-ceramide). Ceramide generation started immediately after cessation of HS treatment and peaked with approximately 155% increase 2 h after treatment. The levels of phospholipids phosphate after HS treatment did not change if the cell number in each sample was same. Similar increase of ceramide after HS treatment was obtained by a different method from DGK assay. As shown in Fig. 4 (A and B), ceramide contents after HS treatment increased by approximately 180% as compared with the control when the cells were labeled by [14C]serine, and labeled ceramide was separated on TLC plate and measured by BAS system.


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  		Fig. 2.   Effects of HS on sphingomyelin and ceramide levels. The cells were harvested immediately after HS treatment for 30 min at the indicated temperatures. The levels of sphingomyelin labeled by [14C]choline (A) and the contents of ceramide (B) were measured by DGK assay as described under "Experimental Procedures." The results are shown as changes relative to the cells treated without HS and were obtained from three different experiments. The bars indicate 1 S.D.


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  		Fig. 3.   Time course of ceramide increase by HS. The cells were treated with HS for 30 min at 42 °C at the initial concentration of 5.0 × 105 cells/ml, resuspended at 37 °C, and then harvested after the indicated times. Contents of intracellular ceramide and exogenous C2-ceramide (40 nmol) were measured by DGK assay as described under "Experimental Procedures." Contents of phospholipids phosphate were measured by ammonium molybdate/ascorbic acid method as described elsewhere. The results are shown as changes relative to the cells treated without HS and were obtained from three different experiments. The bars indicate 1 S.D.


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  		Fig. 4.   Increase of ceramide levels labeled with [14C]serine by HS. The cells were labeled with [14C]serine for 24 h in serum-free medium, harvested, and treated with or without HS for 30 min at 42 °C. Two hours after HS treatment, the lipids were extracted by Bligh and Dyer method and separated on TLC plates with standard lipids such as ceramide, glucosylceramide (GlcCer), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM) (A). After autoradiography, ceramide changes were measured by BAS system as described under "Experimental Procedures" (B). The results are shown as changes relative to the cells treated without HS and were obtained from three different experiments. The bars indicate 1 S.D.

We next examined changes of sphingomyelinase activity after HS, because neutral and/or acid sphingomyelinase were involved in ceramide generation induced by many kinds of stresses. As shown in Fig. 5A, the activity of neutral, magnesium-dependent sphingomyelinase significantly increased from 1.3 to 1.6 nmol/h/mg protein 2 h after HS treatment. Acid and neutral-magnesium-independent sphingomyelinase did not increase after HS treatment (data not shown). To confirm the involvement of sphingomyelinase in ceramide generation by HS, we also examined whether mass of ceramide generation was compatible with that of decreased SM. The amounts of SM changed from 43.8 ± 3.2 to 33.4 ± 3.4 pmol/106 cells 2 h after HS, whereas ceramide increased from 11.3 ± 0.6 to 18.1 ± 1.2 pmol/106 cells (Fig. 5B). The decrease in SM (10.4 ± 3.9 pmol/106 cells) was fairly close to 6.8 ± 1.1 pmol/106 cells increase of ceramide in HS-treated cells. These results suggest that sphingomyelin hydrolysis via neutral, magnesium-dependent sphingomyelinase activation is closely involved in ceramide generation after HS in HL-60 cells.


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  		Fig. 5.   Sphingomyelinase activity increased by HS (A) and comparison of mass changes between increased ceramide and decreased sphingomyelin (B). The cells were treated with or without HS for 30 min at 42 °C. Two hours after treatment, the cells were harvested and the proteins and lipids were extracted as described under "Experimental Procedures." A, sphingomyelinase activity was measured at 37 °C for 60 min in the reaction mixture containing 0.1 M Tris-HCl (pH 7.5), 60 nmol of [methyl-14C]sphingomyelin (specific activity = 1.74 GBq/mmol), 10 mM MgCl2, 0.2% Triton X-100, and 200 µg of enzyme source, and after stopping the reaction the radioactivity of cleaved [14C]cholinephosphate in the aqueous phase made by Folch's lipid extraction method was quantitated by scintillation counter. B, ceramide and SM were quantitated by DGK assay and phosphate measurement after TLC separation as described under "Experimental Procedures." The results were obtained from more than three different experiments. The bars indicate 1 S.D.

Ceramide Generation after HS in HS-resistant HL-60 Cells-- As shown in Fig. 6A, in a subline of HL-60 cells (HL-60/HS), HS for 30 min at 42 °C did not show the induction of apoptosis judged by DAPI staining method up to 4 h after treatment. In this condition, ceramide generation increased by 150% in HS-sensitive HL-60 cells but not in HS-resistant cells, and this HS resistance was overcome by the addition of C2-ceramide with HS simultaneously (Fig. 6, B and C). C2-Ceramide treatment alone in the concentratios up to 20 µM did not induce apoptosis (data not shown). These results demonstrated that ceramide generation is, at least, involved in downstream signaling of HS-induced apoptosis.


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  		Fig. 6.   Ceramide changes and apoptosis induction by HS in HS-resistant cells. A, HL-60 cells and HS-resistant HL-60 cells (HL-60/HS) were harvested at the indicated times after treatment with HS at 42 °C for 30 min. Apoptosis was examined by DAPI method as described under "Experimental Procedures." B, Ceramide generation was examined by DGK assay method as described under "Experimental Procedures." C, HL-60/HS cells were harvested 4 h after simultaneous treatment with HS and C2-ceramide. Apoptosis was examined by DAPI method as described under "Experimental Procedures." The results were obtained from more than three different experiments. The bars indicate 1 S.D.

Increase of c-jun and c-fos mRNAs by Heat Shock or Ceramide-- Since we previously reported that c-jun/AP-1 signal was indispensable to ceramide-induced apoptosis (10), changes of c-jun and c-fos expression were examined in HL-60 cells treated with HS or ceramide. When the cells were treated with 30 min of HS at 42 °C or 10 µM C2-ceramide, the levels of c-jun and c-fos mRNAs increased 1-2 h after treatment and then returned to the control levels (Fig. 7). By the same treatment, the levels of beta -actin expression and 28 S ribosomal RNA were not affected significantly, suggesting the specific increase of c-jun and c-fos expression by HS or ceramide. As apoptotic cell death by HS increased 4 h after treatment and increase of ceramide generation began immediately after HS treatment, these results suggested that HS- or ceramide-increased c-jun expression seemed to precede the execution of apoptosis.


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  		Fig. 7.   Increase of the levels of c-jun and c-fos mRNAs by HS or ceramide. The cells were harvested at the indicated times after treatment with HS treatment at 42 °C for 30 min or 10 µM C2-ceramide. Total RNA was extracted and Northern blotting analysis for c-jun, c-fos, and beta -actin mRNA levels was performed as described under "Experimental Procedures." Each lane contains 20 µg of total RNA, and equal loading was confirmed by the 28 S ribosomal RNA contents stained by methylene blue dye. The results were representative of three different experiments.

Inhibition of HS- or Ceramide-induced Apoptosis by Antisense Oligodeoxynucleotides of c-jun/c-fos-- To confirm whether ceramide-increased c-jun expression is indispensable for HS-induced apoptosis, we examined whether antisense oligodeoxynucleotides of c-jun/c-fos genes could inhibit HS- and ceramide-induced apoptosis. After 24 h of preincubation with various concentrations of sense or antisense oligodeoxynucleotides of c-jun and c-fos genes, the cells were treated with 5 µM C2-ceramide for 24 h. The percentage of apoptotic cells induced by C2-ceramide decreased from 45% to 5% of the control level by 50 µg/ml antisense oligodeoxynucleotides (Fig. 8A). Similarly, the percentage of apoptosis induced by 30 min of HS treatment at 42 °C decreased from 43% to 20% by the addition of 50 µg/ml antisense oligodeoxynucleotides (Fig. 8B). Sense oligodeoxynucleotides did not inhibit HS- or ceramide-induced apoptosis. As shown in Fig. 8 (C and D), HS-induced morphological changes and DNA fragmentation compatible with apoptosis were clearly inhibited with antisense oligodeoxynucleotides but not with sense oligodeoxynucleotides. These results clearly demonstrated that c-jun and/or c-fos expression increased by HS or ceramide was required to induce apoptosis, suggesting that ceramide-induced c-jun expression mediates HS-induced pro-apoptotic signal.


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  		Fig. 8.   Inhibition of ceramide- or HS-induced apoptosis by antisense deoxyoligonucleotides of c-jun and c-fos genes. A, HL-60 cells were pretreated with or without various concentrations of antisense (AS) or sense deoxyoligonucleotides (S) of c-jun and c-fos genes 1 day before treatment with 5 µM C2-ceramide and harvested 24 h after treatment. B, the cells were pretreated with or without 50 µg/ml AS or S of c-jun and c-fos genes 1 day before HS at 42 °C for 30 min and harvested 4 h after HS treatment. The percentages of morphologically apoptotic cells stained by May-Giemsa dye were measured under light microscope. C, the pictures were taken under light microscope (original magnification, ×400) to show the morphological changes compatible with apoptosis. D, apoptosis was also confirmed by nucleosomal DNA fragmentation as described under "Experimental Procedures." (M, phi X174/HaeII size marker; 1, HS; 2, HS with sense of c-fos/c-jun; 3, HS with antisense of c-fos/c-jun). The results were the representative of two different experiments. The bars indicate 1 S.D.

Requirement of Caspase-3 for HS or Ceramide-induced Apoptosis and c-jun Expression-- Before using DMQD-CHO, a synthetic tetrapeptide, we examined the effect on activation of caspase-3, -4, -6, -7, and 8 to confirm its specificity as an inhibitor of caspase-3. The method using YV(bio)KD-aomk was established to assess the formation of active forms of caspase as we described before (32). When we examined DMQD-CHO inhibitory effects on the activities of different kinds of caspases induced by anti-Fas antibody by using this method, 10 µM DMQD-CHO was enough to inhibit caspase-3 activity. In contrast, more than 100 µM DMQD-CHO was required to inhibit the activity of other caspases (caspase-4, -6, -7, and -8) (data not shown). In fact, DMQD-CHO inhibited casapase-3-like activity induced by HS or ceramide in a dose-dependent manner (Fig. 9). To investigate the relation of caspase-3 with ceramide generation and c-jun expression in HS-induced apoptosis, we examined the effects of DMQD-CHO on HS-induced ceramide generation. As shown in Fig. 10, the increase of ceramide generation was not affected significantly by pretreatment with DMQD-CHO for 1 h before HS treatment, suggesting that caspase-3 is a downstream signal of HS-increased ceramide. We, therefore, investigated whether ceramide-activated caspase-3 was required for the increase of c-jun expression to mediate HS-induced apoptosis. The increase of c-jun expression, which peaked 1-2 h after treatment with 30 min of HS at 42 °C or 10 µM C2-ceramide, was completely inhibited by 1 h of pretreatment with 200 µM DMQD-CHO (Fig. 11, A and B). Moreover, pretreatment with 200 µM DMQD-CHO inhibited HS or C2-ceramide-induced apoptotic from 50% and 70% to 23% and 30%, respectively, when the cells were stained by DAPI (Fig. 12A). As seen in Fig. 12B, it was clearly shown that HS- or ceramide-induced apoptotic morphological changes were inhibited by DMQD-CHO. In summary, these results suggested that activation of caspase-3 by HS or ceramide played a crucial role in induction of apoptosis through c-jun expression.


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  		Fig. 9.   Inhibition of HS or C2-ceramide-induced caspase-3-like activity by DMQD-CHO. HL-60 cells were pretreated with or without 50, 100, and 200 µM DMQD-CHO 1 h before treatment with 10 µM C2-ceramide or HS for 30 min at 42 °C and harvested 6 h after C2-ceramide or 4 h after HS treatment. Caspase-3-like activity was measured by DEVD-MCA cleavage as described under "Experimental Procedures." The results were obtained from three different experiments. The bars indicate 1 S.D.


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  		Fig. 10.   No inhibition of HS-induced ceramide generation by DMQD-CHO. HL-60 cells were pretreated with or without 200 µM DMQD-CHO 1 h before treatment with HS for 30 min at 42 °C and harvested at the indicated times. Ceramide contents were measured by DGK assay as described under "Experimental Procedures." The results were obtained from two different experiments. The bars indicate 1 S.D.


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  		Fig. 11.   Inhibition of HS- or ceramide-induced c-jun expression by DMQD-CHO. HL-60 cells were pretreated with or without 200 µM DMQD-CHO 1 h before treatment with HS for 30 min at 42 °C (A) or 10 µM C2-ceramide (B) and harvested at the indicated times. Total RNA was extracted, and Northern blotting analysis for c-jun mRNA levels was performed as described under "Experimental Procedures." Each lane contains 20 µg of total RNA, and equal loading was confirmed by the 28 S ribosomal RNA contents stained by methylene blue dye. The results were representative of three different experiments.


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  		Fig. 12.   Inhibition of HS or C2-ceramide-induced apoptosis by DMQD-CHO. HL-60 cells were pretreated with or without 200 µM DMQD-CHO 1 h before treatment with 10 µM C2-ceramide or HS for 30 min at 42 °C and harvested 6 h after C2-ceramide or 4 h after HS treatment. Apoptosis was measured by DAPI staining as described under "Experimental Procedures." A, the percentages of morphologically apoptotic cells stained by DAPI were measured by judging at least 200 cells under fluorescent microscope. The results were obtained from three different experiments. The bars indicate 1 S.D. B, the pictures were taken under fluorescent microscope (original magnification, ×400) to show nuclear morphological changes compatible with apoptosis.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previously, it was reported that HS induced a small HS protein, alpha B-crystallin with ceramide generation in Swiss 3T3 cells (33), but the mechanism of ceramide generation by HS was not clarified. We here investigated whether HS increased ceramide generation through sphingomyelin hydrolysis by the activation of sphingomyelinase in hematopoietic HL-60 cells, since many kinds of stresses such as TNF-alpha and Fas cross-linkage were known to activate neutral and/or acid sphingomyelinase for generating ceramide (15, 16) As shown in Fig. 1-3, HS induced apoptosis with ceramide generation in parallel with a decrease of sphingomyelin content in a temperature- and time-dependent manner, and this increase of ceramide measured by DGK assay method was also confirmed by an independent [14C]serine labeling method. We next examined the involvement of sphingomyelinase in sphingomyelin hydrolysis and ceramide generation by directly measuring the enzyme activity. HS increased neutral, magnesium-dependent sphingomyelinase activity significantly, as shown in Fig. 5A. Moreover, the increase of ceramide (10.4 ± 3.9 pmol/106 cells) was fairly close to the decrease of sphingomyelin mass amount (6.8 ± 1.1 pmol/106 cells), when mass changes of ceramide and sphingomyelin were measured 2 h after HS by DGK assay and phosphate assay after TLC separation methods, respectively. These results strongly suggested the involvement of sphingomyelinase activation in HS-induced ceramide generation, even though it is unable to eliminate completely the role of other enzymes including phospholipase D type of sphingomyelinase and ceramide:phosphatidylcholine phosphocholine transferase in HS-induced apoptosis. Another pathway available for ceramide generation is de novo ceramide synthesis. In bacteria or yeast, HS activated ceramide synthase to generate ceramide from dihydrosphingosine via the action of serine palmitoyl-CoA transferase (30, 34). However, ceramide synthase does not seem to be involved in HS-induced ceramide generation in HL-60 cells, because an inhibitor of ceramide synthase, fumonisin B1, did not inhibit HS-induced ceramide generation and apoptosis (data not shown).

Recently, another group (35) reported that E. coli DGK assay, which we usually use for ceramide measurement in the cells, was not an appropriate method because of changes of DGK activity during the experimental procedures. To confirm the propriety of this method, we added a known amount of C2-ceramide (40 nmol) into the reaction mixture of DGK assay as an internal standard. The results showed that the amounts of phosphorylated C2-ceramide did not change, whereas those of phosphorylated physiological ceramide in the cells significantly increased by HS treatment in a time-dependent manner. In addition, no significant change of phospholipid phosphate in the extracted lipids was detected when the same numbers of living cells were examined, suggesting the proper generalization of ceramide content in the samples by using phospholipid phosphate. These results suggested that DGK assay method was, at least in this experiments, appropriate for measuring intracellular ceramide contents because no significant changes of enzyme activity itself were detected at the different time points after HS treatment and, in addition, similar increase of ceramide was confirmed by an independent [14C]serine labeling method.

We showed that the levels of c-jun mRNA increased by ceramide with a subsequent enhancement of AP-1 DNA binding activity, suggesting that the increase of c-jun expression plays a role in ceramide-induced apoptosis (10). Therefore, we here investigated whether c-jun expression increased by endogenous ceramide is required to mediate HS-induced apoptotic signalings. HS generated ceramide immediately after cessation of treatment and HS or ceramide increased c-jun expression 1-2 h after treatment. Both HS- and ceramide-induced apoptosis were significantly blocked by the inhibition of c-jun expression with antisense deoxyoligonucleotides of c-jun gene. These results suggested that ceramide-increased c-jun expression was required to mediate HS-induced apoptosis.

TNF-alpha , Fas cross-linkage, and anti-cancer reagents increased mainly the activity of caspase-3 among many kind of caspases (36-38). Ceramide was also shown to cleave poly(ADP)-ribose polymerase and pro-caspase-3, and increased the activity of caspase-3 to induce apoptosis (39, 40). In addition, very recently, the overexpression of HSP-70 was reported to inhibit ceramide-induced caspase-3 and apoptosis (41), suggesting that caspase-3 is a pro-apoptotic signal even in HS-induced apoptosis as well as other stresses. Therefore, we investigated the signaling relation between ceramide-increased caspase-3 and c-jun expression in HS-induced apoptosis. To block caspase-3-activated signal, we used a synthetic tetrapeptide, DMQD-CHO, which was more specific inhibitor of caspase-3 than DEVD-CHO and inhibited most effectively the cleavage of pro-caspase-3 into active form than other caspases such as caspase-4, -6, -7, and -8 as shown elsewhere (41). As shown in Figs. 10-12, DMQD-CHO inhibited both c-jun expression and apoptotic cell death increased by HS or ceramide but not ceramide generation by HS, suggesting that caspase-3 mediates an increase of c-jun expression downstream of ceramide generation by HS.

How are the mechanisms by which caspase-3 increases c-jun expression? Since HS treatment activates JNK and active JNK could increase c-jun expression in an autophosphorylation manner, one possibility of the mechanism that caspase-3 increases c-jun expression may be the activation of JNK. However, HS-induced apoptosis was not blocked by inhibiting JNK signal in a knock-out mouse of MKK4 gene (43), and dexamethasone and ultraviolet were reported to mediate apoptosis in JNK-independent and caspase-3-dependent mechanism in monoblastic leukemia U-937 cells (44, 45). Therefore, it is unlikely that ceramide increases c-jun expression via caspase-3 by JNK-dependent mechanism and the precise mechanism of caspase-3 to increase c-jun expression is, at present, unknown.

We here showed the possibility that HS induced the increase of c-jun expression as a pro-apoptotic signal by caspase-3 activation probably via endogenous ceramide generation. The precise role of c-jun expression in apoptotic signaling is, however, intriguing because many kinds of anti-apoptotic signals such as 12-O-tetradecanoylphorbol-13-acetate, epidermal growth factor, platelet-derived growth factor receptor, serum and other cytokines are also known to increase c-jun expression. If it is possible to dissect the difference of signaling to increase c-jun expression by modulating caspase-3 activity or ceramide generation, the regulation of intracellular signalings between apoptosis and proliferation/cell survival may be understood more profoundly in the future.

    ACKNOWLEDGEMENT

We are grateful to Hideaki Hori for excellent technical assistance.

    FOOTNOTES

* This work was supported by a grant-in-aid from the Japanese Ministry of Education, Science and Culture and grants from Ono Pharmaceutical Co. Ltd. (to T. O.), and the Osaka Dental University Research Foundation (to 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.

§ The first two authors contributed equally to this work.

** To whom correspondence should be addressed. Tel./Fax: 81-75-751-3154; E-mail: toshiroo@kuhp.kyoto-u.jp.ac.

    ABBREVIATIONS

The abbreviations used are: HS, heat shock; HSP, heat shock protein; DGK, diacylglycerol kinase; TNF, tumor necrosis factor; SM, sphingomyelin; JNK, c-Jun N-terminal kinase; AS, antisense; FCS, fetal calf serum; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DAPI, 4',6-diamidino-2-phenylindole.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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