©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Requirement of AP-1 for Ceramide-induced Apoptosis in Human Leukemia HL-60 Cells (*)

(Received for publication, June 19, 1995; and in revised form, August 23, 1995)

Hirofumi Sawai (1) Toshiro Okazaki (2)(§) Hirotaka Yamamoto (3) Hakuro Okano (3) Yasushi Takeda (1) Masaro Tashima (1) Hiroyoshi Sawada (1) Minoru Okuma (1) Hiroto Ishikura (4) Hisanori Umehara (2) Naochika Domae (2)

From the  (1)First Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, Kyoto 606, the Departments of (2)Medicine and (3)Oral Surgery, Osaka Dental University, Osaka 540, and the (4)Division of Transfusion, Shimane Medical College, Shimane 693, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Ceramide has emerged as a novel lipid mediator in cell proliferation, differentiation, and apoptosis. In this work, we demonstrate that the levels of c-jun mRNA, c-Jun protein, and DNA binding activity of a nuclear transcription factor AP-1 to 12-o-tetradecanoylphorbol 13-acetate responsive elements all increased following treatment with the cell-permeable ceramide, N-acetylsphingosine in human leukemia HL-60 cells. N-Acetylsphingosine (1-10 µM) increased the levels of c-jun mRNA in a dose-dependent manner, and maximal expression was achieved 1 h after treatment. Increase of c-jun expression treated with 5 µMN-acetyldihydrosphingosine, which could not induce apoptosis, was one third of that with 5 µMN-acetylsphingosine. Ceramide-induced growth inhibition and DNA fragmentation were both prevented by treatment with curcumin, 1,7-bis[4-hydroxy-3-methoxy-phenyl]-1,6-heptadiene-3,5-dione (an inhibitor of AP-1 activation), or antisense oligonucleotides for c-jun. These results suggest that the transcription factor AP-1 is critical for apoptosis in HL-60 cells and that an intracellular sphingolipid mediator, ceramide, modulates a signal transduction inducing apoptosis through AP-1 activation.


INTRODUCTION

Sphingolipids, which have been considered only as structural elements of the cell membrane, are now recognized as a new group of intracellular second messengers(1, 2) . We found that sphingomyelin levels transiently decreased and concomitantly ceramide increased when human leukemia HL-60 cells were treated with 1alpha,25-dihydroxyvitamin D(3) to differentiate into monocytic cells and that synthetic cell-permeable ceramide could induce monocytic differentiation of HL-60 cells(3, 4) . A new signal transduction pathway termed ``sphingomyelin cycle'' was proposed by our group and subsequently similar sphingomyelin hydrolysis was reported to be induced by many other cytokines, including tumor necrosis factor-alpha (TNF-alpha), (^1)-interferon(5, 6) , interleukin-1(7, 8) , and nerve growth factor(9) , and by cross-linking of Fas(10) . Ceramide, a product of sphingomyelin hydrolysis, is postulated as an intracellular mediator of apoptosis, because it was shown to induce DNA fragmentation and typical morphological change to apoptosis following the inhibition of cell growth(11, 12) .

To exert its biological effects, ceramide seems to transduce the intracellular signals by activating protein kinase(s) (13, 14) or protein phosphatase(s) (15, 16) and by inducing the down-regulation of c-myc(5) and the activation of nuclear translocation of nuclear factor NF-kappaB(17, 18, 19) . The proto-oncogene c-jun encodes another transcription factor that is a component of AP-1 (reviewed in (20) ). c-Jun is a member of the bZip transcription factor family and possesses a basic DNA-binding domain and a leucine-zipper dimerization motif(21) . The c-Jun homodimers or the heterodimers with c-Jun and c-Fos family members bind to the DNA consensus sequence TGA(C/G)TCA (called the 12-o-tetradecanoylphorbol 13-acetate-responsive element) found in the promoter regions of several genes. Induction of the c-jun gene is an early response event during activation of quiescent fibroblasts by serum (22, 23) or mitogens such as phorbol esters(24) . Furthermore, c-jun gene expression is induced by cytokines including TNF-alpha(25) , nerve growth factor(26) , and interleukin-1(27) . Induction of c-jun during cell differentiation is also reported(28, 29, 30, 31) . Recent studies have demonstrated that c-jun expression is induced by apoptosis-inducing agents such as ionizing radiation(32) , UV light (33) , and cytotoxic drugs including etoposide(34) , 1-beta-D-arabinofuranosylcytosine(35, 36) , and cis-diamminedichloroplatinum (II) (37) or by growth factor deprivation(38) . These and other data (39, 40, 41, 42) suggest that c-jun/AP-1 is a very important factor in the regulation of cell growth, differentiation, and apoptosis.

In this study, we examined whether ceramide induces c-jun/AP-1 activation and whether its activation is required for the induction of apoptosis by ceramide in human leukemia HL-60 cells, because c-jun/AP-1 activation seems to play a role in inducing apoptosis as well as other early responsive genes such as NF-kappaB and c-myc. Our results show that c-jun/AP-1 is activated by ceramide in the early process of apoptosis and that the impairment of AP-1 rescues apoptosis. These data suggest that ceramide is crucially involved in the signal transduction pathway leading to apoptosis through the activation of c-jun/AP-1.


EXPERIMENTAL PROCEDURES

Materials

Human leukemia HL-60 cells were kindly provided by Dr. M. Saito (Jichi Medical College). C(2)-ceramide and C(2)-dihydroceramide were kindly provided by Dr. Y. Hannun (Duke University). C(2)-ceramide was also purchased from Matreya, Inc. [-P]ATP (6000 Ci/mmol) and [alpha-P]UTP (3000 Ci/mmol) were purchased from Amersham Corp. DNase and RNasin were purchased from Promega. Other chemicals were obtained from Sigma.

Cell Culture

HL-60 cells were maintained in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum and 80 mg/liter kanamycin at 37 °C in a humidified atmosphere of 5% CO(2). Before the experiments, the cells were washed with RPMI 1640 medium containing transferrin (5 mg/liter) and insulin (5 mg/liter) instead of serum and incubated overnight in the serum-free medium at a concentration of 5 times 10^5/ml if not described particularly.

Northern Blot Analysis

Total cellular RNA was isolated using ISOGEN (Nippon Gene) according to the manufacturer's protocol. Northern blotting was performed as described previously(43) . Human c-jun oligonucleotide probe (Oncogene Science) was 5`-end labeled with a Kination kit (Toyobo Co., Ltd). Briefly, 4 pmol of oligonucleotide was mixed with 1 µl of 10 times protruding end kinase buffer and 5 µl of [-P]ATP, incubated at 37 °C for 30 min, and then purified through Sephadex G-25 spun column twice. Hybridizations were performed at 42 °C for 24 h, and then the membranes were washed in 2 times SSC/0.1% SDS (1 times SSC, 0.15 M Nacl, 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 intensifying screens at -80 °C for 2 days. Equal loading of RNA was confirmed by methylene blue staining of the membrane.

Nuclear Run-on Assay

Nuclear run-on assay was performed as described (28) with modification. The cells (5 times 10^7) were collected, resuspended in 4 ml of ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl(2), 0.5% Nonidet P-40), and vortexed gently for 20 s and left on ice for 5 min. Nuclei were pelleted by centrifugation at 500 times g for 5 min and resuspended in 100 µl of glycerol buffer (50 mM Tris-HCl, pH 8.3, 40% glycerol, 5 mM MgCl(2), 0.1 mM EDTA). An equal volume of reaction buffer (100 mM KCl, 0.5 mM each of ATP, CTP, and GTP) was added to the nuclear suspension, and the reaction mixture was incubated with 100 µCi of [alpha-P]UTP (3000 Ci/mmol) at 26 °C for 30 min. Then the reaction was terminated by the addition of 100 µl of stop buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl, 20 mM MgCl(2), 150 units/ml RNasin, 40 units/ml DNase) and incubated at 28 °C for 15 min. Then proteinase K (750 µg/ml) and 1% SDS were added and incubated at 37 °C for 30 min. RNA was isolated by phenol/chloroform extractions and precipitated in ethanol and 2.5 M ammonium acetate. RNA was further purified through a Sephadex G-50 spun column equilibrated and eluted with column buffer (0.3 M NaCl, 0.1% SDS, 1 mM EDTA, 10 mM Tris-HCl, pH 7.5).

Rat c-jun cDNA (obtained from RIKEN DNA Bank, Japan) or beta-actin cDNA (2.5 µg each) was denatured in 100 µl of 0.1 N NaOH for 30 min, neutralized by the addition of an equal volume of buffer containing 0.5 M Tris-HCl, pH 7.0 and 3 M NaCl, and then blotted onto nylon membrane (Biodyne, Pall Corp.) using a slot-blot apparatus (Schleicher & Schuell). The membrane was prehybridized in 5 times Denhardt's solution, 40% formamide, 4 times SSC, 5 mM EDTA, 0.4% SDS, and 100 µg/ml yeast tRNA at 42 °C for 2 h. Hybridization was performed with 10^7 cpm of P-labeled RNA/ml of hybridization buffer at 42 °C for 72 h. Then the membrane was washed in 2 times SSC with 0.1% SDS at 37 °C for 30 min, 10 µg/ml RNase A in 2 times SSC at 37 °C for 30 min, and 0.1 times SSC with 0.1% SDS at 42 °C for 30 min. Autoradiography was performed with intensifying screens at -80 °C for 5-14 days.

Nuclear Extract Preparation

Nuclear extracts were prepared as previously described (44) with modification. Briefly, 5 times 10^6 cells were collected, washed once in phosphate-buffered saline, resuspended in 400 µl cold buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 3 mM MgCl(2), 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride), allowed to swell for 15 min on ice, and then lysed by gently passing through a 27-gauge needle. The nuclei were collected by centrifugation at 400 times g for 5 min and resuspended in 50 µl of buffer C (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride). The tube was vigorously rocked on a shaking platform at 4 °C for 30 min and centrifuged at 12,000 times g for 5 min at 4 °C, and the supernatant was used as the nuclear extract. The protein concentration of the extract was determined by using the Bio-Rad protein assay.

Electrophoretic Mobility Shift Assay

Nuclear extract (6 µg) was mixed with 4 µg of poly(dIbulletdC)bulletpoly(dIbulletdC) (Pharmacia Biotech Inc.) in a binding buffer (5 mM HEPES, pH 7.9, 5 mM MgCl(2), 50 mM KCl, 1 mM dithiothreitol, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride; final volume, 20 µl) for 10 min on ice. AP-1 consensus oligonucleotide (5`-CGCTTGATGACTCAGCCGGAA-3`) (Santa Cruz Biotechnology, Inc.) was 5`-end labeled as described above. The labeled probe (2.5 times 10^4 cpm) was added, and the reaction mixture was incubated for 30 min on ice. Then the samples were separated by 4% native polyacrylamide gel electrophoresis in 1 times TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA). The gel was dried under vacuum and subjected to autoradiography overnight at -80 °C. Competition studies were performed by adding 10- or 50-fold molar excess of unlabeled AP-1 consensus oligonucleotide or AP-1 mutant oligonucleotide (5`-CGCTTGATGACTTGGCCGGAA-3`) (Santa Cruz Biotechnology, Inc.) or Sp1 consensus oligonucleotide (5`-ATTCGATCGGGGCGGGGCGAGC-3`) (Promega).

Western Blot Analysis

Cells were suspended in lysis buffer containing 50 mM Tris-HCl, pH 7.6, 0.5% Triton X-100, 300 mM NaCl, 5 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride. After incubation on ice for 30 min, the lysates were centrifuged at 10,000 times g for 10 min, and the supernatants were recovered. The supernatants were boiled in SDS-containing sample buffer and subjected to SDS-polyacrylamide gel electrophoresis(46) . Separated proteins were electroblotted to Immobilon polyvinylidene difluoride transfer membrane (Millipore). c-Jun was detected by ECL Western blotting detection system (Amersham Corp.) using anti-c-jun/AP-1(N) antibody (Santa Cruz Biotechnology).

Oligonucleotides

c-jun antisense (5`-CGTTTCCATCTTTGCAGT-3`) and sense (5`-ACTGCAAAGATGGAAACG-3`) oligonucleotides corresponding to the first 18 bases after the AUG sequence of c-jun mRNA in the phosphorothioate modified condition were synthesized or obtained from BioServe Biotechnologies, MD.

Analysis of DNA Fragmentation

DNA was isolated by using a GENOME kit (Bio 101, CA), electrophoresed through 3% NuSieve agarose (FMC BioProducts) mini-gel in 1 times TAE buffer at 50 V for 1.5 h and visualized under UV light after ethidium bromide staining.


RESULTS

c-jun Expression Induced by Ceramide

As previously reported(11, 12) , 5 µM C(2)-ceramide apparently induced DNA fragmentation of HL-60 cells in 3 h. Because other studies suggest that c-jun expression may be related to apoptosis(32, 33, 34, 35, 36, 37, 38) , we examined the change of c-jun mRNA levels in the cells treated with C(2)-ceramide by Northern blot analysis. As shown in Fig. 1A, 5 µM C(2)-ceramide showed a rapid and transient increase at the positions of 2.7- and 3.2-kilobases corresponding to c-jun mRNA molecular sizes, and the elevation of c-jun mRNA began to be detected 30 min after the addition of C(2)-ceramide. Maximal induction of c-jun expression was achieved at 1 h, and then the levels of c-jun mRNA decreased gradually to the basal level 4 h after C(2)-ceramide addition. Induction of c-jun expression by C(2)-ceramide (1-10 µM) was in a dose-dependent manner (Fig. 1B). These concentrations, at which c-jun mRNA increased, correspond to those at which ceramide induces its biological effects, such as growth inhibition and apoptosis. Although a 15-fold increase in c-jun expression was induced by 10 µM C(2)-ceramide 1 h after treatment, HL-60 cells began to die within 2 h and completely died 24 h after treatment with 10 µM C(2)-ceramide. In most following experiments, a C(2)-ceramide concentration of 5 µM or less was therefore used to detect the biological effects of C(2)-ceramide after a long term treatment.


Figure 1: Induction of c-jun mRNA by C(2)-ceramide. A, time course of induction. HL-60 cells were treated with 0.1% ethanol vehicle (C) or 5 µM C(2)-ceramide (C) in serum-free medium for the indicated times, and then total RNA was extracted. B, dose-dependence of induction. HL-60 cells were treated with the indicated concentrations of C(2)-ceramide for 1 h. C, specificity of the effect of ceramide. HL-60 cells were treated with 0.1% ethanol vehicle (C), 5 µM C(2)-ceramide (C) or 5 µM C(2)-dihydroceramide (DHC) for 1 h. Northern blot analysis for c-jun mRNA was performed as described under ``Experimental Procedures.'' Each lane contains 15 µg of total RNA and equal loading was confirmed by the amount of 28 S ribosomal RNA stained with methylene blue dye. The upper panel and the lower panel in each figure show c-jun mRNA levels and 28 S ribosomal RNA levels, respectively.



A previous report (45) showed the specificity of biological activity of ceramide compared with dihydroceramide whose chemical structure is the same as that of ceramide except for the lack of a double bond between the fourth and fifth carbons. We found that 5 µM C(2)-dihydroceramide failed to induce apoptosis and that the increase of c-jun expression by 5 µM C(2)-dihydroceramide was approximately one-third of 5 µM C(2)-ceramide or corresponding to the effect of 1 µM C(2)-ceramide (Fig. 1C), which could not induce apoptosis, when measured by the densitometer (data not shown). It is clearly shown that C(2)-ceramide with biological effects in HL-60 cells but not C(2)-dihydroceramide induced a significant increase of c-jun mRNA.

Regulation of c-jun Induction by Ceramide at Both Transcriptional and Post-transcriptional Levels

Next, we examined whether ceramide induced c-jun expression by a transcriptional or post-transcriptional mechanism. Actinomycin D and cycloheximide were used as inhibitors of RNA transcription and protein synthesis, respectively. As shown in Fig. 2A, the treatment with actinomycin D completely inhibited c-jun expression, whereas cycloheximide, which itself slightly increased c-jun mRNA, superinduced c-jun expression when treated with ceramide. A nuclear run-on assay showed that 5 µM C(2)-ceramide increased the rate of c-jun transcription (Fig. 2B). As shown in Fig. 2C, an approximately 4-fold increase of c-jun transcription level was found by treatment with ceramide compared with the control when the increase rate was generalized by the transcriptional level of beta-actin, which is recognized as a housekeeping gene. We confirmed that the levels of beta-actin mRNA did not change significantly in our experimental condition (Fig. 2D). These studies suggested that c-jun induction by ceramide is regulated at both transcriptional and post-transcriptional levels.


Figure 2: Regulation of c-jun induction by C(2)-ceramide at transcriptional and post-transcriptional levels. A, effects of actinomycin D or cycloheximide on c-jun induction by C(2)-ceramide. HL-60 cells were treated with 5 µg/ml actinomycin D or 10 µg/ml cycloheximide in the presence or absence of 5 µM C(2)-ceramide for 1 h. lane 1, control; lane 2, treated with C(2)-ceramide; lane 3, treated with actinomycin D alone; lane 4, treated with actinomycin D and C(2)-ceramide; lane 5, treated with cycloheximide alone; lane 6, treated with cycloheximide and C(2)-ceramide. Northern blot analysis for c-jun mRNA was performed as described under ``Experimental Procedures.'' Each lane contains 15 µg of total RNA, and equal loading was confirmed by the amount of 28 S ribosomal RNA stained with methylene blue dye. The upper panel and the lower panel show c-jun mRNA levels and 28 S ribosomal RNA levels, respectively. B, analysis of c-jun gene transcription in HL-60 cells treated with C(2)-ceramide. HL-60 cells were either untreated (control) or treated with 5 µM C(2)-ceramide for 1 h (ceramide). Nuclear run-on assay for c-jun and beta-actin was performed as described under ``Experimental Procedures.'' This is representative of two different experiments. C, fold increase of c-jun transcriptional levels by C(2)-ceramide. Relative values were calculated by generalizing the density for c-jun compared with beta-actin transcriptional levels in B. The densities were assessed by an Epson GT-8000 scanner using NIH Image version 1.47 software for the Macintosh computer. The bars indicate S.D. D, changes of beta-actin mRNA levels. HL-60 cells were treated with 5 µM C(2)-ceramide for the indicated times, and the levels of beta-actin mRNA were examined by Northern blot analysis (upper panel). The lower panel shows 28 S ribosomal RNA stained with methylene blue dye.



AP-1 DNA Binding Activity Induced by Ceramide

Because induction of c-jun mRNA does not always mean the activation of AP-1(42) , we examined whether AP-1 DNA binding activity was increased by ceramide. By the electrophoretic mobility shift assay (Fig. 3), we found that C(2)-ceramide increased the binding of the nuclear extract to synthetic oligonucleotides containing AP-1 consensus sequence. As shown in Fig. 3A, the prominent increase of AP-1 DNA binding activity was detected 1 h after treatment with 5 µM C(2)-ceramide, and the activity was still elevated after 4 h. Competition studies were performed to confirm the specificity of the binding complexes (Fig. 3B). By addition of 10- or 50-fold molar excess of unlabeled AP-1 consensus oligonucleotides, AP-1 complex induced by ceramide disappeared. On the other hand the addition of 10-fold molar excess of unlabeled oligonucleotides containing AP-1 mutant sequence or Sp1 consensus sequence did not affect the binding ability. Although 50-fold molar excess of those seemed to slightly decrease the binding, it may be because of nonspecific effect by high concentration. These studies demonstrate that C(2)-ceramide can specifically induce AP-1 DNA binding activity after the induction of c-jun mRNA.


Figure 3: Effect of C(2)-ceramide on AP-1 DNA binding activity. A, time course of AP-1 binding. HL-60 cells were treated with 0.1% ethanol vehicle (C) or 5 µM C(2)-ceramide (C) for the indicated times. B, competition assay for the specific DNA binding of AP-1. Electrophoretic mobility shift assay was performed using nuclear extracts from cells treated with 5 µM C(2)-ceramide for 6 h. Protein DNA binding reactions were carried out in the absence of competitors(-) or in the presence of 10- or 50-fold molar excess of unlabeled AP-1 consensus oligonucleotide (AP-1), AP-1 mutant oligonucleotide (mutant), or Sp1 consensus oligonucleotide (Sp1). Electrophoretic mobility shift assay was performed as described under ``Experimental Procedures.''



Rescue of Ceramide-induced Apoptosis by the Inhibition of c-jun/AP-1 Pathway

To investigate whether AP-1 plays a crucial role in the induction of apoptosis by ceramide in HL-60 cells, we tried to block AP-1 activation by treatment with curcumin, 1,7-bis[4-hydroxy-3-methoxy-phenyl]-1,6-heptadiene-3,5-dione. Curcumin, a dietary pigment responsible for the yellow color of curry, was reported to inhibit c-jun expression and to block AP-1 activation induced by phorbol esters(47, 48) . When we treated the cells with 0.5 µM curcumin for 12 h before adding ceramide, curcumin indeed decreased the protein level of c-Jun to 50% of the control and returned the 2-fold increase of c-Jun protein induced by C(2)-ceramide to the control level by approximately 50% inhibition (Fig. 4A). As shown in Fig. 4B, the viable cell number treated with 2 µM ceramide, 0.5 µM curcumin, and both 2 µM ceramide and 0.5 µM curcumin were 2.6 times 10^5/ml, 3.5 times 10^5/ml, and 3.5 times 10^5/ml, respectively, on day 3. These results showed that the growth inhibition induced by C(2)-ceramide was recovered to the levels treated with curcumin alone when the cells were treated with ceramide and curcumin. Therefore, we next investigated whether the growth recovery by curcumin meant a rescue from apoptosis. Assay of DNA fragmentation, a characteristic of apoptosis, was performed 3 h after treatment with ceramide or both ceramide and curcumin (Fig. 4C). Curcumin alone did not induce DNA fragmentation at concentrations up to 2 µM. Addition of only 0.5 µM curcumin inhibited DNA fragmentation induced by 4 µM C(2)-ceramide.


Figure 4: Rescue of ceramide-induced apoptosis by curcumin. A, inhibition of ceramide-induced increase of c-Jun protein by curcumin. HL-60 cells were treated with 0.5 µM curcumin (Cur) 12 h before treatment with 0.1% ethanol vehicle (C) or 2 µM C(2)-ceramide (C). Analysis of c-Jun protein was done 24 h after treatment by Western blot analysis as described under ``Experimental Procedures.'' Nuclear extracts of A431 cells treated with phorbol ester (12-o-tetradecanoylphorbol 13-acetate) or epidermal growth factor (EGF) (Santa Cruz Biotechnology) were used as standard for c-Jun protein. B, rescue of ceramide-induced growth inhibition by curcumin. HL-60 cells were treated with 0.5 µM curcumin 12 h before treatment with 2 µM C(2)-ceramide. The viable cell numbers were counted at the indicated times by trypan blue dye exclusion. The results were obtained from three different experiments. The bars indicate S.D. C, rescue of ceramide-induced DNA fragmentation by curcumin. HL-60 cells were treated with various concentrations of curcumin 12 h before treatment with 4 µM C(2)-ceramide. Analysis of DNA fragmentation was performed 3 h after treatment with C(2)-ceramide as described under ``Experimental Procedures.''



In another experiment, we used antisense c-jun oligonucleotides instead of curcumin in order to further confirm the role of c-jun/AP-1 in the induction of apoptosis(38) . HL-60 cell growth was little affected by antisense or sense c-jun oligonucleotides alone (data not shown). As shown in Fig. 5A, the treatment with 8 µM ceramide in serum-containing medium showed about 65% of growth inhibition compared with the control on day 2. When the cells were treated with both ceramide and antisense c-jun oligonucleotides, the growth recovered according to the increase of oligonucleotide concentrations. By the treatment with 50 µg/ml antisense c-jun, the viable cell numbers treated with 8 µM C(2)-ceramide increased from 2.7 ± 0.3 times 10^5/ml to 7.3 ± 1.0 times 10^5/ml, whereas the number of the control was 7.5 ± 0.5 times 10^5/ml (Fig. 5A). Sense c-jun oligonucleotides, however, did not rescue the growth inhibition induced by ceramide. Moreover, DNA fragmentation induced by 20 µM C(2)-ceramide in serum-containing medium was partially inhibited when the cells were treated with 50 µg/ml antisense c-jun oligonucleotides for 2 days before treatment with C(2)-ceramide, and pretreatment with sense c-jun oligonucleotides had no significant effect on ceramide-induced DNA fragmentation (Fig. 5B). These results strongly suggest that ceramide-induced apoptosis in HL-60 cells is inhibited by blocking AP-1 signal. In other words, AP-1 activation seems to be required in the early process of signal transduction for inducing apoptosis by ceramide in human leukemia HL-60 cells.


Figure 5: Rescue of ceramide-induced apoptosis by antisense c-jun oligonucleotides. A, rescue of ceramide-induced growth inhibition by antisense c-jun oligonucleotides. HL-60 cells at an initial concentration of 2.5 times 10^5 cells/ml were either left untreated (control) or treated with 50 µg/ml antisense (AS) or sense (S) c-jun oligonucleotides in 10% fetal calf serum containing medium 12 h before treatment with 0.1% ethanol vehicle (control) or 8 µM C(2)-ceramide (ceramide). The viable cell numbers were counted 2 days after treatment by trypan blue dye exclusion. The results were obtained from the average of two independent experiments by three different determinations. The bars indicate S.D. B, rescue of ceramide-induced DNA fragmentation by antisense c-jun oligonucleotides. HL-60 cells at an initial concentration of 1 times 10^5 cells/ml were either untreated (C) or treated with 50 µg/ml antisense (AS) or sense (S) c-jun oligonucleotides in 10% fetal calf serum-containing medium for 2 days before the cells were treated with 0.1% ethanol vehicle (C) or 20 µM C(2)-ceramide (C) for 16 h. Analysis of DNA fragmentation was performed as described under ``Experimental Procedures.'' Similar results were obtained in four different experiments.




DISCUSSION

Sphingolipids and sphingolipid breakdown products are emerging as a new class of bioactive molecules that affect cell growth, differentiation, and apoptosis(1, 2) . The sphingomyelin cycle was discovered in HL-60 human myelogenous leukemia cells in response to 1alpha,25-dihydroxyvitamin D(3)(3, 4) . Briefly, 1alpha,25-dihydroxyvitamin D(3) induced the hydrolysis of sphingomyelin with the concomitant generation of ceramide through the activation of neutral and cytosolic sphingomyelinase(49) . TNF-alpha was also found to induce the sphingomyelin cycle (5, 6) and to activate the transcription factor NF-kappaB(12, 13, 14) . Several studies support the idea that ceramide is a biological second messenger for apoptosis and that it mediates some effects through nuclear factor(s). Although c-Jun/AP-1 is an important transcription factor as well as NF-kappaB (20) and the expression of c-jun gene is induced by serum, phorbol esters, or several cytokines including TNF-alpha(22, 23, 24, 25, 26, 27) , the relation between AP-1 and ceramide in apoptosis has not been described. In this study, we demonstrated that ceramide could induce c-jun expression and activate AP-1 binding activity. Although in our study c-jun is transiently induced by C(2)-ceramide as shown in Fig. 1A, TNF-alpha induced prolonged activation of c-jun expression (25) . The reason for the different time course of c-jun induction between ceramide and TNF-alpha is unclear, but it may be due to the difference of the cell type or to the complexity of AP-1 dimer formation corresponding to multiple functions of TNF-alpha. TNF-alpha was reported to activate sphingomyelin cycle to generate ceramide and AP-1 binding activity, respectively(41) . On the other hand, here we have shown that ceramide induced c-jun expression and AP-1 binding activity in HL-60 cells. These results suggest that in some functions of TNF-alpha, including the induction of apoptosis, ceramide may be the intracellular signal to activate AP-1.

The pathway of c-jun/AP-1 activation by ceramide remains to be examined. Among many kinds of serine/threonine kinases, MAP kinase was reported to be activated by ceramide(50) . Recently, the existence of another MAP kinase family termed JNK (c-Jun N-terminal kinase) has been reported(51, 52) . JNK, not MAP kinase, can stimulate c-Jun transcriptional activity by N-terminal phosphorylation(53) . These and our studies suggest to us the idea that ceramide may mediate the signal transduction to induce apoptosis by the activation of AP-1 through JNK, because TNF-alpha and UV light, which seemed to increase intracellular ceramide levels, have been shown to activate JNK(52, 54) . However, as an effector of ceramide signal a novel serine/threonine kinase(s) (13, 14) and phosphatase(s) (15, 16) were also proposed. Although the involvement of those enzymes in c-jun induction by ceramide has not been eliminated, at least serine/threonine phosphatase(s) may not play a role because 10 ng/ml okadaic acid, a potent inhibitor of protein phosphatase 1 and 2A, did not inhibit the increase of c-jun mRNA. (^2)Moreover, the involvement of protein tyrosine kinase(s) and phosphatase(s) in ceramide-induced signals also remained to be clarified.

AP-1 has been shown to have multiple functions in cellular regulation including proliferation(39, 41) , differentiation(40) , and apoptosis (38) . To confirm the requirement of AP-1 activation for apoptosis by ceramide, in this work we used curcumin, 1,7-bis[4-hydroxy-3-methoxy-phenyl]-1,6-heptadiene-3,5-di- one, and antisense c-jun oligonucleotide as specific inhibitors of c-jun/AP-1. Curcumin was reported to possess an inhibitory effect on 12-o-tetradecanoylphorbol 13-acetate-induced AP-1 activity(47) . We found that curcumin actually decreased the protein level of c-Jun (Fig. 4A) and demonstrated that the ceramide-induced growth inhibition was recovered following the inhibition of DNA fragmentation by curcumin (Fig. 4, B and C). Moreover, antisense c-jun oligonucleotide prevented the growth inhibition and DNA fragmentation induced by C(2)-ceramide (Fig. 5, A and B). These data indicate that inhibition of c-jun/AP-1 blocks the growth inhibitory effect of C(2)-ceramide. In other words, C(2)-ceramide seems to induce, at least in part, apoptosis via activation of c-jun/AP-1 in HL-60 cells. Colotta et al.(38) also demonstrated that antisense c-fos and/or c-jun oligonucleotides protect cells from apoptosis induced by growth factor deprivation. In their experiments it was unknown what kind of intracellular signals were involved in between growth factor deprivation and c-jun induction. For the first time we here reported that ceramide is an intracellular physiological molecule to transduce an apoptotic signal through AP-1.

It has been recently shown that sphingosine 1-phosphate, a metabolite of sphingolipids, stimulates AP-1 DNA binding activity in quiescent Swiss 3T3 fibroblasts(55) . In the above report, DL-threo-dihydrosphingosine, a competitive inhibitor of sphingosine kinase, inhibited AP-1 DNA binding activity induced by sphingosine. Because ceramide could be converted to sphingosine by ceramidase and further converted to sphingosine 1-phosphate by sphingosine kinase, we checked the effect of DL-threo-dihydrosphingosine on ceramide-induced c-jun expression. We found that DL-threo-dihydrosphingosine rather increased c-jun expression,^2 suggesting that sphingosine 1-phosphate is not involved in c-jun induction by ceramide. Moreover, we previously reported that C(2)-ceramide was not converted to sphingosine in HL-60 cells within 1 h(4) , suggesting that ceramide exerted the effect without converting to sphingosine or sphingosine 1-phosphate.

In the present work, we demonstrated that ceramide induced apoptosis through the activation of c-jun/AP-1. Because the AP-1 complex may have different functions according to the different components (Jun, JunB, JunD, Fos, FosB, etc.), further study on the components of AP-1 activated by ceramide and the relationship between the composition and function of AP-1 complex will be required.


FOOTNOTES

*
This work was supported by Grants 06267221 and 06670174 from the Japanese Ministry of Education, Science, and Culture and by funds from the Ono Medical Research Fund (to T. O.), the Science Research Promotion Fund of the Japan Private School Promotion Foundation, and the Osaka Dental University Research Foundation (to T. O., H. U., H. O., and N. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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, 1-5-17 Otemae, Cyuo-ku, Osaka, Japan 540. Tel.: 81-6-943-6521; Fax: 81-6-943-8051.

(^1)
The abbreviations used are: TNF-alpha, tumor necrosis factor-alpha; C(2)-ceramide, N-acetylsphingosine.

(^2)
H. Sawai and T. Okazaki, unpublished data.


REFERENCES

  1. Hannun, Y. A., and Bell, R. M. (1989) Science 243, 500-507 [Medline] [Order article via Infotrieve]
  2. Hannun, Y. A. (1994) J. Biol. Chem. 269, 3125-3128 [Free Full Text]
  3. Okazaki, T., Bell, R. M., and Hannun, Y. A. (1989) J. Biol. Chem. 264, 19076-19080 [Abstract/Free Full Text]
  4. Okazaki, T., Bielawska, A., Bell, R. M., and Hannun, Y. A. (1990) J. Biol. Chem. 265, 15823-15831 [Abstract/Free Full Text]
  5. Kim, M.-Y., Linardic, C., Obeid, L., and Hannun, Y. (1991) J. Biol. Chem. 266, 484-489 [Abstract/Free Full Text]
  6. Dressler, K., Mathias, S., and Kolesnick, R. N. (1992) Science 255, 1715-1718 [Medline] [Order article via Infotrieve]
  7. Ballou, L. R., Chao, C. P., Holness, M. A., Barker, S. C., and Raghow, R. (1992) J. Biol. Chem. 267, 20044-20050 [Abstract/Free Full Text]
  8. Mathias, S., Younes, A., Kan, C.-C., Orlow, I., Joseph, C., and Kolesnick, R. N. (1993) Science 259, 519-522 [Medline] [Order article via Infotrieve]
  9. Dobrowsky, R. T., Werner, M. H., Castellino, A. M., Chao, M. V., and Hannun, Y. A. (1994) Science 265, 1596-1599 [Medline] [Order article via Infotrieve]
  10. Cifone, M. G., Maria, R. D., Roncaioli, P., Rippo, M. R., Azuma, M., Lanier, L. L., Santoni, A., and Testi, R. (1994) J. Exp. Med. 177, 1547-1552
  11. Obeid, L. M., Linardic, C. M., Karolak, L. A., and Hannun, Y. A. (1993) Science 259, 1769-1771 [Medline] [Order article via Infotrieve]
  12. Jarvis, W. D., Kolesnick, R. N., Fornari, F. A., Traylor, R. S., Gewirtz, D. A., and Grant, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 73-77 [Abstract]
  13. Mathias, S., Dressler, K. A., and Kolesnick, R. N. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10009-10013 [Abstract]
  14. Liu, J., Mathias, S., Yang, Z., and Kolesnick, R. N. (1994) J. Biol. Chem. 269, 3047-3052 [Abstract/Free Full Text]
  15. Dobrowsky, R. T., and Hannun, Y. A. (1992) J. Biol. Chem. 267, 5048-5051 [Abstract/Free Full Text]
  16. Dobrowsky, R. T., Kamibayashi, C., Mumby, M. C., and Hannun, Y. A. (1993) J. Biol. Chem. 268, 15523-15530 [Abstract/Free Full Text]
  17. Schutze, S., Potthoff, K., Machleidt, T., Berkovic, D., Wiegmann, K., and Kronke, M. (1992) Cell 71, 765-776 [Medline] [Order article via Infotrieve]
  18. Dbaibo, G. S., Obeid, L. M., and Hannun, Y. A. (1993) J. Biol. Chem. 268, 17762-17766 [Abstract/Free Full Text]
  19. Yang, Z., Costanzo, M., Golde, D. W., and Kolesnick, R. N. (1993) J. Biol. Chem. 268, 20520-20523 [Abstract/Free Full Text]
  20. Angel, P., and Karin, M. (1991) Biochim. Biophys. Acta 1072, 129-157 [CrossRef][Medline] [Order article via Infotrieve]
  21. Mitchell, P. J., and Tijan, R. (1989) Science 245, 371-378 [Medline] [Order article via Infotrieve]
  22. Ryseck, R. P., Hirai, S. I., Yaniv, M., and Bravo, R. (1988) Nature 334, 535-537 [CrossRef][Medline] [Order article via Infotrieve]
  23. Ryder, K., and Nathans, D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8464-8467 [Abstract]
  24. Lamph, W. W., Wamsley, P., Sassone-Corsi, P., and Verma, I. M. (1988) Nature 334, 629-631 [CrossRef][Medline] [Order article via Infotrieve]
  25. Brenner, D. A., O'Hara, M., Angel, P., Chojkier, M., and Karin, M. (1989) Nature 337, 661-663 [CrossRef][Medline] [Order article via Infotrieve]
  26. Wu, B., Fodor, E. J., Edwards, R. H., and Rutter, W. J. (1989) J. Biol. Chem. 264, 9000-9003 [Abstract/Free Full Text]
  27. Muegge, K., Williams, T. M., Kant, J. A., Chui, R., Karin, M., Schmidt, A., Young, H. A., and Durrum, S. K. (1989) Science 246, 249-251 [Medline] [Order article via Infotrieve]
  28. Sherman, M. L., Stone, R. M., Datta, R., Bernstein, S. H., and Kufe, D. W. (1990) J. Biol. Chem. 265, 3320-3323 [Abstract/Free Full Text]
  29. Gaynor, R., Simon, K., and Koeffler, P. (1991) Blood 77, 2618-2623 [Abstract]
  30. Mollinedo, F., and Naranjo, J. R. (1991) Eur. J. Biochem. 200, 483-486 [Abstract]
  31. Mollinedo, F., Gajate, C., Tugores, A., Flores, I., and Naranjo, J. R. (1993) Biochem. J. 294, 137-144 [Medline] [Order article via Infotrieve]
  32. Sherman, M., Datta, R., Hallahan, D., Weichselbaum, R., and Kufe, D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5663-5666 [Abstract]
  33. Devary, Y., Gottlieb, R. A., Lau, L. F., and Karin, M. (1991) Mol. Cell. Biol. 11, 2804-2811 [Medline] [Order article via Infotrieve]
  34. Rubin, E., Kharbanda, S., Gunji, H., and Kufe, D. (1991) Mol. Pharmacol. 39, 697-701 [Abstract]
  35. Kharbanda, S. M., Sherman, M. L., and Kufe, D. W. (1990) J. Clin. Invest. 86, 1517-1523 [Medline] [Order article via Infotrieve]
  36. Brach, M. A., Herrmann, F., and Kufe, D. W. (1992) Blood 79, 728-734 [Abstract]
  37. Rubin, E., Kharbanda, S., Gunji, H., Weichselbaum, R., and Kufe, D. (1992) Cancer Res. 52, 878-882 [Abstract]
  38. Colotta, F., Polentarutti, N., Sironi, M., and Mantovani, A. (1992) J. Biol. Chem. 267, 18278-18283 [Abstract/Free Full Text]
  39. Smith, M. J., and Prochownik, E. V. (1992) Blood 79, 2107-2115 [Abstract]
  40. Lord, K. A., Abdollahi, A., Hoffman-Liebermann, B., and Liebermann, D. A. (1993) Mol. Cell. Biol. 13, 841-851 [Abstract]
  41. Brach, M. A., Gruss, H.-J., Sott, C., and Herrmann, F. (1993) Mol. Cell. Biol. 13, 4284-4290 [Abstract]
  42. Goldstone, S. D., and Lavin, M. F. (1994) Oncogene 9, 2305-2311 [Medline] [Order article via Infotrieve]
  43. Ogawa, K., Tashima, M., Toi, T., Sawai, H., Sawada, H., Fujita, J., Maruyama, Y., and Okuma, M. (1994) Exp. Hematol. 22, 45-51 [Medline] [Order article via Infotrieve]
  44. Schreiber, E., Matthias, P., Muller, M. M., and Schaffner, W. (1989) Nucleic Acids Res. 17, 6419 [Medline] [Order article via Infotrieve]
  45. Bielawska, A., Crane, H. M., Liotta, D., Obeid, L. M., and Hannun, Y. A. (1993) J. Biol. Chem. 268, 26226-26232 [Abstract/Free Full Text]
  46. Umehara, H., Minami, Y., Domae, N., and Bloom, E. T. (1994) Int. Immunol. 6, 1071-1080 [Abstract]
  47. Huang, T.-S., Lee, S.-C., and Lin, J.-K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5292-5296 [Abstract]
  48. Hanazawa, S., Takeshita, A., Amano, S., Semba, T., Nirazuka, T., Katoh, H., and Kitano, S. (1993) J. Biol. Chem. 268, 9526-9532 [Abstract/Free Full Text]
  49. Okazaki, T., Bielawska, A., Domae, N., Bell, R. M., and Hannun, Y. A. (1994) J. Biol. Chem. 269, 4070-4077 [Abstract/Free Full Text]
  50. Raines, M. A., Kolesnick, R. N., and Golde, D. W. (1993) J. Biol. Chem. 268, 14572-14575 [Abstract/Free Full Text]
  51. Hibi, M., Lin, A., Smeal, T., Minden, A., and Karin, M. (1993) Genes & Dev. 7, 2135-2148
  52. Derijard, B., Hibi, M., Wu, I.-H., Barrett, T., Su, B., Deng, T., Karin, M., and Davis, R. J. (1994) Cell 76, 1025-1037 [Medline] [Order article via Infotrieve]
  53. Minden, A., Lin, A., Smeal, T., Derijard, B., Cobb, M., Davis, R., and Karin, M. (1994) Mol. Cell. Biol. 14, 6683-6688 [Abstract]
  54. Sluss, H. K., Barrett, T., Derijard, B., and Davis, R. J. (1994) Mol. Cell. Biol. 14, 8376-8384 [Abstract]
  55. Su, Y., Rosenthal, D., Smulson, M., and Spiegel, S. (1994) J. Biol. Chem. 269, 16512-16517 [Abstract/Free Full Text]

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