(Received for publication, June 19, 1995; and in revised form, August 23, 1995)
From the
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.
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
1,25-dihydroxyvitamin D
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-
(TNF-
), (
)
-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-B(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-
(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-
-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-B 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.
Rat c-jun cDNA
(obtained from RIKEN DNA Bank, Japan) or -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
Denhardt's solution, 40% formamide, 4
SSC, 5 mM EDTA, 0.4% SDS, and 100 µg/ml yeast tRNA at 42 °C for 2 h.
Hybridization was performed with 10
cpm of
P-labeled RNA/ml of hybridization buffer at 42 °C for
72 h. Then the membrane was washed in 2
SSC with 0.1% SDS at 37
°C for 30 min, 10 µg/ml RNase A in 2
SSC at 37 °C
for 30 min, and 0.1
SSC with 0.1% SDS at 42 °C for 30 min.
Autoradiography was performed with intensifying screens at -80
°C for 5-14 days.
Figure 1:
Induction
of c-jun mRNA by C-ceramide. A, time
course of induction. HL-60 cells were treated with 0.1% ethanol vehicle (C) or 5 µM C
-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
-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
-ceramide (C
) or 5
µM C
-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-dihydroceramide failed to induce
apoptosis and that the increase of c-jun expression by 5
µM C
-dihydroceramide was approximately
one-third of 5 µM C
-ceramide or corresponding
to the effect of 1 µM C
-ceramide (Fig. 1C), which could not induce apoptosis, when
measured by the densitometer (data not shown). It is clearly shown that
C
-ceramide with biological effects in HL-60 cells but not
C
-dihydroceramide induced a significant increase of
c-jun mRNA.
Figure 2:
Regulation of c-jun induction by
C-ceramide at transcriptional and post-transcriptional
levels. A, effects of actinomycin D or cycloheximide on
c-jun induction by C
-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
-ceramide for
1 h. lane 1, control; lane 2, treated with
C
-ceramide; lane 3, treated with actinomycin D
alone; lane 4, treated with actinomycin D and
C
-ceramide; lane 5, treated with cycloheximide
alone; lane 6, treated with cycloheximide and
C
-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
-ceramide. HL-60 cells were
either untreated (control) or treated with 5 µM
C
-ceramide for 1 h (ceramide). Nuclear run-on
assay for c-jun and
-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
-ceramide. Relative values
were calculated by generalizing the density for c-jun compared
with
-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
-actin mRNA levels. HL-60 cells were
treated with 5 µM C
-ceramide for the indicated
times, and the levels of
-actin mRNA were examined by Northern
blot analysis (upper panel). The lower panel shows 28
S ribosomal RNA stained with methylene blue
dye.
Figure 3:
Effect
of C-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
-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
-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.''
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-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
-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
-ceramide. Analysis of DNA fragmentation was
performed 3 h after treatment with C
-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-ceramide increased from 2.7 ± 0.3
10
/ml to 7.3 ± 1.0
10
/ml, whereas the number of the control was 7.5 ±
0.5
10
/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
-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
-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
10
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
-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
10
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
-ceramide (C
) for 16 h. Analysis of DNA
fragmentation was performed as described under ``Experimental
Procedures.'' Similar results were obtained in four different
experiments.
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 1,25-dihydroxyvitamin
D
(3, 4) . Briefly,
1
,25-dihydroxyvitamin D
induced the hydrolysis of
sphingomyelin with the concomitant generation of ceramide through the
activation of neutral and cytosolic sphingomyelinase(49) .
TNF-
was also found to induce the sphingomyelin cycle (5, 6) and to activate the transcription factor
NF-
B(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-
B (20) and the expression of c-jun gene is
induced by serum, phorbol esters, or several cytokines including
TNF-
(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
-ceramide as shown in Fig. 1A, TNF-
induced prolonged activation of c-jun expression (25) . The reason for the different time course of c-jun induction between ceramide and TNF-
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-
.
TNF-
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-
, 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- 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. (
)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-ceramide (Fig. 5, A and B). These data indicate that inhibition of
c-jun/AP-1 blocks the growth inhibitory effect of
C
-ceramide. In other words, C
-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, suggesting that sphingosine
1-phosphate is not involved in c-jun induction by ceramide.
Moreover, we previously reported that C
-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.