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(Received for publication, June 28, 1995; and in revised form, August 2, 1995) From the
Tumor necrosis factor
TNF TNF
We investigated the role of ceramide in TNF
Figure 1:
Stimulation of JNK/SAPK activity by
sphingomyelinase (SM) and D-e-C
Increases in JNK/SAPK activity are observed at
C Because C The effect of ceramide on
the induction of NF
Figure 2:
TNF
TNF
Figure 3:
MAP kinase activity is stimulated by
sphingomyelinase but not ceramide. An immune complex MAP kinase assay
was performed with HL-60 cell extracts prepared following cell
treatment (Tx) with the indicated agonists for 10 min (lanes 1-5) or 2 min (lanes 6-9). C5 and C10, 5 and 10 µMD-e-C
The ability of ceramide to activate JNK/SAPK predicts that ceramide
would activate downstream targets of this kinase cascade. The c-jun gene contains two non-consensus AP-1 binding sites in its
promoter, which are recognized with high affinity by protein complexes
containing c-Jun and ATF-2(37, 38) . The
transcriptional activation domains of both c-Jun (11) and ATF-2 (12) are phosphorylated and activated by JNK/SAPK. TNF
Figure 4:
Stimulation of c-jun transcriptional activity. A, c-jun mRNA levels
are increased following TNF
To
further assess the functional sequela of stimulation by TNF TNF Recent studies have delineated the steps immediately
upstream of JNK/SAPK activation(46, 47, 48) ,
but connections between these cytoplasmic and nuclear kinases and
upstream messengers leading to their activation are unresolved. This
study demonstrates that ceramide is a link between binding of TNF TNF TNF The evidence is
pointing to the existence of a novel signal transduction pathway that
may be specifically involved in the stress response. A number of agents
of systemic stress (e.g. TNF
Volume 270,
Number 39,
Issue of September 29, pp. 22689-22692, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(TNF) activates the
stress-activated protein kinases (SAPKs, also known as Jun nuclear
kinases or JNKs) resulting in the stimulation of AP-1-dependent gene
transcription and induces the translocation of NF
B to the nucleus
resulting in the stimulation of NFB-dependent gene transcription.
A potential second messenger for these signaling pathways is ceramide,
which is generated when TNF activates sphingomyelinases. We show
that treatment of HL-60 human promyelocytic cells with exogenous
sphingomyelinase leads to rapid stimulation of JNK/SAPK activity, an
effect not mimicked by treatment with phospholipase A
, C,
or D. Further, JNK/SAPK activity is stimulated 2.7- and 2.8-fold,
respectively, in cells exposed to C-ceramide (5
µM) or TNF (10 ng/ml). The prolonged stimulation of
this kinase activity by C-ceramide is similar to that
previously reported for TNF. In contrast, the related
mitogen-activated protein kinases ERK1 and ERK2 are weakly stimulated
following TNF treatment (1.5-fold) and are inhibited by
C-ceramide treatment. TNF also potently stimulates
NF-B DNA binding activity and transcriptional activity, but these
effects are not mimicked by addition of C-ceramide or
sphingomyelinase to intact cells. Furthermore, TNF,
sphingomyelinase, and C-ceramide induce c-jun, a
gene that is stimulated by the ATF-2 and c-Jun transcription factors.
These data suggest that ceramide may act as a second messenger for a
subset of TNF's biochemical and biological effects.
(
)is a multifunctional cytokine involved in
inflammation, infection, and cancer(1, 2) . TNF
has potent biological effects on cultured cells, including induction of
apoptosis and differentiation(3, 4) . TNF signals
via unknown second messengers leading to activation of NFB (5, 6) and JNKs/SAPKs(7, 8, 9) and a weaker stimulation of MAP kinase
activity(7, 8) . Each of these pathways leads to
activation of a specific set of transcription factors; JNK/SAPK
phosphorylates and activates c-Jun (10, 11) and ATF-2 (12) , NF-B translocates to the nucleus where it functions
as a transcription factor, and MAP kinase phosphorylates and activates
Elk-1 (13, 14) and other transcription factors. stimulates sphingomyelinase activity, which results in the
generation of ceramide(15, 16) . Treatment of cells
with the cell-permeable C-ceramide mimics TNF in the
induction of apoptosis(4) , differentiation(15) , and
in the activation of a ceramide-activated protein phosphatase activity
similar to PP2A(17, 18) . A ceramide-activated protein
kinase that is proline-directed has been described, but its specific
target(s) is unknown(19) . Thus, many of the biological and
biochemical effects of TNF are mimicked by ceramide treatment, but
the molecular links between TNF-induced ceramide production and
transcriptional activation are not clear.
Reagents
All reagents were obtained from Sigma
unless otherwise specified. Recombinant human TNF (for HL-60
cells) and murine TNF (for L929 cells) were obtained from R&
Systems, Minneapolis, MN. Sphingomyelinase (Bacillus cereus)
was obtained from Boehringer Mannheim.
(2S,3R)-D-erythro-N-Acetylsphingosine (D-e-C) was prepared by acetylation of
(2S,3R)-sphingosine as described (20) . DL-erythro-Dihydro-C-ceramide was
prepared by acetylation of DL-erythro-dihydrosphingosine (20) (obtained
from Sigma). Aliquots were resuspended at 20 mM in ethanol
before use. Radionucleotides were obtained from ICN Biomedicals
(Irvine, CA). [-
P]ATP used for in vitro kinase assays was >4000 Ci/mmol.
Tissue Culture
HL-60 cells were maintained at a
density of <1 
10
cells/ml for less than 1 month
before discarding. Cells were cultured in RPMI 1640 (Life Technologies,
Inc.) with 10% heat-inactivated fetal bovine serum (Gemini Bioproducts,
Calabasas, CA) in a humidified atmosphere of 5% CO, 95%
air. For treatment with agonists, cells were resuspended in serum-free
medium as described (18) at a density of 250,000 cells/ml and
incubated for at least 2 h before treatment. L929 fibrosarcoma cells (21) were cultured in Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) supplemented with 5% fetal bovine
serum (Gemini).Extract Preparation and in Vitro Kinase
Assays
Whole cell extracts were prepared as described
previously(22) . For assessment of JNK/SAPK activity, 50 µg
of extract was used in a solid state in vitro kinase assay as
described (23) with GST-c-Jun (10) as the substrate.
For assessment of MAP kinase activity, whole cell extracts were
immunoprecipitated with antisera to ERK2, which cross-reacts with ERK1
(SC-94, Santa Cruz Biotechnology, Santa Cruz, CA). Immune complexes
were collected with protein A/G-agarose (Oncogene Science, Uniondale,
NY), washed extensively, and used in an in vitro kinase assay
with 50 µM ATP (5 µCi of
[-
P]ATP), 10 mM MgCl
,
1 mM dithiothreitol, 0.1 mM Na
VO
, 25 mM 
-glycerophosphate, 10 mMp-nitrophenyl
phosphate for 20 min at 30 °C. 2 µg of GST-ElkC (a glutathione S-transferase fusion protein containing the C terminus of
Elk-1, which contains multiple MAP kinase phosphorylation sites) was
used as substrate(14, 24) . Proteins were fractionated
on 12.5% SDS-polyacrylamide gel electrophoresis and subjected to
autoradiography.Gel Mobility Shift Assays
NF-B gel mobility
shift assays were performed as described previously(3) .
Antibodies used in binding assays (1 µl/reaction) were obtained
from Santa Cruz Biotechnology.Plasmids and Transfection Analysis
Transient
transfection of L929 cells was performed by calcium phosphate
coprecipitation as described previously(25) . Jun-luciferase
consists of -1.1 to +740 of human c-jun(26) linked to luciferase. Gal-ElkC (13) is
cotransfected with 5 Gal-luciferase as described(25) .
(KB)3-luciferase consists of three consensus NF-B sites (27) linked to luciferase. Luciferase assays were performed as
previously described (28) .Northern Blot Analysis
Total RNA was prepared from
HL-60 cells and electrophoresed as described previously(29) .
After blotting to nitrocellulose, the RNA was probed with a HindIII-PstI fragment of human c-jun(30) labeled by random priming (Stratagene, San Diego, CA)
with [-P]dCTP (3000 Ci/mmol, ICN, Irvine,
CA). Final wash stringency was 0.1
SSC, 0.1% SDS at 55 °C.
The blot was stripped and reprobed with a glyceraldehyde-phosphate
dehydrogenase cDNA probe (pHcGAP, ATCC 57090) to assess sample loading.
-stimulated
signal transduction by comparing the effects of TNF,
sphingomyelinase, and C-ceramide on JNK/SAPK activity,
NFB activity, and MAP kinase activity. Exogenous bacterial
sphingomyelinase catalyzes the hydrolysis of cell membrane
sphingomyelin and the formation of ceramide when added to intact HL-60
human myelocytic leukemia cells(31, 32) . Treatment of
cells with exogenous sphingomyelinase potently activates JNK/SAPK
activity, while equivalent amounts of the lipases phospholipase C,
phospholipase A, or phospholipase D do not (Fig. 1A). Exogenously added C-ceramide or
TNF stimulates JNK/SAPK activity in HL-60 cells to comparable
levels (Fig. 1A). The activation of JNK/SAPK activity
by C-ceramide is first detected in cell extracts after 10
min (Fig. 1B). In contrast to agents that transiently
stimulate JNK/SAPK activity, such as epidermal growth factor and
phorbol esters(8) , ceramide elicits a prolonged activation
(more than 2-fold over background levels) at least 2 h after
stimulation. The kinetics of JNK/SAPK activation by ceramide parallel
those of TNF stimulation (data not shown and (8) ).
-ceramide. A, HL-60 cells were cultured as described (18) and
treated with D-e-C-ceramide (C2, 5
µM, lane2) or ethanol only (con, lane1) or the enzymes indicated (300
milliunits/ml, lanes4-7) or vehicle (con, lane3) or TNF (10 ng/ml, lane8) for 20 min. The single prominent band visible
following 10% SDS-polyacrylamide gel electrophoresis and
autoradiography represents phosphorylated GST-c-Jun. Equal substrate
loading was confirmed in all cases by Coomassie Blue staining of the
gels prior to autoradiography. PL, phospholipase. B,
time course of JNK/SAPK induction following D-e-C-ceramide addition (5 µM) to
HL-60 cells. The substrate (sub) for the kinase assays was
GST-cJun or GST alone as control. C, response of SAPK activity
to D-e-C-ceramide treatment. All cells received
the indicated concentration in an equal volume of ethanol or ethanol
alone 5(di), 5 µM dihydro-C-ceramide. Extracts
were prepared after 10 min of treatment. The substrate for the kinase
assay was wild-type GST-cJun (lanes 2-9) or GST-cJun(AA) (lane1) in which Ser-63 and Ser-73 are mutated to
Ala(10) .
-ceramide concentrations of 1 µM and above (Fig. 1C). 5 µM C-ceramide
stimulates JNK/SAPK activity 2.7-fold over untreated cell levels
(average of seven experiments), while TNF (10 ng/ml) stimulates
JNK activity 2.8-fold (average of four experiments). Uptake of
C-ceramide applied at 3 µM(16, 20) results in an intracellular
C-ceramide concentration that is equimolar to the
concentration of ceramides generated endogenously following cell
treatment with TNF(15) .-ceramide
is an amphiphilic lipid analog that may have nonspecific activities, it
was important to establish the specificity of its activation of
JNK/SAPK, a family of kinases activated by a wide variety of stress
stimuli(7) . DL-erythro-dihydro-C-ceramide is a close
structural analog of C-ceramide lacking the trans-unsaturated bond in the sphingosine moiety. Previous
studies (20) demonstrated that the uptake and metabolism of
radiolabeled C-ceramide and dihydro-C-ceramide
are similar. DL-erythro-dihydro-C-ceramide fails to
activate JNK/SAPK activity (Fig. 1C, lane8). Therefore, the effects of C-ceramide on
JNK/SAPK are specific and suggest a specific interaction of ceramide
with a component of the JNK/SAPK pathway.B activity is unresolved, in that ceramide
treatment of intact cells induces binding activity in one report (31) but not others(3, 33, 34) .
TNF potently stimulated NF-B DNA binding activity (Fig. 2A, lane7), with the
predominant form of NFB consisting of p65/p50 heterodimers (Fig. 2B, lane3). However, treatment
with bacterial sphingomyelinase (Fig. 2A, lanes5 and 6) or C-ceramide (Fig. 2A, lanes 2-4) failed to induce
NFB binding activity.
, but not sphingomyelinase or D-e-C-ceramide, activates NF-B binding
activity. A, HL-60 cells were treated with vehicle, 0.1, 1, or
10 µM C-ceramide (lanes 1-4),
or with 10 or 100 milliunits/ml sphingomyelinase (SM) (B.
cereus, Boehringer Mannheim) (lanes5 and 6) or recombinant TNF (T, 10 nM, lane7) for 30 min prior to nuclear extract
preparation and gel mobility shift analysis using a consensus NF-B
binding site probe. ns, nonspecific shifted complex; p50-65, heterodimeric NF-B. B, extracts
from TNF-treated HL-60 cells were used in binding assays, with
antibodies to p50 (lane2), p65 (lane3), or c-Rel (lane4), or with a
50-fold excess of unlabeled wild-type (wt, lane5) or mutant NF-B binding site (mut, lane6) included in the reaction. After incubation on
ice for 1 h, products were resolved on non-denaturing polyacrylamide
gels. SS, supershifted NF-B; ns, nonspecific
shifted complex (not competed by cold excess
oligonucleotide).
is a weak inducer of MAP kinase
(ERK1 and ERK2) activity relative to its induction of JNK/SAPK or
relative to the induction of MAP kinases by growth factors such as
epidermal growth factor(7, 8) . In HL-60 cells, high
concentrations of TNF weakly activate MAP kinase activity using
recombinant GST-Elk activation domain as a substrate in an immune
complex kinase assay (1.5-fold over unstimulated levels) (Fig. 3, lane5). As reported
elsewhere(35) , high concentrations of exogenous
sphingomyelinase activate MAP kinase activity (Fig. 3, lane7). However, treatment of cells with
C-ceramide almost completely attenuates basal MAP kinase
activity in a concentration-dependent manner (Fig. 3, lanes
2, 3, and 8), and this effect was not observed
with dihydro-C-ceramide-treated cells (Fig. 3, lanes4 and 9). Similar results are obtained
using myelin basic protein as a substrate for MAP kinase in an immune
complex kinase assay or using an in-gel MAP kinase assay (36) with MBP incorporated into the gel (data not shown).
-ceramide; C5(di), 5 µMDL-erythro-dihydro-C-ceramide; SM, 100 milliunits/ml Staphylococcus aureus sphingomyelinase; TNF, 50 ng/ml recombinant human
TNF; con, control. GST-ElkC was used as the substrate.
Equal substrate loading was confirmed in all cases by Coomassie Blue
staining of the gels prior to
autoradiography.
,
C-ceramide, and sphingomyelinase stimulate c-jun mRNA levels in HL-60 cells (Fig. 4A). Thus, all
three agents that stimulate JNK/SAPK activity also lead to enhanced
expression of the c-jun gene. The heightened ability of
sphingomyelinase to activate c-jun transcription may be due to
its activation of Elk-1 (Fig. 3), which can stimulate c-fos transcription and hence increase total AP-1 activity.
, sphingomyelinase, and
C-ceramide treatment. HL-60 cells were incubated with
TNF (TNF, 20 ng/ml), sphingomyelinase (SM, 100
milliunits/ml), C-ceramide (CER, 10
µM), or untreated (NONE) for 1 h, and total RNA
was prepared followed by electrophoresis and Northern blotting. The
blot was hybridized first with a radioactively labeled human c-jun probe (upperpanel) and then with a labeled
human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe.
Blots were quantitated by phosphor image analysis, and -fold induction
of c-jun mRNA normalized to GAPDH mRNA (FOLD) is
indicated. B, activity of luciferase reporter genes in the
L929 fibrosarcoma cell line. Jun-luciferase (Luc), or
5XGal-luciferase plus Gal-ElkC, or (KB)3-luciferase (three copies of
the consensus NF-B response element linked to luciferase) was
transfected into L929 cells. Cells were then treated with TNF (20
ng/ml) or sphingomyelinase (100 milliunits/ml) for 5 h, and extracts
were prepared for determination of luciferase activity. -Fold
activation represents reporter activity relative to cells treated with
vehicle only. Results from one representative experiment performed in
duplicate are shown (±S.E.). Results were consistent over at
least three experiments.
or
bacterial sphingomyelinase, we measured transcriptional activities with
reporter gene assays in L929 fibrosarcoma cells, which are efficiently
transfected and are responsive to TNF(21) . TNF and
sphingomyelinase are equally effective in stimulating the reporter gene
driven by the c-jun promoter (Jun-Luc, Fig. 4B, toppanel), consistent with
their stimulation of JNK/SAPK activity (Fig. 1) and c-jun mRNA level (Fig. 4A). The transcriptional activity
of a fusion protein consisting of the Gal4 DNA binding domain and the
Elk-1 transactivation domain (Gal-ElkC(14) ; a MAP kinase
substrate), measured by a reporter plasmid containing a luciferase gene
with Gal4 binding sites (5XGal-luciferase), is activated by
sphingomyelinase and to a lesser degree by TNF (Fig. 4B, middlepanel), in accord
with their stimulation of MAP kinase activity in vitro (Fig. 3). TNF but not sphingomyelinase markedly
stimulated an NF-B-responsive reporter gene ((KB)3-luciferase, Fig. 4B, bottompanel), consistent
with the effect of these agents on NF-B DNA binding activity (Fig. 2). signals through two cell surface
receptors, TNF-R1 (p55) and TNF-R2 (p75), which contain no apparent
catalytic activity and whose intracellular domains are not homologous
to characterized signaling proteins. Two proteins, TRAF1 and TRAF2,
bind to the cytoplasmic domain of TNF-R2(39) . An unrelated
protein, TRADD, binds to the TNF-R1-associated death domain and might
be involved in TNF-induced apoptosis and NF-B
activation(40) . Most of TNF's biological effects
are mediated by TNF-R1(41, 42, 43) , but the
mechanisms by which second messengers are recruited are unknown.
Several of TNF's second messengers are activated via TNF-R1,
including the activation of a sphingomyelinase, protein kinase C, and
phospholipase A(41) . By using differences in the
species specificity of TNF(44, 45) , we have
found that TNF also activates JNK/SAPK through TNF-R1 (data not
shown).
to TNFR1 and activation of a cytoplasmic kinase cascade that results in
stimulation of JNK/SAPK activity and c-jun expression.
TNF and C-ceramide activate JNK/SAPK to the same
degree (Fig. 1) and stimulate activity with similar kinetics in
HL-60 cells. We have previously demonstrated that JNK1 (also known as
SAPK) is a major component of TNF
-activated
JNK/SAPK(8) . By activating JNK/SAPK, TNF and ceramide
activate a subset of AP-1 transcription factors, such as c-Jun and
ATF-2, which in turn will preferentially induce genes with specific
non-consensus AP-1 binding sites, such as the c-jun gene
itself (Fig. 4). Therefore, this study provides strong evidence
that ceramide functions as the second messenger in TNF signaling
resulting in the activation of JNK/SAPK and c-jun.
is not a potent activator of MAP kinases(7, 8) , and
we demonstrate here that C-ceramide actually decreases MAP
kinase activity in HL-60 cells. Inhibition of MAP kinase activity may
result from the activity of a ceramide-activated protein phosphatase (17, 18) acting on a component of the MAP kinase
cascade. Ceramide-activated protein phosphatase is a phosphatase of the
PP2A class, and PP2A activity on both MAP kinases and MEKs has been
described (reviewed in (49) ). The weak activation of MAP
kinase activity by TNF may be the result of ceramide-independent
activation of a component (MEK or MAP kinase itself) downstream of
Raf-1 and/or by activation of protein kinase C(50) . is a potent inducer of NFB (5, 51) ( Fig. 2and Fig. 4), perhaps through the activation of
protein kinase C (52) , and ceramide potentiates the
stimulation of NF
B by TNF(3) . Ceramide stimulates
NF-B binding activity in permeabilized cells, which has been
attributed to the activation of an acidic
sphingomyelinase(32) . However, extracts derived from intact
HL-60 cells treated with exogenous sphingomyelinase or
C-ceramide have elevated JNK/SAPK activity but not
NF-B DNA binding activity. Perhaps these different results reflect
the existence of separate ceramide pools, such that
C-ceramide and the ceramide generated by exogenous
bacterial sphingomyelinase are in a different cellular compartment than
the ceramide generated by acidic sphingomyelinase.) and tissue injury (hypoxia,
UV irradiation, and chemotherapeutic agents) activate both a neutral
sphingomyelinase and JNK/SAPK. This study provides a link between the
sphingomyelinase/ceramide pathway and the JNK/SAPK pathway by
demonstrating that ceramide functions upstream of JNK/SAPK, resulting
in activation of nuclear downstream effects.
), tumor necrosis factor ; JNK, Jun
nuclear kinase; SAPK, stress-activated protein kinase; MAP,
mitogen-activated protein; PP2A, protein phosphatase 2A; D-e-C,
(2S,3R)-D-erythro-N-acetylsphingosine;
GST, glutathione S-transferase; MEK, MAP kinase kinase.
We thank G. Franzoso for advice on extract
preparation, C. Hauser for providing the (KB)3-luciferase reporter
plasmid, R. Treisman for Gal-ElkC, A. Lin for GST-ElkC, and C. Bradham
for comments on the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 V. M.-D. Mas, C. Bezombes, A. Quillet-Mary, A. Bettaïeb, A. D. T. D'orgeix, G. Laurent, and J.-P. Jaffrézou Implication of Radical Oxygen Species in Ceramide Generation, c-Jun N-Terminal Kinase Activation and Apoptosis Induced by Daunorubicin Mol. Pharmacol., November 1, 1999; 56(5): 867 - 874. [Abstract] [Full Text]
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 Y. Nagata and K. Todokoro Requirement of Activation of JNK and p38 for Environmental Stress-Induced Erythroid Differentiation and Apoptosis and of Inhibition of ERK for Apoptosis Blood, August 1, 1999; 94(3): 853 - 863. [Abstract] [Full Text] [PDF]
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R. Li, E. J. Blanchette-Mackie, and S. Ladisch Induction of Endocytic Vesicles by Exogenous C6-ceramide J. Biol. Chem., July 23, 1999; 274(30): 21121 - 21127. [Abstract] [Full Text] [PDF]
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