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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 217-225
Ceramide and Cyclic Adenosine Monophosphate (cAMP) Induce cAMP
Response Element Binding Protein Phosphorylation via Distinct Signaling
Pathways While Having Opposite Effects on Myeloid Cell Survival
By
Michael P. Scheid,
Ian N. Foltz,
Peter R. Young,
John W. Schrader, and
Vincent Duronio
From the Department of Medicine and The Biomedical Research Centre,
University of British Columbia and Vancouver Hospital and Health
Sciences Centre, Vancouver, Canada; and SmithKline Beecham, King of
Prussia, PA.
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ABSTRACT |
The role of ceramide as a second messenger is a subject of great
interest, particularly since it is implicated in signaling in response
to inflammatory cytokines. Ceramide induces apoptosis in both
cytokine-dependent MC/9 cells and factor-independent U937 cells.
Elevation of cyclic adenosine monophosphate (cAMP) levels inhibits
apoptosis induced by ceramide and several other treatments. One target of cAMP-mediated signaling is the transcription factor CREB
(cAMP response element binding protein), and recently CREB phosphorylation at an activating site has been shown to also be mediated by a cascade involving p38 mitogen-activated protein kinase
(MAPK), one of the stress-activated MAP kinases. Because no role for
p38 MAPK in apoptosis has been firmly established, we examined the
relationship between p38 MAPK and CREB phosphorylation under various
conditions. Ceramide, or sphingomyelinase, like tumor necrosis
factor- (TNF-) or the hematopoietic growth factor, interleukin-3
(IL-3), was shown to activate p38 MAPK, which in turn activated MAPKAP
kinase-2. Each of these treatments led to phosphorylation of CREB (and
the related factor ATF-1). A selective p38 MAPK inhibitor,
SB203580, blocked TNF-- or ceramide-induced CREB phosphorylation,
but had no effect on the induction of apoptosis mediated by these
agents. The protective agents cAMP and IL-3 also led to CREB
phosphorylation, but this effect was independent of p38 MAPK, even
though IL-3 was shown to activate both p38 MAPK and MAPKAP kinase-2.
Therefore, the opposing effects on apoptosis observed with cAMP and
IL-3, compared with ceramide and TNF-, could not be explained on the
basis of phosphorylation of CREB. In addition, because SB203580 had no
effect of TNF- or ceramide-induced apoptosis, our results strongly
argue against a role for p38 MAPK in the induction of TNF-- or
ceramide-induced apoptosis.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
CERAMIDE IS GENERATED intracellularly by
sphingomyelinase (SMase)-mediated breakdown of
sphingomyelin, and has gained attention as a potential second messenger
in response to various extracellular stimuli.1,2
Intracellular levels of ceramide have been shown to increase in
response to inflammatory cytokines3 and cellular
stress,4 and its actions have been reported to affect both
differentiation5,6 and apoptosis1,4 in target cells. Ceramide may act via the activation of specific
kinases7,8 and/or phosphatases,9,10
although not until recently was a cDNA clone identified that
corresponded to a ceramide-activated kinase.11 Ceramide has
been shown to induce activation of a stress-activated protein kinase
(SAPK), and the activation of SAPK has been proposed to be a necessary
event in the induction of apoptosis.12 While several
downstream events regulated by ceramide have been characterized,
potential nuclear events affected by ceramide that might contribute to
its biologic effect have not been as well described.
Another second messenger that has been suggested to regulate apoptosis,
both positively and negatively, is cyclic adenosine monophosphate
(cAMP). For example, cAMP has been shown to induce apoptosis in
thymocytes,13,14 but inhibit apoptosis in PC12 cells, in
which cAMP has the additional property of inducing
differentiation.15 Lanotte et al16 found that
cAMP analogs, cholera toxin, and prostaglandins could lead to apoptosis
in a rat myelocytic leukemic cell line.
In our recent work, we have studied the role of key enzymes in
signaling pathways that are important in regulation of apoptosis in
myeloid cells. We17 and others18,19 have shown
that activation of phosphatidylinositol (PI) 3-kinase is important,
since use of PI 3-kinase inhibitors can cause apoptosis in many,
although not all, conditions. Recent studies have also implicated
activation of the protein kinase PKB/AKT, downstream of PI
3-kinase as a key event for inhibiting apoptosis.20,21
We have found that elevation of cAMP levels is able to inhibit
apoptosis induced by several means, including ceramide treatment, in
myeloid cells. This study was designed to characterize downstream phosphorylation events regulated in response to ceramide and cAMP, as
well as other agonists known to promote or inhibit apoptosis. We
focused our efforts on p38 mitogen-activated protein kinase (MAPK),22 since this kinase has been demonstrated to be an
upstream signaling component of one pathway leading to cAMP response
element binding protein (CREB) phosphorylation.27
Additionally, conflicting results have been published regarding a role
for p38 MAPK in the execution of apoptosis in hematopoietic cells. We
were therefore interested in the activation state of p38 MAPK in
response to each of the conditions that lead to either apoptosis or
survival.
Treatment of cells with exogenous C2-ceramide, natural endogenous
ceramide generated by bacterial sphingomyelinase, or tumor necrosis
factor- (TNF-), all led to activation p38 MAPK, and subsequent
activation of MAPKAP kinase-2, as well as inducing phosphorylation at
activating sites of CREB and a related transcription factor,
ATF-1. CREB and ATF-1 phosphorylation in cells with
elevated ceramide or following treatment with TNF- could be blocked
by a selective inhibitor of p38 MAPK, SB203580. In contrast,
phosphorylation of CREB and ATF-1 induced by interleukin-3 (IL-3)
treatment, or by elevated cAMP levels, was not blocked by SB203580,
although IL-3 also activated MAPKAP kinase-2 via a p38 MAPK-dependent
mechanism. CREB and ATF-1 phosphorylation can therefore be considered
downstream targets following p38 MAPK activation in response to
elevation of ceramide, but IL-3-induced CREB phosphorylation is
independent of p38 MAP kinase. It is not clear at present what the
potential physiologic role of CREB and ATF-1 may be in myeloid cells,
but our studies show that the contrasting effects of various agents on
apoptosis cannot be explained solely by their effects either on a p38
MAPK pathway, or on phosphorylation of CREB and ATF-1.
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MATERIALS AND METHODS |
Cell culture and reagents.
MC/9 and U937 cells were obtained from the American Type Culture
Collection (Rockville, MD). Both cell lines were
maintained in RPMI media supplemented with 10% fetal bovine serum at
37°C in a humidified atmosphere containing 5% CO2.
Additionally, WEHI-3-conditioned media (10% vol/vol) was added to
MC/9 cultures as a source of IL-3. Before experiments, cells were
washed several times with phosphate-buffered saline (PBS) and
resuspended in RPMI containing 20 mmol/L HEPES-HCl, pH 7.4, and
incubated for a minimum of 1 hour at 37°C.
C2-ceramide and dihydro-C2-ceramide were obtained from Calbiochem (La
Jolla, CA); Staphylococcus aureus SMase and forskolin were from
Sigma (St Louis, MO), and TNF- from R & D Systems (Minneapolis, MN).
Antibodies to MAPKAP kinase-2, CREB, and phospho-CREB were from Upstate
Biotechnology (Lake Placid, NY). ATF-2 (1-96) and Hsp25 were obtained
from Santa Cruz Biotechnology (Santa Cruz, CA).
C2-ceramide and sphingomyelinase treatments.
MC/9 or U937 cells (2 to 10 × 106) suspended in 500 µL RPMI media supplemented with 20 mmol/L HEPES-HCl, pH 7.4, were
either treated with 2 µL dimethyl sulfoxide (DMSO) or an equal volume of DMSO containing SB203580 for a final concentration of 1 µmol/L, for 20 minutes. Cells were then treated for various times with bacterial sphingomyelinase (100 mU/mL), C2-ceramide (25 to 50 µmol/L), or dihydro-C2-ceramide (50 µmol/L). In some experiments, U937 cells were stimulated with recombinant human TNF- (10 ng/mL) or
forskolin (40 µmol/L). In addition, MC/9 cells were treated with 10 µg/mL synthetic IL-3, previously shown to give maximal activation of
tyrosine phosphorylation, or forskolin (40 µmol/L), for various
times.
cAMP determinations.
cAMP was measured using an enzyme linked immunosorbent assay kit from
Amersham (Montreal, Canada; RPM-225). Cells treated with
various stimuli were lysed in ice-cold ethanol:H2O (65:35), dried under nitrogen, and resuspended in assay dilution buffer according to the manufacturer's protocol. cAMP measured in each unknown was quantitated using a standard curve generated using known
quantities of cAMP.
Cell extract preparation and immunoblot analysis.
For analysis of phospho-CREB levels, cells were pelleted following
treatments and solubilized in 200 µL of sodium dodecyl sulfate (SDS)
sample buffer containing 6 mol/L urea, followed by boiling for 5 minutes. Lysates were then briefly probe-sonicated to shear DNA.
Samples were separated by SDS-polyacrylamide gel electrophoresis
(PAGE), transferred by semidry blotting to nitrocellulose membranes,
and blocked for several hours with 3% skim milk in Tris-buffered
saline. Membranes were probed overnight with anti-phospho-CREB (UBI)
and detected by chemiluminesence (ECL) according to the manufacturer
(Amersham). In some cases, membranes were incubated in stripping buffer
(60 mmol/L Tris-HCl, pH 6.6, 2% SDS, and 100 mmol/L 2-mercaptoethanol)
for 20 minutes at 50°C, reblocked, and reprobed with anti-CREB
(UBI).
For analysis of poly(ADP-ribose) polymerase (PARP) cleavage, cells were
harvested following treatments with C2-ceramide for various times, in
the presence or absence of forskolin (10 µmol/L) and
isobutylmethylxanthine (IBMX; 50 µmol/L). Cell lysates were prepared
as for CREB analysis, fractionated by SDS-PAGE (10% 30:1 acrylamide:bisacrylamide), transferred to nitrocellulose, and immunoblotted with an antibody raised against full-length PARP (#422;
Enzyme System Products, La Jolla, CA) to detect the
unprocessed 118-kD form and the processed 86-kD fragment.
p38 MAPK and MAPKAP kinase-2 in vitro kinase assays.
For kinase assays, cells were pelleted and lysed with ice-cold
solubilization buffer (50 mmol/L Tris-HCl, pH 7.4, 10% [vol/vol] glycerol, 1% [vol/vol] Triton X-100, 100 mmol/L NaCl, 25 mmol/L  -glycerophosphate, 10 mmol/L NaF, 1 mmol/L sodium molybdate, 0.2 mmol/L sodium vanadate, 40 µg/mL phenylmethylsulfonylfluoride, 1 µmol/L pepstatin, 0.5 µg/mL leupeptin, and 10 µg/mL soybean trypsin inhibitor), and nuclei removed by brief centrifugation at
4°C. Supernatants were incubated with either anti-p38 antibody and
10 µL of protein A-Sepharose beads or anti-MAPKAP kinase-2 antibody
with 10 µL protein G-Sepharose beads for 1 hour at 4°C with
continual mixing. Beads were washed twice with solubilization buffer
and twice with kinase buffer (20 mmol/L HEPES-HCl, pH 7.4, 25 mmol/L
MgCl2, 25 mmol/L -glycerophosphate, 1 mmol/L
dithiothreitol [DTT], and the same quantities of phosphatase and
protease inhibitors as in the solubilization buffer). Beads were
incubated with 25 µL of kinase buffer containing 50 µmol/L
adenosine triphosphate (ATP), 2 µg/sample ATF-2 peptide
(corresponding to amino acids 1-96; for p38 assays), 2 µg/sample
Hsp25 (for MAPKAP kinase-2 assays), and 10 µCi/sample
 -[32P]-ATP at 30°C for 15 minutes. Reactions were
stopped with 6 µL 5× SDS sample buffer and boiling for 5 minutes. Samples were separated by SDS-PAGE, transferred to
nitrocellulose membranes, and visualized by autoradiography. Membranes
were then blocked with 3% (wt/vol) bovine serum albumin (BSA) in
Tris-buffered saline for several hours and probed with
antiphosphotyrosine antibody (4G10). Immunoblot analysis of the
stripped membranes was performed with anti-p38 antibody to verify equal
amounts of immunoprecipitated protein in each sample.
DNA fragmentation assay.
Cell extracts were prepared and DNA fragments were separated by agarose
gel electrophoresis exactly as described previously.17
Surface phosphatidylserine measurements.
For quantitation of apoptosis, phosphatidylserine exposure on cells
under various conditions was quantitated using annexin-V-FITC (Pharmingen, Mississauga, Ontario, Canada) according to
the manufacturer's protocol. Data reported here are cells that stain
positive for annexin-V-FITC, but exclude PI, indicative of early
apoptosis.
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RESULTS |
cAMP protects cells from apoptosis induced under a variety of
conditions.
Treatment of cells of the murine IL-3-dependent mast cell line, MC/9,
grown in the presence of IL-3, with 50 µmol/L
N-acetylsphingosine (hereby referred to as C2-ceramide) for 6 hours resulted in DNA fragmentation typical of cells undergoing
apoptosis and similar to cells deprived of cytokine (Fig
1). We investigated the ability of elevated
cAMP levels to promote survival of these cells and, as shown in Fig 1,
cells that were simultaneously incubated with forskolin to activate
endogenous adenylate cyclase, along with ceramide or removal of
cytokine, showed much less DNA fragmentation. The cells in cAMP also
appeared normal morphologically compared with the cells undergoing
apoptosis. Although not shown here, similar effects were observed by
using a cAMP analog, CPT-cAMP, and elevation of cAMP levels could also
inhibit apoptosis observed in the presence of PI 3-kinase inhibitors
(M.P.S. and V.D., manuscript in preparation).
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| Fig 1.
Forskolin rescues myeloid cells from ceramide-induced
apoptosis. MC/9 cells were treated with IL-3 or left unstimulated
("starved"). Forskolin (20 µmol/L) and C2-ceramide (50 µmol/L) were then added to cells where indicated. Cells treated with
C2-ceramide were also incubated in the presence of IL-3. After 6 hours
at 37°C, cells were isolated, lysed, and DNA fragments resolved on
2% agarose gels and visualized by ethidium bromide staining.
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Ceramide in these cells also induced cleavage of PARP, characteristic
of caspase activation (Fig 2). Treatment
with cAMP-elevating agents inhibited this processing, with only a
modest degradation of the 118-kD full-length PARP occurring between 11 and 14 hours after the addition of C2-ceramide. This contrasts with a
rapid degradation of PARP as early as 3 hours following exposure to C2-ceramide in the absence of cAMP elevating agents. We found that a
signature 86-kD fragment of PARP was visible early during this
treatment and quickly disappeared, suggesting further processing by
proteases.
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| Fig 2.
PARP cleavage induced by ceramide is prevented by
elevation of cAMP. MC/9 cells grown in IL-3 were washed and resuspended
in RPMI-1640 supplemented with 10% fetal calf serum. C2-ceramide (50 µmol/L) and forskolin and IBMX (10 µmol/L and 50 µmol/L,
respectively) or vehicle (DMSO) were added. At the hours indicated, an
aliquot of cells were harvested and lysed in sample buffer containing 6 mol/L urea, followed by sonication and boiling. Samples were
fractionated by SDS-PAGE (10%) and transferred to nitrocellulose.
Immunoblot analysis was performed to detect PARP (118 kD) and the
corresponding 86-kD fragment. This lower fragment appeared to be
cleaved further at later time points.
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As an additional measure of apoptosis, the presence of
phosphatidylserine at the cell surface, an early event in cells
undergoing apoptosis, was detected by binding of fluorescein-conjugated
annexin-V. Figure 3 shows the annexin-V
staining of MC/9 cells treated with ceramide or ceramide along with
cAMP-elevating agents. The data demonstrate that cAMP-elevating agents
prevent the symmetrical distribution of phosphatidylserine induced by
ceramide.
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| Fig 3.
Phosphatidylserine exposure on myeloid cells induced by
ceramide is prevented by cAMP-elevating agents. MC/9 cells growing in
IL-3 were treated with vehicle (DMSO;  ), C2-ceramide (50 µmol/L;
 ), C2-ceramide plus forskolin and IBMX (10 µmol/L and 50 µmol/L,
respectively;  ), or only forskolin and IBMX ( ). At the times
indicated, cells were harvested, washed twice with PBS, and resuspended
in buffer containing annexin-V-FITC and PI. Cells stained with
annexin-V-FITC but excluding PI were quantitated by flow cytometry.
Representative experiment of 3 is shown, with each point being the mean
of duplicate determinations, and with error bars representing the
range.
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Ceramide and IL-3 activation of p38 MAPK.
C2-ceramide was added to MC/9 cells and it caused a marked elevation of
p38 MAPK activity, comparable to the level of stimulation induced by
IL-3 (Fig 4). Activation of p38 MAPK by
ceramide was rapid and transient, with activity returning to
unstimulated levels within 20 minutes. An inactive ceramide analog,
N-acetyl-dihydro-sphingosine (DH-C2-ceramide) was without
effect on p38 MAPK. C2-ceramide, as well as IL-3, also induced tyrosine
phosphorylation of p38 MAPK, which is characteristic of its activation
by dual-specificity kinases such as MKK3/MKK6,23-25 which
act upstream of MAPK family members. An effect of ceramide on p38 MAPK
activation was also observed independently in U937 human monoblastic
leukemia cells or in hepatocytes from a liver regeneration model
system.25a,25b Our recent studies have shown that several
cytokines, including IL-3, are able to activate p38 MAPK in MC/9
cells.26
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| Fig 4.
C2-ceramide stimulates p38 MAPK activity . MC/9 cells
were treated with either synthetic IL-3 (10 µg/mL) for 5 minutes,
C2-dihydroceramide (DH; 50 µmol/L for 5 minutes, or C2-ceramide (50 µmol/L) for the indicated times. Cells were then
detergent-solubilized and p38 MAPK was immunoprecipitated from the
lysates with -p38 MAPK antibody bound to protein A-Sepharose beads.
(A) Activity of the washed immunoprecipitates was determined by
32P incorporation into a peptide corresponding to amino
acids 1-96 of ATF-2. Immunoblot analysis was performed to detect
phosphotyrosine (4G10; B) and with -p38 MAPK (C) to confirm equal
amounts of p38 MAPK in the immunoprecipitates.
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CREB phosphorylation induced by ceramide, IL-3, and cAMP.
In light of a recent report describing p38 MAPK-dependent
phosphorylation of CREB,27 we examined CREB phosphorylation
at serine 133, as determined by immunoblotting with a specific antibody recognizing CREB phosphorylated at this site.28,29
Increased CREB phosphorylation was observed following treatment with
C2-ceramide (Fig 5), but not with
dihydro-C2-ceramide (results not shown). Pretreatment with a selective
p38 MAPK inhibitor, SB203580, at concentrations as low as 0.1 µmol/L,
resulted in partial inhibition of CREB phosphorylation, with almost
complete inhibition between 0.5 and 1.0 µmol/L, consistent with the
reported potency of SB203580 on inhibition of p38 MAPK
activation.22 As reported elsewhere,27 the
anti-phospho-CREB antibody also detected phosphorylation of the
CREB-related transcription factor, ATF-1, due to similarity in the
sequence surrounding the serine phosphorylation site. In all cases,
changes in phosphorylation of CREB were mirrored by changes in
phosphorylation of ATF-1.
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| Fig 5.
C2-ceramide stimulates CREB phosphorylation. MC/9 cells
treated with the indicated concentrations of SB203580 for 20 minutes
were stimulated with C2-ceramide (50 µmol/L) or vehicle alone for 5 minutes. Whole-cell lysates were separated on SDS-PAGE and immunoblot
analysis was performed to detect phospho-CREB (A) and reprobed to
detect CREB (B).
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IL-3 also induced phosphorylation of CREB and ATF-1 (Fig 4). The levels
of CREB phosphorylation in response to IL-3 or C2-ceramide were
comparable. However, pretreatment with SB203580 had little or no effect
on IL-3-induced CREB phosphorylation, demonstrating that IL-3-induced
activation of p38 MAPK was not a necessary event for CREB
phosphorylation, or that there was an alternate pathway activated by
IL-3 independent of p38 MAPK. Addition of IL-3 and C2-ceramide together
resulted in an additive effect on CREB phosphorylation (Fig
6A), which could be reduced to
IL-3-stimulated levels by SB203580 pretreatment. Phosphorylation of
CREB induced by suboptimal doses of IL-3 (data not shown), or by IL-3
treatment for various times (Fig 6B), was also not affected
significantly by SB203580 pretreatment.
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| Fig 6.
IL-3 or forskolin stimulated phosphorylation of
CREB occurs independently of p38 MAPK and MAPKAP kinase-2. (A) MC/9
cells were treated with SB203580 (1 µmol/L) or vehicle alone for 20 minutes, followed by treatments with either 10 µg/mL synthetic IL-3,
50 µmol/L C2-ceramide, or both, for 5 minutes. Whole-cell lysates
were separated on SDS-PAGE and immunoblot analysis was performed to
detect phospho-CREB. (B) MC/9 cells were treated with 10 µg/mL
synthetic IL-3 for the times indicated following pretreatment with
SB203580 or vehicle alone for 20 minutes. Immunoblot analysis was
performed to detect phospho-CREB (top) and CREB (bottom). (C) MC/9
cells were treated with 10 µg/mL IL-3 for the times indicated,
following treatment with SB203580 (1 µmol/L) or vehicle alone for 20 minutes. Lysates were incubated with 1 µg anti-MAPKAP kinase-2
antibody and protein G-Sepharose beads, and kinase activity was
determined from the washed immunoprecipitates by measuring the
32P transferred to Hsp25. (D) Cells were treated with the
indicated concentrations of forskolin for 10 minutes following a
20-minute treatment with SB203580 (1 µmol/L) or vehicle alone.
Immunoblot analysis was performed to detect phospho-CREB (top) and CREB
(bottom).
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IL-3 was also shown to activate MAPKAP kinase-2 activity (Fig 6C),
which was blocked in the presence of SB203580. This result shows that
the inability of the inhibitor to block CREB phosphorylation was not
due to activation of MAPKAP kinase-2 via a p38 MAPK-independent pathway, and confirms that the compound was able to block some IL-3-dependent responses. As expected, direct activation of PKA by
elevation of cAMP using forskolin, a direct activator of adenylate cyclase, or addition of a cAMP analog, CPT-AMP (data not shown) also
resulted in phosphorylation of CREB (Fig 6D). The cAMP-mediated activation was also independent of p38 MAPK (Fig 6D), since SB203580 had no effect on phosphorylation of CREB. It was also shown that cAMP
elevation had no effect on activation of p38 MAPK (data not shown).
We also tested whether IL-3 may be mediating phosphorylation of CREB
through cAMP elevation, thus bypassing the need for p38 MAPK activation
by using a cAMP-dependent pathway. In experiments in which cells were
treated with IL-3 or cAMP-elevating agents, we found that IL-3 did not
significantly change the level of cAMP compared with unstimulated
cells, whereas forskolin/IBMX induced a 35- to 40-fold increase of
cAMP.
Ceramide and TNF- effects on p38 MAPK and CREB phosphorylation.
We next examined a human monoblastic leukemia cell line, U937, in which
TNF- has also been shown to induce apoptosis (data not shown).
TNF- has been reported to generate ceramide in these cells as part
of the signaling pathway downstream of its receptor.3 Elevation of endogenous ceramide levels in U937 cells was achieved by
treatment of cells with bacterial SMase, which catalyses the hydrolysis
of sphingomyelin to ceramide. Sphingomyelinase and TNF- treatment
each induced rapid elevation of p38 MAPK activity and increased
tyrosine phosphorylation of the enzyme (Fig
7A). In addition, these treatments also
activated MAPKAP kinase-2 (Fig 7B). Sphingomyelinase (Fig
8) and TNF- (Fig
9A) also rapidly induced CREB and ATF-1
phosphorylation. Preincubation with SB203580 blocked the effects of
ceramide and TNF- on CREB and ATF-1, suggesting a p38 MAPK-dependent
pathway leading to CREB and ATF-1 phosphorylation in response to both
of these treatments. As expected, phosphorylation of CREB following
direct activation of PKA by cAMP, generated following treatment of
cells with forskolin, was unaffected by SB203580 treatment (Fig 9B).
Therefore, in this cell system, a cytokine agonist known to elevate
endogenous ceramide levels, as well as artificial elevation of ceramide
levels, activated the same signaling pathways leading to CREB
phosphorylation.
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| Fig 7.
Sphingomyelinase and TNF- stimulate p38 MAPK and
MAPKAP kinase-2 activity. (A) U937 cells were stimulated with 10 ng/mL
TNF- (T) for 5 minutes or 100 mU/mL bacterial sphingomyelinase for
the indicated duration. Detergent-solubilized cell lysates were
incubated with -p38 MAPK and protein A-Sepharose beads. Activity
(top) of the washed immunoprecipitates were determined by
32P incorporation into a peptide corresponding to amino
acids 1-96 of ATF-2. Immunoblot analysis was performed to detect
phosphotyrosine (4G10; middle) and with -p38 MAPK (bottom) to
confirm equal amounts of p38 MAPK in the immunoprecipitates. (B) U937
cells were preincubated with SB203580 (1 µmol/L) or vehicle alone for
20 minutes and then stimulated with either TNF- (10 ng/mL; T) or
bacterial sphingomyelinase (100 mU/mL) for the indicated duration and
detergent-solubilized. Lysates were incubated with 1 µg -MAPKAP
kinase-2 antibody and protein G-Sepharose beads, and kinase activity
was determined from the washed immunoprecipitates by measuring the
32P transferred to Hsp25.
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| Fig 8.
Sphingomyelinase stimulates p38 MAPK-dependent CREB
phosphorylation. U937 cells were treated with bacterial
sphingomyelinase (100 mU/mL) for the indicated times, lysed in sample
buffer, and immunoblot analysis performed to detect phospho-CREB (top)
and reprobed to detect CREB (middle). In a separate experiment, U937
cells were preincubated with SB203580 (1 µmol/L) for 20 minutes,
which significantly blocked the ability of several concentrations of
bacterial sphingomyelinase (50 to 200 mU/mL for 5 minutes) to stimulate
CREB phosphorylation (bottom).
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| Fig 9.
TNF- stimulates p38 MAPK-dependent CREB
phosphorylation. (A) U937 cells were treated with SB203580 or vehicle
for 20 minutes and then stimulated with TNF- (10 µg/mL) for the
indicated times. Whole-cell lysates were separated by SDS-PAGE and
immunoblot analysis to detect phospho-CREB (top) was performed and
reprobed to detect CREB (bottom). (B) Cells were treated with SB203580
(1 µmol/L) or vehicle alone for 20 minutes and stimulated with either
TNF- (10 µg/mL; T) for 5 minutes or forskolin (40 µmol/L) for 10 minutes.
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Effect of SB203580 on cell responses.
Ceramide-induced cell death in MC/9 was not effected by p38 MAPK
inhibition, nor was the ability of IL-3 to suppress apoptosis (Fig
10). In similar experiments with TNF-
on U937 cells, SB203580 again was unable to prevent apoptosis (data not
shown).
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| Fig 10.
SB203580 does not prevent ceramide-induced apoptosis.
MC/9 cells growing in IL-3 were treated with SB203580 (1 µmol/L; )
or vehicle () for 15 minutes, and then treated with C2-ceramide (50 µmol/L; and , respectively). At the indicated times, cells
were removed and the presence of phosphatidylserine on the cell surface
was quantitated by annexin-V-FITC staining as described in the Methods.
Cells staining positive for PI were not included in the analysis. The
percentage of annexin-V-FITC-positive/PI-negative staining cells never
surpassed 35%, although total cell death (annexin-V-FITC- and
PI-positive cells) steadily climbed and reached about 60% by 9 hours
in the C2-ceramide or C2-ceramide plus SB203580 treated cells.
Representative experiment of 3 is shown, with each point being the mean
of duplicate determinations, and with error bars representing the
range.
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| |
DISCUSSION |
The study of signal transduction events important in both induction and
inhibition of apoptosis in various cell types is currently of intense
interest. It is clear that a better understanding of these events will
help explain the regulation of cell numbers attributable to regulation
of apoptosis. In the case of myeloid cells, these types of events have
obvious importance in various blood disorders resulting in either a
lack of or an overabundance of cells, as well as in inflammatory
conditions in which the longevity of inflammatory cells may cause side
effects that result from the persistence of normally short-lived cells.
In our studies, we focused on the signal transduction pathways involved
in either induction or inhibition of apoptosis. In previous work, we
showed that PI 3-kinase is an important enzyme in the pathways
downstream of cytokines that inhibit apoptosis.17 However,
that same study showed that one cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), was able to bypass a need for PI
3-kinase, thereby inhibiting apoptosis even in the presence of PI
3-kinase inhibitors. We have speculated that GM-CSF (and other
cytokines whose antiapoptosis effect is not affected by PI 3-kinase
inhibitors) can activate a signaling pathway that is independent of PI
3-kinase. Recent studies have shown the importance of PKB/akt as a
downstream kinase activated by PI 3-kinase that is important for
inhibition of apoptosis in several systems.20,21 The PI
3-kinase-independent inhibition of apoptosis could therefore be
mediated by activation of a kinase that phosphorylates the same
target(s) as PKB/akt. The search for alternate pathways that inhibit
apoptosis led us to the finding that cAMP elevation in our model
systems could inhibit apoptosis as determined by three different
criteria: DNA fragmentation (Fig 1), PARP cleavage by caspases (Fig 2),
and phosphatidylserine exposure on the plasma membrane (Fig 3), thereby
implicating cAMP-dependent protein kinase (PKA) as one possible element
in a pathway that is blocking apoptosis.
In contrast, it is also important to delineate the signaling pathways
used downstream of potential mediators of apoptosis such as ceramide.
We have demonstrated in this study that elevation of intracellular
ceramide can lead to activation of p38 MAPK, although as discussed
later, we have shown that blocking p38 MAPK does not block the ability
of ceramide to induce apoptosis. In addition, treatment of cells with
TNF- or IL-3 under conditions where they induce or inhibit
apoptosis, respectively, also activates the same stress-activated
kinase. Furthermore, in the case of ceramide or TNF- , but not IL-3,
activity of p38 MAPK was required for subsequent phosphorylation of
potentially important nuclear targets, the CREB and ATF-1 transcription
factors. Phosphorylation of CREB and ATF-1 on Ser 133 and Ser 63, respectively, was also observed in response to elevated cAMP in both
MC/9 and U937 cells, as expected. Our results with TNF- and ceramide
are consistent with the report implicating p38 MAPK and MAPKAP kinase-2
upstream of CREB and ATF-1 phosphorylation, with MAPKAP kinase-2 having been identified as the most likely CREB kinase in response to FGF.27
Consistent with the study of Tan et al,27 we also found the
CREB and ATF-1 phosphorylation in response to TNF- or SMase treatment was sensitive to a selective inhibitor of p38 MAPK, SB203580.
The cAMP-mediated phosphorylation of CREB was insensitive to the p38
MAPK inhibitor and cAMP had no effect on p38 MAPK activity. The
inhibitor was also unable to block IL-3-mediated CREB phosphorylation, even though it did block IL-3-mediated p38 MAPK and MAPKAP kinase-2 activation, so it is clear that the ability of IL-3 to stimulate CREB
and ATF-1 phosphorylation functions independently of the p38 MAPK
pathway. We found also that IL-3 could not induce elevation of cAMP at
multiple time points after IL-3 treatment, thereby showing that CREB
phosphorylation can be mediated by at least three different signaling
pathways in myeloid cells, one via p38 MAPK and MAPKAP kinase-2, one
via cAMP-dependent protein kinase, and a third unidentified pathway in
response to IL-3 stimulation. In the case of EGF treatment of PC12
cells, it was recently shown that the CREB kinase was
rsk-2,30 which is activated downstream of erk's. Because
IL-3 activates both erk and p38 MAPK pathways, it is likely that IL-3
may be working preferentially via the erk pathway to stimulate CREB
phosphorylation. However, the question of why blocking p38 MAPK has
little or no effect on IL-3-mediated CREB and ATF-1 phosphorylation is
intriguing.
In contrast to our results, a recent study showed that in endothelial
cells, TNF- activated p38 MAPK, while treatment of the cells with
SMase to generate endogenous ceramide was not able to mimic this
effect, thereby suggesting that only some actions of TNF- are
ceramide-dependent.31 Our results in hematopoietic cells
contrast with those in the latter study, since the activation of p38
MAPK by TNF- correlates with the ability of ceramide to activate
this enzyme in U937 cells. There may be multiple pathways of varying
importance leading to activation of the p38 MAPK and SAPK pathways,
which could vary among different cell types in response to different
agonists.
One question that arises from these studies is whether activation of
p38 MAPK in a ceramide-dependent fashion is involved in pathways
leading to apoptosis. Treatment of the cells used in this study with
C2-ceramide, SMase, or TNF- can lead to apoptosis. Invariably,
addition of concentrations of SB203580 that blocked p38 MAPK activity,
did not block apoptosis (Fig 10). In addition, the observation that a
growth-promoting cytokine, IL-3, also induces p38 MAPK activation (this
study)26 also supports the suggestion that p38 MAPK
activation is not involved in pathways leading to apoptosis.
Furthermore, addition of SB203580 had no effect on the ability of IL-3
to stimulate growth and survival of MC/9 cells. Together, these results
suggest that p38 MAPK activation does not play an active part in the
execution of apoptosis, although one must also consider that other,
parallel pathways may be operating under the conditions tested. It is
possible that p38 MAPK activation could play a role in apoptosis in the
case of ceramide and TNF-, but when inhibited by SB203580, apoptosis
could still occur as a result of the activation of other pathways.
Likewise, prosurvival pathways activated downstream of growth-promoting
agonists may overcome the proapoptotic function of p38 MAPK. For
example, PI 3-kinase activation has been demonstrated to provide
antiapoptotic signaling, and activation of PI 3-kinase by IL-3 may
nullify the proapoptotic activity of p38 MAPK. This would be consistent
with the finding of Berra et al, who showed that in the absence of serum-stimulated PI3K activity, p38 MAPK inhibition led to increased apoptosis.32 Activation of p38 MAPK may
normally be a redundant event with respect to both apoptosis (via
ceramide) and survival (via IL-3), but it could play a role under very
specialized conditions.
A role for another stress-activated kinase, SAPK, in mediating
apoptosis has been demonstrated recently in U937 cells and bovine
aortic endothelial cells.12 However, in a different model system examining apoptosis of murine thymocytes, the upstream activator
of SAPK, SEK1, was found to protect from apoptosis.33 It is
interesting that these studies suggest that the SAPK pathway can have
opposite effects in myeloid versus lymphoid cells. We have also
observed an opposite effect of cAMP in myeloid compared with lymphoid
cells, since we find that in the former cells, cAMP can inhibit
apoptosis, while others have shown that in lymphoid cells it can
promote apoptosis.13,14
The role of SAPK activation in response to TNF receptor 1 (p55)
activation was also dissociated from effects on apoptosis in a recent
study.34 These studies were performed by a cotransfection assay of various forms of the receptor and the appropriate signaling molecules in MCF-7 cells. The ability of TNF to induce apoptosis was
found to require the signal transducer FADD binding to
the receptor death domain, independently of SAPK activation. Another recent study identified a novel protein, FAN, that
couples the TNF receptor to neutral sphingomyelinase, using a discrete
domain of the receptor that is distinct from the death
domain.35 However, activation of acid SMase requires a
domain of the TNF receptor that at least partially overlaps with the
death domain of the receptor.36 Other recent
studies have also shown a clear requirement for acid SMase in induction
of apoptosis, because loss of the gene for acid SMase results in
defective radiation-induced apoptosis and a normal response is
reconstituted by transfection of acid SMase cDNA.37 At
present, the potential linkage between ceramide generation in response
to TNF and induction of apoptosis remains unresolved. However, we can
postulate that activation of the p38 MAPK by TNF may be dependent on
ceramide production, either via acid or neutral SMase activation.
In summary, we have demonstrated that ceramide, as well as TNF- and
cytokines such as IL-3, can all activate p38 MAPK and the downstream
kinase, MAPKAP kinase-2. Each of these treatments led to
phosphorylation of CREB and ATF-1 at activating sites, but
IL-3-mediated CREB phosphorylation, unlike the other agents, was not
affected by a p38 MAPK inhibitor. Not unexpectedly, cAMP also
stimulated CREB and ATF-1 phosphorylation, and this was not affected by
the p38 MAPK inhibitor. Since cAMP and IL-3 are shown to mediate cell
survival, while TNF- and ceramide cause apoptosis in the myeloid
cells we have been studying, we can conclude that the phosphorylation
of CREB and ATF-1 alone are not able to explain the biologic effects of
cAMP and ceramide. However, we cannot rule out a role for CREB or ATF-1
transcriptional activity, since it is now clear that phosphorylation of
these factors is not sufficient for their activity, and association
with CREB binding protein (CBP) family members will determine the
ultimate response. In addition, phosphorylation of the transcription
factors at other sites could contribute to regulation of their
activity. Future experiments will have to address the effect of the
second messengers in controlling CRE-driven gene expression and in
inducing phosphorylation of CREB and ATF-1 at other sites, to determine
whether opposite effects can be observed at that level of regulation. A
role for the stress-activated kinase, p38 MAPK, in inducing apoptosis
might be proposed based on the fact that this enzyme is activated in response to ceramide and other stresses known to induce apoptosis, including TNF- treatment. However, the fact that the selective inhibitor of p38 MAPK has neither a positive or negative effect on
apoptosis, coupled with the fact that numerous growth-promoting cytokines can also activate the enzyme, argues against a specific role
for p38 MAPK as an inducer of apoptosis.
| |
FOOTNOTES |
Submitted April 28, 1998;
accepted August 24, 1998.
Supported by grants from the Medical Research Council (MRC) of Canada
and the British Columbia (BC) Health Research Foundation. M.P.S. was
supported by a Cancer Research Society studentship and I.N.F. was
supported by a Medical Research Council of Canada studentship. V.D. was
the recipient of a MRC Canada/BC Lung Association Scholarship.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Vincent Duronio, PhD,
Department of Medicine, University of British Columbia, The Jack Bell
Research Centre, 2660 Oak St, Vancouver, BC, V6H 3Z6 Canada.
| |
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Top
Abstract
Introduction