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INTRODUCTION |
In addition to their role in membrane structure, sphingolipids
play a critical role in cellular signal transduction pathways, particularly in response to stress stimuli (1-6). Diverse stress stimuli, including chemotherapeutic drug treatment, irradiation, and
growth factor withdrawal, promote the generation of ceramide, which
more often than not results in cell death or cell growth arrest (1, 2,
6). Clearly, ceramide regulates stress-signaling pathways by multiple
actions (e.g. activation of stress kinases, inhibition of
protein kinase C, activation of protein phosphatases) (1-6). However,
a role for ceramide has yet to be determined in the stress-mediated
arrest of protein synthesis that has recently been found to precede
apoptosis (7).
The most well characterized mechanism for regulating protein synthesis
involves the reversible phosphorylation of the
-subunit of
eukaryotic initiation factor-2
(eIF2
)1 (8-10). The
phosphorylation of eIF2
at serine 51 prevents protein translation
initiation events (11-13). A physiologic eIF2
kinase and key
regulator of this process is the double-stranded RNA-activated protein
kinase, PKR (14-16). However, until recently, much of what is known
about PKR has been derived from its role in inhibiting host cell
protein synthesis in response to viral infection, thereby activating
the host antiviral defense mechanism (17, 18). Recent data, however,
suggest that PKR may play a more expanded role in regulating protein
synthesis in response to cellular stresses such as IL-3 growth factor
withdrawal (7), serum deprivation (19), tumor necrosis factor-
(20,
21), or lipopolysaccharide treatment (21). Recently, RAX
(PKR activator X) (22) and its human homolog, PACT (23), were independently discovered as the first
known cellular activators of PKR (22, 23). Consistent with this role,
RAX/PACT has been shown to activate PKR in response to stress
applications such as IL-3 withdrawal, sodium arsenite treatment, and
peroxide treatment (22, 24, 25). In response to stress stimuli, RAX is
phosphorylated by an unknown stress-activated protein kinase (SAPK)
(22). Phosphorylated RAX associates with PKR, resulting in PKR
activation and inhibition of protein synthesis (22). Thus, RAX appears
to be directly involved in the regulation of PKR during diverse
cellular stress events.
Ceramide is a naturally occurring sphingolipid that has emerged as an
important second messenger molecule in apoptosis signaling (26,
27). Ceramide is produced during diverse stress stimuli, including
chemotherapeutic drug treatment (28), ischemia/reperfusion injury (29),
FAS antigen activation (30), corticosteroid treatment (31), and
irradiation (31). Indeed, the generation of ceramide is so common
during apoptosis that it has been considered a universal feature of
this process (1, 3). Ceramide has been demonstrated to activate a
number of stress-activated kinases, including the mitogen-activated
protein kinases JNK1 and JNK2 (4, 32, 34). Considering that
ceramide is produced during diverse apoptotic stress applications and
has been demonstrated to activate stress-activated kinases, it is
possible that ceramide may regulate RAX. The findings presented
indicate a novel signal pathway linking ceramide to the regulation of
protein translation, via a mechanism involving RAX. These data point to
a unique level of cellular homeostatic control involving this important
second messenger molecule.
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EXPERIMENTAL PROCEDURES |
Materials--
All reagents used were purchased from commercial
sources unless otherwise stated.
Cell Lines--
Murine leukemia NSF/N1.H7 cells and
NSF/N1.H7 cells stably transfected with HA-RAX or vector control
plasmid were maintained as previously described (22).
Analysis of Cell Viability and Apoptosis--
Cells were treated
with increasing doses of C2-ceramide (Calbiochem) or 50 µM C2-dihydroceramide (Calbiochem) for 3, 6, or 24 h. Where appropriate, cells were treated with increasing
doses of 2-aminopurine (Sigma) for 24 h. Cell viability was
measured by trypan blue dye exclusion, and apoptosis was determined by detecting DNA laddering in apoptotic cells as previously described (7,
22).
Analysis of RAX Phosphorylation--
Cells (1 × 107 cell eq) were lysed, and HA-RAX was immunoprecipitated
with an anti-HA antibody as previously described (22). Where indicated,
cells were treated with 1, 10, or 50 µM
C2-ceramide for 30 min. The immunoprecipitated protein was
resuspended in sample buffer, and protein was subjected to vertical
slab isoelectric focusing and immunoblot analysis with an anti-RAX
polyclonal antibody as described (22).
Metabolic Labeling, Immunoprecipitation, and Immunoblot
Analysis--
Cells were labeled with
[32P]orthophosphoric acid, and eIF2
was analyzed by
immunoprecipitation as previously described (22). Where indicated,
cells were treated with 25 µM C2-ceramide for 30 min. Samples were electrophoresed on a 0.1% SDS-12% acrylamide gel, transferred to nitrocellulose, and exposed to Kodak X-Omat film at
80 °C. The same blot was used for Western blotting with anti-eIF2
antiserum and developed using an ECL kit (Amersham Pharmacia Biotech) as previously described (22).
Co-immunoprecipitation Analysis--
Cells were treated with
increasing doses of C2-ceramide for 30 min and lysed, and
PKR was immunoprecipitated with an anti-PKR antibody as previously
described (22). The immunoprecipitated protein was electrophoresed on a
0.1% SDS-12% acrylamide gel and transferred to nitrocellulose, and
Western blot analysis was performed with an anti-RAX polyclonal
antibody as described (22).
Measurement of Cellular Protein Synthesis--
Cells were
treated with C2-ceramide (0, 1, 10, or 50 µM)
or C2-dihydroceramide (50 µM) for 30 min.
Where indicated, cells were treated with 1 mM 2-AP for 30 min. Measurement of protein synthesis was performed as previously
described (7). After treatment, ~1 × 106 cell eq
were incubated with L-U-14C-labeled amino acid
mixture (Amersham Pharmacia Biotech) at 2 µCi/ml for 10 min at
37 °C. The reaction was terminated by the addition of 20% (w/v)
trichloroacetic acid, and the radioactivity in the acid-precipitable
fraction was measured in a scintillation counter.
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RESULTS |
RAX Overexpression Potentiates Ceramide-induced Cell Killing via a
Mechanism Involving PKR--
Protein synthesis, which is required for
cell growth, is inhibited by activated PKR (7). Ceramide is a potent
apoptotic agent and inhibitor of cell growth (3-5), yet the effect of
ceramide on protein translation is unknown. To determine whether RAX,
the first cellular activator of PKR to be discovered, can participate in a ceramide-mediated pathway, the effect of ceramide treatment was
compared in cells stably expressing exogenous HA-RAX versus vector-only or parental control cells. Overexpression of RAX has previously been shown to accelerate the cell death of murine
IL-3-dependent myeloid cells following IL-3 withdrawal or
after treatment with apoptotic agents such as peroxide and sodium
arsenite in the presence of IL-3 (22). Since ceramide is generated
during virtually all known stress stimuli (2, 3), we tested whether
stress activation of RAX may be mediated by ceramide. If ceramide can
participate in RAX-mediated stress, it is predicted that RAX may
sensitize cells to ceramide-induced cell death in a mechanism similar
to that by which RAX sensitizes factor-dependent cells to
killing following factor deprivation (22). The results revealed that cells expressing exogenous HA-RAX were at least 2.5-fold more sensitive
to active C2-ceramide killing compared with
vector-transfected or untransfected cells (Fig.
1A). The IC50 for
the untransfected or vector-transfected cells treated with ceramide for
24 h was >50 µM, whereas the IC50 for
cells overexpressing RAX was dramatically reduced to 18 µM. Cell killing resulted from apoptosis as determined by
the classic pattern of DNA fragmentation observed (Fig. 1B) (35, 36). As a further control, cells overexpressing HA-RAX were also
treated with an inactive C2-ceramide analog,
C2-dihydroceramide (2), at 50 µM. The
inactive analog had no effect on cell viability (Fig.
2). Although a role for RAX in
ceramide-induced cell killing is suggested but not proven by these
findings, it was initially unclear whether PKR is involved. Therefore,
2-AP, a serine/threonine kinase inhibitor that has previously been
shown to inhibit PKR (37, 38), was used. A high concentration of 2-AP
(10 mM) was found to have no effect on cell viability.
However, in cells overexpressing HA-RAX, 2-AP was found to protect
cells from C2-ceramide-induced cell killing in a
dose-dependent manner (Fig. 2). Using a concentration of
C2-ceramide that approximates the IC50 at
24 h (i.e. 18 µM), cells treated with
1
mM 2-AP were protected from ceramide-induced cell killing.
These data strongly suggest a novel mechanism for ceramide-mediated
apoptosis involving both RAX and PKR.

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Fig. 1.
RAX potentiates ceramide-induced cell
killing. A, NSF/N1.H7 parental cells ( ) or cells
stably transfected with either empty vector ( ) or HA-RAX ( ) were
treated for 24 h with increasing concentrations of
C2-ceramide. Cell viability was determined by trypan blue
staining. Data shown are means ± S.D. of triplicate samples from
a representative clone (clone 13) of four phenotypically similar clones
that have been previously described (22). B, NSF/N1.H7 cells
transfected with HA-RAX were treated for 6 h with 25 µM C2-ceramide, and DNA fragmentation was
analyzed as described under "Experimental Procedures."
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Fig. 2.
The PKR inhibitor 2-AP blocks
ceramide-induced cell killing. NSF/N1.H7 cells overexpressing
HA-RAX were treated for 24 h with 18 µM
C2-ceramide or 50 µM
C2-dihydroceramide. Varying concentrations of 2-AP were
introduced to cells at the same time as C2-ceramide
addition. Cell viability was determined by trypan blue staining. Data
shown are means ± S.D. of triplicate samples.
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Ceramide Promotes RAX Phosphorylation in a
Dose-dependent Manner--
Ceramide is known to activate
serine/threonine stress-activated kinases, including JNK (3-5, 26,
27). Since RAX is serine-phosphorylated by an as yet unidentified
stress-activated kinase (22), it is possible that a mechanism by which
ceramide can induce PKR activation and inhibit protein synthesis is by
promoting RAX phosphorylation and activation. Therefore, whether
C2-ceramide could induce RAX phosphorylation was assessed
in vivo. Previously, isoelectric focusing analysis revealed
that exponentially growing NFS/N1.H7 cells contain predominantly a
single unphosphorylated species of RAX with an approximate pI of 8.6, whereas stress applications to cells, including IL-3 deprivation or
treatment of cells with sodium arsenite or thapsigargin, induces a
prominent acidification (i.e. via phosphorylation) of RAX
with a pI 8.3 (22). Thus, cells not exposed to stress stimuli contain
primarily the unphosphorylated form of RAX, whereas stress
treatment induces phosphorylation of RAX. The results demonstrated that
ceramide could also induce the phosphorylation of RAX in a
dose-dependent manner (Fig.
3). Although little, if any, of the
phosphorylated form of RAX (observed as the slower migrating band
following vertical slab isoelectric focusing electrophoresis) could be
detected by Western blot analysis of protein lysates from untreated
cells or cells treated with a low concentration of
C2-ceramide (i.e. 1 µM), cells
treated with >10 µM C2-ceramide expressed
virtually all RAX as a phosphoprotein (Fig. 3). Thus, ceramide appears
to activate a RAX kinase.

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Fig. 3.
Ceramide promotes phosphorylation of
RAX. NSF/N1.H7 cells overexpressing HA-RAX were treated with
varying concentrations of C2-ceramide for 30 min. Protein
lysates (100 µg) were separated by isoelectric focusing and Western
blot analysis performed with anti-RAX antiserum as described under
"Experimental Procedures."
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Ceramide Promotes PKR Activation--
Since RAX phosphorylation
promotes the activation of PKR by RAX (22), it is likely that ceramide
activates PKR. Co-immunoprecipitation studies have previously
demonstrated that cellular activation of PKR strongly correlates with
the association of PKR with RAX (22). To determine whether ceramide can
promote RAX/PKR association, protein extracts from cells treated with
increasing doses of C2-ceramide were immunoprecipitated
with anti-PKR antiserum as previously described (22). RAX was detected
by Western analysis using an anti-RAX polyclonal antibody that was
produced as previously described (22). The results indicated that
ceramide promoted RAX/PKR association in a dose-dependent
manner (Fig. 4). However, whether
ceramide induced PKR activation was not clear. Previously, it has been demonstrated that stress application induces RAX phosphorylation and
promotes RAX/PKR association concomitant with PKR activation and
eIF2
phosphorylation (22, 24). Since the biogenesis of ceramide can
result from many diverse stress stimuli, we tested whether ceramide may
trigger this cascade by inducing PKR activation. To determine whether
ceramide can promote the activation of PKR in a
RAX-dependent manner, the effect of C2-ceramide
treatment on eIF2
phosphorylation was assessed. Cells were
metabolically radiolabeled with [32P]orthophosphoric
acid, treated with C2-ceramide for 30 min, and lysed in
detergent buffer, and the phosphorylation status of eIF2
was
assessed as described under "Experimental Procedures." 25 µM C2-ceramide was used since this is roughly
the IC50 for these cells (Fig. 1A). In addition,
it was expected that most, if not all, of the RAX would be
phosphorylated since only >10 µM C2-ceramide was shown to induce RAX phosphorylation (Fig. 3).
C2-ceramide was found to promote the increased
phosphorylation of eIF2
(Fig. 5),
suggesting a novel mechanism whereby ceramide can promote RAX
phosphorylation and activation.

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Fig. 4.
Ceramide promotes RAX/PKR association.
NSF/N1.H7 cells overexpressing HA-RAX were treated with varying
concentrations of C2-ceramide for 30 min. Cells were lysed,
and protein was immunoprecipitated (IP) with an anti-PKR
antibody. Western blot analysis was performed using anti-RAX antiserum
to observe co-immunoprecipitated RAX protein.
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Fig. 5.
Ceramide promotes phosphorylation of
eIF2 . NSF/N1.H7 cells
overexpressing HA-RAX were treated with C2-ceramide for 30 min. Cells (2 × 107 cell eq) were metabolically
labeled with [32P]orthophosphate and then
immunoprecipitated with an anti-eIF2 antibody. Phosphorylation was
observed by autoradiography. Western blot analysis was performed to
confirm that the detected bands were eIF2 . Pre,
pre-immune sera control.
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Ceramide Inhibits Protein Synthesis in a Dose-dependent
Manner--
However, whether ceramide-activated signaling actually
couples to a RAX pathway that involves regulation of protein synthesis was not clear. To determine the effect of ceramide on protein synthesis, the rate of nascent protein synthesis in vivo was
assessed. Cells were treated with increasing concentrations of
C2-ceramide for 30 min, followed by incubation with a
14C-radiolabeled amino acid mixture for 10 min at
37 °C and precipitation of protein by the addition of
trichloroacetic acid as previously described (7). Protein synthesis
rates were determined by measuring the amount of radioactivity
incorporated in the acid-precipitable fraction. The results indicated
that ceramide inhibited protein synthesis in a
dose-dependent manner (Fig.
6). Cells that were treated with
10
µM C2-ceramide exhibited a markedly reduced
level (i.e. ~60%) of nascent protein synthesis compared
with untreated cells. Collectively, these findings strongly support a
mechanism whereby ceramide can promote RAX activation and inhibit
protein synthesis.

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Fig. 6.
Ceramide inhibits protein synthesis.
NSF/N1.H7 cells overexpressing HA-RAX were treated with varying
concentrations of C2-ceramide, and the protein synthesis
rate was measured as described under "Experimental Procedures."
Data shown are means ± S.D. of triplicate samples.
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Next, a role for PKR in ceramide-mediated inhibition of protein
synthesis was examined using 2-AP. The results revealed that the
inactive ceramide analog (C2-dihydroceramide) failed to
inhibit protein synthesis, whereas 50 µM
C2-ceramide resulted in potent inhibition of protein
synthesis (Fig. 7). Importantly,
ceramide-induced inhibition of protein synthesis was prevented when
cells were co-treated with 2-AP (Fig. 7). These results indicate a role
for PKR in this process and suggest a novel role for ceramide in the regulation of protein synthesis involving both RAX and PKR.

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Fig. 7.
The PKR inhibitor 2-AP blocks ceramide
inhibition of protein synthesis. NSF/N1.H7 cells overexpressing
HA-RAX were treated with C2-dihydroceramide or
C2-ceramide in the absence or presence of 2-AP. The protein
synthesis rate was measured as described under "Experimental
Procedures," and the data are presented as a percentage of the rate
of protein synthesis in control cells (i.e. cpm treated
cells/cpm control cells). Data shown are means ± S.D. of
triplicate samples.
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DISCUSSION |
The role of PKR in viral host defense is well known (17, 18). In
response to viral double-stranded RNA, host PKR is activated, resulting
in the phosphorylation of eIF2
with a shutdown of protein synthesis
to prevent viral replication (13-16). Yet it is only recently that
cellular activation of PKR has been established (7). The identification
of RAX/PACT as a cellular PKR activator and its participation in
programmed cell death suggest an important link between the regulatory
mechanisms that mediate cell survival and protein synthesis (22-25).
But how these pathways may be linked remains unclear. One mechanism for
linkage would be via a second signal molecule that is rapidly generated
such as ceramide (1-5). Considering that ceramide production is
associated with most, if not all, stress stimuli (1, 3), we examined
whether ceramide may provide the link between these pathways. Our
findings clearly indicate that ceramide can regulate cell survival by
multiple mechanisms involving diverse signal pathways. For example,
ceramide can affect transcriptional mechanisms (e.g.
c-Jun) (4, 34). In addition, ceramide was found to regulate
stress-signaling pathways involved in apoptosis by stimulating
dephosphorylation of Bcl2 and Bad (36, 39, 40). Now we have discovered
a novel mechanism whereby ceramide can regulate protein synthesis by a
mechanism(s) involving both PKR and RAX. PKR is likely involved since
2-AP can reverse ceramide-induced killing (Fig. 2) and ceramide-induced inhibition of protein synthesis (Fig. 7). In addition, ceramide promotes RAX/PKR association, which strongly correlates with PKR activation and inhibition of protein synthesis (Fig. 4) (22). Although
it was not formally tested whether RAX may also activate another
cellular eIF2
kinase such as HRI
(heme-regulated inhibitor of
translation) (9) or PERK (41), an association of RAX with any other
eIF2
kinase has yet to be reported. Thus, the findings presented
here strongly suggest that the upstream event triggering the inhibition
of protein synthesis involves RAX activation of PKR.
The precise mechanism by which phosphorylated RAX may activate PKR
remains to be elucidated. PKR is a ribosomal protein that is apparently
activated when it is released from the ribosome (42, 43). Recently, PKR
has been shown to interact with the L18 ribosomal protein (44).
Interestingly, L18 inhibits PKR autophosphorylation, and
phosphorylation of eIF2
is blocked in vitro and in
vivo (44). Since ribosome-associated PKR is in a monomeric
(inactive) form (45), it is possible that activation of PKR coincides
with displacement from the ribosome (43). Since RAX can associate with
and activate PKR, it is tempting to speculate that at least one
mechanism by which RAX may activate PKR is by blocking L18 binding to
PKR. A role for ceramide in this potential mechanism remains to be determined.
An elaborate homeostatic regulatory mechanism for apoptosis involving
ceramide is beginning to emerge. On the one hand, during stress that
can lead to cell death, ceramide activates protein phosphatases such as
protein phosphatase 2A that can promote the functional
dephosphorylation and inactivation of anti-apoptotic signal molecules,
including Bcl2 (36), Akt (46), and protein kinase C (33). On the other
hand, ceramide can directly activate SAPKs such as JNK (Fig. 4) (4,
32). Although it remains to be determined which SAPK(s) may be
responsible for RAX phosphorylation, a role for ceramide in inhibiting
protein synthesis may involve activation of a ceramide-activated SAPK
to phosphorylate RAX and lead to PKR activation, eIF2
phosphorylation, inhibition of protein synthesis, and apoptosis (Fig.
8). Further studies are necessary to
identify a physiologic RAX kinase to fully understand the mechanism(s) by which protein synthesis may be negatively regulated under
stress.

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Fig. 8.
Model of ceramide regulation of various
stress-signaling pathways. During growth conditions
(e.g. factor-dependent cells in the presence of
IL-3), signal pathways such as those mediated by phosphatidylinositol
3-kinase (PI3K) and protein kinase C (PKC)
promote survival by mechanisms including the phosphorylation of Bcl2
and Bad. During stress (e.g. IL-3 deprivation), SAPKs such
as JNK and protein phosphatases such as protein phosphatase 2A
(PP2A) are activated and can promote apoptotic pathways.
Ceramide can also inhibit protein synthesis by activation of an as yet
unknown RAX kinase. PKB, protein kinase B.
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