1 Department of Pharmacology, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania 17033; and 2 Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6
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ABSTRACT |
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We have previously shown that interleukin-1
receptor-generated ceramide induces growth arrest in smooth muscle
pericytes by inhibiting an upstream kinase in the extracellular
signal-regulated kinase (ERK) cascade. Here, we now report the
mechanism by which ceramide inhibits ERK activity. Ceramide renders the
human embryonic kidney 293 cells (HEK 293) resistant to the mitogenic
actions of growth factors and activators of protein kinase C (PKC). A role for PKC to mediate ceramide inhibition of growth factor-induced ERK activity and mitogenesis is suggested, as exogenous ceramide directly inhibits both immunoprecipitated and recombinant PKC- activities. To confirm that PKC-
is necessary for ceramide-inhibited ERK activity, HEK 293 cells were transfected with a dominant-negative mutant of PKC-
(
PKC-
). These transfected cells respond to
insulin-like growth factor I (IGF-I) with a significantly decreased ERK
activity that is not further reduced by ceramide treatment.
Coimmunoprecipitation studies reveal that the treatment with IGF-I
induces the association of ERK with PKC-
but not with PKC-
.
Ceramide treatment significantly inhibits the IGF-I-induced PKC-
interaction with bioactive phosphorylated ERK. Ceramide also inhibits
IGF-I-induced PKC-
association with Raf-1, an upstream kinase of
ERK. Together, these studies demonstrate that ceramide exerts
anti-mitogenic actions by limiting the ability of PKC-
to form a
signaling complex with Raf-1 and ERK.
protein kinase C; extracelular signal-regulated kinase; mitogen-activated protein kinase; ceramide; Raf-1
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INTRODUCTION |
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INFLAMMATORY
CYTOKINES, including interleukin-1 (IL-1), tumor necrosis
factor-, and interferon-
, activate sphingomyelinases, resulting
in increased cellular ceramide concentration (6, 15, 19).
Ceramide is a sphingolipid-derived second messenger molecule implicated
as an inducer of cellular differentiation, growth inhibition, and
apoptosis (12, 13). We and others demonstrated that the inhibitory action of ceramide in cell growth involves inhibition of extracellular signal-regulated kinase (ERK) activity, a
member of the mitogen-activated protein kinase (MAPK) family (7, 22). To date, however, the precise role of ceramide in inhibition of ERK activation and cell growth has not been determined. It is likely that the active site of ceramide is upstream of ERK, since
ceramide does not directly regulate immunoprecipitated ERK activity in
cell-free systems.
Activation of the ERK signal pathway is characterized by a cascade of protein kinases that are recruited to the plasma membrane. Specifically, GTP-dependent activation of Ras recruits Raf-1 to the plasma membrane, where it is phosphorylated and activated (34). Activated Raf-1 directly phosphorylates and activates mitogen/extracellular signal-regulated kinase (MEK), which in turn directly activates ERK. In addition to Ras, protein kinase C (PKC) has also been shown to activate the Raf-1-MEK-ERK signaling pathway (4).
In response to growth factors, such as insulin-like growth factor-I
(IGF-I) and platelet-derived growth factor, PKC is activated through
phospholipase C-generated diacylglycerol (DAG). At least 12 distinct
isotypes of the PKC family have now been identified and subdivided into
the following three classes: conventional (DAG dependent and calcium
sensitive), novel (DAG dependent and calcium insensitive), and atypical
(DAG independent and calcium insensitive). Among these, DAG-regulated
PKC-, a member of the novel class of PKC isotypes, activates Raf-1
kinase (28, 31). Overexpression of active PKC-
overcame
the inhibitory effects of dominant-negative Ras, suggesting that
PKC-
-induced activation of the Raf-1-MEK-ERK signaling cascade is
independent of Ras activation (4, 32). We have previously
reported that the activity of PKC-
is inhibited significantly by
IL-1 treatment in rat mesangial cells (17). Furthermore,
we have demonstrated that the cell-permeable ceramide analog,
C6-ceramide, mimicked the effect of IL-1 to inhibit both
tyrosine kinase receptor- and G protein receptor-linked
mitogenesis (7, 17). Because ceramide is structurally
similar to DAG, the endogenous cofactor for PKC-
activation, it is
possible that the inhibitory action of ceramide upon growth
factor-induced ERK activation and subsequent cell growth inhibition may
be due to the antagonistic action of ceramide displacing DAG on
PKC-
.
In this study, we demonstrate that IGF-I treatment induces PKC-
activation in HEK 293 cells. Upon activation, PKC-
selectively interacts with Raf-1 and ERK. We also demonstrate that ceramide inhibits IGF-I-stimulated ERK activation and cell growth by direct inhibition of PKC-
activation and subsequent interaction with Raf-1
and ERK. Together, these findings suggest a novel role of ceramide in
modulation of the physical interactions between signaling elements in
PKC/MAPK complexes.
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MATERIALS AND METHODS |
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Human embryonic kidney 293 (HEK 293) cells were obtained from
American Type Culture Collection (Rockville, MD). Anti-PKC-, -PKC-
, -PKC-
, -Raf-1, and -ERK antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Purified, recombinant PKC-
was obtained from Panvera (Madison, WI).
1-Oleoyl-2-acetyl-sn-glycerol (OAG) was purchased from Sigma
(St. Louis, MO). DAG (1,2-diolein) was purchased from Serdary Research
Laboratories (Ontario, Canada). Cell-permeable C6-ceramide,
physiological C18:1-ceramide, and the inactive analog
dihydro-C6-ceramide were obtained from Avanti Polar Lipids
(Alabaster, AL). Human IGF-I and IL-1
recombinant proteins were
purchased from GIBCO (Grand Island, NY). The enhanced chemiluminescence
(ECL) detection kit and Gamma Bind Sepharose were obtained from
Amersham Life Sciences (Arlington Heights, IL).
HEK 293 cell culture.
HEK 293 cells are adenovirus-transformed HEK cells of tubule epithelial
origin. These cells express functional IGF receptors and are an
excellent model for growth factor-induced mitogenesis and inflammatory
cytokine-induced growth arrest (4). Utilizing RT-PCR
protocols, we have shown that these cells express the mRNA for the type
1 form of the IL-1 receptor (data not shown). Western blot analyses
revealed that HEK 293 cells express PKC-, -
, and -
.
HEK 293 cell proliferation assay. Initially, HEK 293 cells were grown to ~50% confluency in DMEM cell culture medium containing 10% FBS in 12-well cell culture plates. The HEK cells were then downregulated by a 48-h incubation in DMEM without FBS. The cells were pretreated with either 1 µM C6-ceramide, 1 µM dihydro-C6-ceramide, or vehicle for 1 h and then were treated with mitogens (IGF-I or OAG) for an additional 18 h. These treated HEK 293 cells were further incubated with 0.3 µCi/ml [3H]thymidine during the last 6 h of treatment. The cells were washed one time with ice-cold PBS and then were washed three times for 5-10 min with 10% TCA. The fixed cells were then solubilized in 0.3 M NaOH-0.1% SDS solution, and [3H]thymidine incorporation into acid-insoluble DNA was quantified by measuring radioactivity using a liquid scintillation counter.
Western blot analysis.
Western blot analysis using anti-PKC- antibody was performed as
previously described (17). Briefly, HEK 293 cells were washed in ice-cold Dulbecco's PBS solution and lysed in 1 ml of ice-cold lysis buffer [20 mM HEPES, 40 mM NaCl, 50 mM NaF, 1 mM EDTA,
1 mM EGTA, 1 mM NaVO4, 0.2% Nonidet P-40 (NP-40), and 1 µg/ml of leupeptin, pepstatin, and aprotinin]. Cell lysates were cleared by centrifugation, and the Bio-Rad protein assay was performed to determine protein concentration. Forty micrograms of protein lysate
per sample were separated on a 10% SDS-PAGE and transferred to Hybond
nitrocellulose membranes. The membranes were blocked in 5% nonfat milk
in Tris-buffered saline (TBS) for 1 h and then were incubated with
the primary anti-PKC-
antibody (1:1,000 dilution in 5% nonfat milk
TBS) for 2 h at room temperature. After incubation, the membranes
were washed three times with TBS for 10 min each. The blots were then
incubated with secondary horseradish peroxidase (HRP)-conjugated goat
anti-rabbit antibody (1:5,000 dilution in 5% nonfat milk in TBS) for
2 h at room temperature. The membranes were then washed three
times with TBS, and PKC-
bands were visualized by ECL and quantified
using laser densitometry.
In vitro reconstitution activity assay for immunoprecipitated or
recombinant PKC-.
Immunoprecipitation of PKC-
and the subsequent reconstitution
activity assay were adapted from previous methods (2, 17, 23). A similar protocol using recombinant PKC-
(50 ng) was used to verify the in vitro effects of physiological ceramide on
immunoprecipitated PKC-
. Briefly, PKC-
was immunoprecipitated from HEK 293 lysates using 0.5 µg of polyclonal rabbit anti-PKC-
antibody. After overnight incubation at 4°C, Gamma Bind Sepharose was
added and rotated for 2 h, and the immunocomplex containing PKC-
was pelleted by brief centrifugation. After three washes, the
pellets were resuspended in kinase buffer (50 mM HEPES, 100 mM NaCl, 10 mM MgCl2, 50 mM NaF, 1 mM NaVO4, 1 mM
dithiothreitol, and 0.1% Tween 20). The in vitro kinase reaction was
initiated by addition of 40 µg/ml phosphatidylserine/reaction, 10 mM
MgCl2, 0.25 mM ATP (cold) and 1 µCi
[
-32P]ATP (10 mCi/mmol), and 10 µg histone IIIS as a
substrate. In selected experiments, 10 µg of myelin basic protein
were used as the exogenous substrate rather than histone IIIS.
Specified samples were treated with DAG (1,2-diolein, 1 µM) and/or
C18:1-ceramide, C6-ceramide, or
dihydro-C6-ceramide (0.1-1 µM). After 20 min of incubation at 37°C, the kinase reactions were terminated by adding SDS-PAGE sample buffer and heating at 95°C for 5 min. Phosphorylated histone IIIS proteins or myelin basic protein was then separated on
12% SDS-PAGE and transferred to Hybond nitrocellulose membranes. The
bands corresponding to phosphorylated histone IIIS were detected by
autoradiography (Kodak X-OMAT). In contrast, the bands corresponding to
phosphorylated myelin basic protein were excised and quantified by
liquid scintillation analysis.
Coimmunoprecipitation of PKC with ERK or Raf-1.
Lysates from HEK 293 cells were incubated with rabbit anti-ERK2 (or
anti-Raf-1) antibody for 16 h at 4°C. The next day, the goat
anti-rabbit antibody conjugated to agarose was added to each sample and
incubated for 2 h at 4°C. Immunocomplexes were then pelleted by
brief centrifugation and washed two times in lysis buffer (20 mM HEPES,
40 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM NaVO4,
0.2% NP-40, and 1 µg/ml of leupeptin, pepstatin, and aprotinin).
Immunoprecipitates were then heated at 95°C for 5 min in SDS-gel
loading buffer and separated on 12% SDS-PAGE. Proteins were
transferred to Hybond nitrocellulose membranes and probed with either
anti-PKC- or PKC-
antibody (1:1,000 dilution in 5% nonfat milk
in TBS). Subsequently, the membranes were incubated with the
HRP-conjugated anti-rabbit IgG antibody (1:5,000), and the bands
corresponding to PKC-
or PKC-
were visualized by ECL. Equal
loading of ERK2 was determined by reprobing the membranes with
anti-ERK2 antibody.
Transfection of HEK 293 cells with either wild-type or
dominant-negative PKC- (
PKC-
) mutant constructs.
HEK 293 cells were transiently transfected with either wild-type or
PKC-
constructs (a generous gift from Dr. I. Bernard Weinstein)
using Superfect (Qiagen). The wild-type construct is a full-length
PKC-
in a pHACE vector, and the dominant-negative mutant construct
is the same full-length PKC-
with a point mutation in the catalytic
domain at the ATP-binding site. Transfection efficiency was
consistently 40-50%, as determined by green fluorescent protein
cotransfection assay. Western blot analysis was performed using lysates
from either wild-type or
PKC-
construct-transfected HEK 293 cells
to determine the expression level of pERK. pERK bands were visualized
with ECL. As a control, HEK cells were transfected with empty vector.
To verify that the transfections with constructs for PKC-
did not
alter protein levels of other PKC isoforms, Western analyses were
performed to assess PKC-
, -
, and -
expression.
Statistical analysis. Independent t-tests were used to determine the significant differences between groups. The P value of the individual components was adjusted for multiple comparisons by the Bonferroni method. The data were expressed as means ± SE. All nonparametric data were analyzed by the Kruskal-Wallis test. In those experiments where the control optical density values were set to 100%, the SE for each of these control values was reported using the nontransformed data.
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RESULTS |
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Ceramide inhibits IGF-I- and OAG-stimulated HEK 293 cell growth.
To initially assess the effect of ceramide on cell growth, HEK 293 cells were treated with either IGF-I (50 ng/ml) or OAG (106 M), a cell-permeable mimetic of DAG, and
[3H]thymidine uptake into acid-insoluble DNA was
measured. As shown in Fig. 1, both IGF-I
and OAG significantly increased HEK 293 cell growth by ~150%
compared with control cells. When HEK 293 cells were pretreated with
C6-ceramide (1 µM), we observed a significant decrease in
cell growth in response to IGF-I or OAG. Specifically, C6-ceramide inhibited IGF-I- and OAG-induced cell growth to
near basal levels. In contrast, the inactive ceramide analog
dihydro-C6-ceramide (1 µM) did not reduce IGF-stimulated
[3H]thymidine incorporation. These results were
consistent with our previous studies, which demonstrated that ceramide
inhibited rat glomerular mesangial and A7r5 vascular smooth muscle cell growth induced by mitogenic stimuli (7). This inhibitory
effect of C6-ceramide on HEK 293 cell growth does not
appear to be caused by cell death, as C6-ceramide, at
concentrations up to 100 µM, did not induce apoptotic or necrotic
cell death, as assessed by lactate dehydrogenase release (unpublished
data). Together, these results demonstrate that bioactive ceramide
potently inhibits HEK 293 cell growth induced by OAG and IGF-I,
activators of the PKC-dependent signaling pathway.
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Ceramide inhibits DAG-dependent PKC- bioactivity.
The fact that ceramide potently inhibited IGF-I- and OAG-stimulated HEK
293 cell growth strongly suggests a possible inhibitory role of
ceramide in PKC-
activation, since IGF-I-induced mitogenesis is
predominantly transduced through PKC-
in a DAG-dependent manner (30). Therefore, we examined the ability of ceramide to
directly and acutely affect the bioactivity of immunoprecipitated
PKC-
by performing in vitro reconstitution activity assays. The
immunocomplexes were treated with physiological DAG
(1,2-diolein) and/or physiological ceramide (C18:1), and
bioactivity was assessed by resolving radiolabeled phosphorylation of
histone IIIS. As shown in Fig. 2, the
bioactivity of PKC-
in DAG-treated immunoprecipitates was
significantly increased (3-fold) compared with the control
immunocomplexes without DAG treatment. This result was consistent with
previous findings demonstrating that DAG is required for PKC-
activation (17). When DAG-treated immunocomplexes were
challenged with the addition of C18:1-ceramide, the
bioactivity of PKC-
was decreased significantly. To confirm these
findings, additional experiments quantified phosphorylation of an
alternative PKC substrate, myelin basic protein, by liquid scintillation analysis (Table 1). Again,
with the use of immunoprecipitated PKC-
, physiological ceramide
significantly inhibited DAG-stimulated phosphorylation of exogenous
substrate. In contrast, the inactive ceramide analog
dihydro-C6-ceramide did not inhibit DAG-stimulated PKC-
activity. Together, these results further suggest an apparent reciprocal relationship between bioactive ceramide and DAG for PKC-
bioactivity.
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Ceramide does not change PKC- expression.
To determine whether the inhibitory actions of ceramide on PKC-
activity are also a consequence of altered protein expression, we
examined the PKC-
protein expression level by performing Western blot analysis using anti-PKC-
antibody. As shown in Fig.
4, when HEK 293 cells were treated with
C6-ceramide, the protein expression level of PKC-
was
not altered compared with the control cells without ceramide treatment.
These results demonstrated that ceramide treatment of HEK 293 cells
does not alter PKC-
protein expression levels. Furthermore, these
results suggest that the inhibitory actions of ceramide may involve a
direct inactivation of PKC-
and not downregulation of protein
expression.
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PKC- is a necessary component for ceramide inhibition of ERK
activity.
To further confirm that ceramide may exert its cell growth inhibitory
actions through inactivation of PKC-
, we examined the effects of
C6-ceramide in HEK 293 cells overexpressing
dominant-negative PKC-
(
PKC-
). Because the activation of ERK
is required for IGF-I-induced mitogenesis, we initially investigated
the involvement of PKC-
in the ERK cascade through the use of
wild-type and
PKC-
mutants. In data not shown, transfection with
wild-type and dominant-negative constructs resulted in equal expression
of PKC-
. However, both of these constructs had a higher level of
PKC-
expression compared with empty vector controls. In contrast to
PKC-
, the cellular levels of other PKC isoforms, including
and
, did not change as a result of transfection with any of the cDNA constructs.
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Ceramide inhibits PKC--ERK interaction.
Because PKC-
activation has been shown to be an upstream kinase of
ERK activation (4, 32), we next performed
coimmunoprecipitation assays to determine whether ceramide can inhibit
the ability of PKC-
to interact with ERK, resulting in a decreased
ERK activity. Because ceramide has also been shown to activate PKC-
(21), we also investigated whether ceramide regulates
PKC-
-ERK interaction. As shown in Fig.
6, HEK 293 cells treated with IGF-I
specifically increased PKC-
, but not PKC-
, association with ERK2.
C6-ceramide treatment abrogated this IGF-I-induced
interaction between ERK and PKC-
. C6-ceramide had no
significant effect by itself on any of these interactions. These
results demonstrate that ceramide specifically prevents the
IGF-I-induced interaction between PKC-
and ERK2.
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Ceramide inhibits PKC- association with pERK.
It is possible that only activated PKC-
may recruit and activate ERK
through phosphorylation. Therefore, the inactivation of PKC-
may
result in blocked ERK recruitment and subsequent activation. To
determine whether ceramide specifically blocks PKC-
interaction with
pERK, we performed coimmunoprecipitation assays between PKC-
and
pERK. As shown in Fig. 7, IGF-I treatment significantly increased (6-fold) the association of PKC-
with pERK
in HEK 293 cells. C6-ceramide, but not
dihydro-C6-ceramide, pretreatment led to a significant
reduction in the IGF-I-stimulated PKC-
association with pERK.
Changes in pERK association with activated PKC-
most likely reflect
the specific interactions between PKC-
and pERK, as equal levels of
PKC-
were observed in the immunoprecipitates from all treatments.
These results clearly demonstrate that bioactive ceramide specifically
inhibits PKC-
interaction with pERK.
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Ceramide inhibits PKC- interaction with Raf-1.
We demonstrated that ceramide blocked ERK activation via selective
inhibition of PKC-
activity. However, it is not clear whether the
selective inhibitory action of ceramide on ERK activity is dependent on
Raf-1, an upstream kinase of ERK. Both Raf-1-dependent and
-independent activation of ERK by PKCs have been demonstrated by
others. Therefore, we next examined if Raf-1 kinase is
coimmunoprecipitated with PKC-
in HEK 293 cells treated with IGF-I.
As shown in Fig. 8, we observed a strong
association of PKC-
with Raf-1 in response to IGF-I treatment. This
result was consistent with other reports (5) that
demonstrated PKC-
association with Raf-1 and that the association
was increased with growth factor treatment. In contrast, HEK 293 cells
treated with either C6-ceramide or IL-1
, a
receptor-mediated inducer of ceramide formation (6), did not induce association of PKC-
with Raf-1. In fact, IGF-I-stimulated Raf-1 association with PKC-
was inhibited significantly by
pretreatment with C6-ceramide or IL-1, demonstrating
that ceramide potently inhibits IGF-I-stimulated PKC-
interaction
with Raf-1. Together these data support our conclusion that direct
inhibition of PKC-
by ceramide inhibits the interaction between
PKC-
and upstream elements of the ERK cascade.
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DISCUSSION |
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The field of signal transduction is now embracing the concept of signaling complexes in which the assembly and interactions of multiple kinases in large-scale aggregates determines the specificity and selectivity of cellular responses. The role of scaffolding and/or adapter proteins such as MEK partner-1, Jun-interacting protein, kinase suppressor of Ras (KSR), and 14-3-3 proteins to assemble these signaling aggregates is only recently being appreciated (9, 27, 29, 33). Adding to this orchestrated complexity, we now elucidate a novel mechanism by which the sphingolipid metabolite ceramide can negatively modulate cellular responses by antagonizing the protein-protein interaction of kinases involved in mitogenic signaling pathways.
The mechanism by which growth factors and their receptors regulate the
assembly of kinase signaling complexes between PKCs and the elements of
the ERK cascade is still unclear. However, the fact that Raf-1 is
activated by PKC- suggests that PKC-
may directly phosphorylate
Raf-1. Supporting this, Ueffing et al. (31) demonstrated
that PKC-
and Raf-1 coimmunoprecipitate from PKC-
-transformed
NIH/3T3 cells, indicating that PKC-
may activate Raf-1 through
direct protein-protein interactions. Our study clearly demonstrates the
novel role for lipid-derived second messengers to modulate these
protein-protein interactions, as ceramide inactivation of PKC-
limits interactions of PKC-
with Raf-1 and ERK.
Our previous studies showed that ether-linked diglyceride species
competitively bound to the DAG-binding site on PKC- and -
without
activating the kinase (17). Because ceramide structurally resembles DAG, it is possible that ceramide competes against DAG for
the putative DAG-binding site. Ceramide could also bind to a secondary
ceramide-binding site, rendering the PKC-
insensitive to activation
by DAG. Alternatively, when ceramide is bound to PKC-
, it may hinder
PKC-
from interacting with other proteins, such as Raf-1 and ERK.
Whether ceramide directly competes with DAG for the putative
C-1-lipid-binding motif within PKC-
is not clear at the present
time. In fact, a radioiodinated photoaffinity-labeled ceramide
analog was unable to directly interact with
immunoprecipitated nonactivated PKC-
(11). Regardless
of the mechanism, our finding of ceramide-induced inactivation of
immunoprecipitated and recombinant PKC-
is supported by previous
studies that demonstrated that ceramide treatment induced the
translocation of PKC-
and -
from the plasma membrane to the
cytosol (14, 26), an event consistent with inactivation.
Ceramide has also been shown to inhibit PKC-
activity
(16), perhaps in a similar mechanism to PKC-
inactivation by ceramide.
Our data indicate that one mechanism by which ceramide decreases ERK
activity is via direct inhibition of PKC- and the subsequent inability to form a signaling complex with Raf-1 and ERK. Other studies have postulated alternative mechanisms by which ceramide regulates the Raf-1/ERK cascade. Ceramide has been shown to bind to
c-Raf (24) and KSR (34). Ceramide binding to
Raf-1 leads to sequestration of Raf-1 into inactive Ras-Raf-1 complexes
(22). Moreover, KSR has been shown to bind to and
functionally inactivate MEK1 (8, 35). All of these studies
are consistent with decreased ERK activity. Finally, a recent study by
Basu et al. (1) has shown that downstream targets, such as
BAD, convert the normally promitogenic ERK cascade into a
ceramide-dependent proapoptotic signal pathway. Thus ceramide may
regulate several mechanisms to inhibit ERK-mediated proliferation.
The finding that ceramide-induced cell growth inhibition is a
consequence of inactivated PKC- clearly suggests the critical role
of PKC-
in mitogenesis. Downregulation of PKC-
has been shown to
inhibit the G1/S transition in vascular smooth muscle cells, an event consistent with IL-1-induced growth arrest (18, 25). Furthermore, overexpression of PKC-
induced
tumorigenicity in fibroblasts (3, 20) and enhanced nerve
growth factor-induced phosphorylation of ERK in PC-12 pheochromocytoma
cells (10). Taken together, the role of ceramide to
selectively limit interactions between PKC-
and Raf-1/ERK may
illustrate one mechanism by which a proinflammatory response can be
maintained in the absence of cell growth. This novel role of ceramide
to regulate protein-protein interactions, including the
PKC-
-Raf-1-ERK interactions, is an attractive hypothesis by which
inflammatory cytokine-induced ceramide formation may inhibit cellular
proliferation in models of nonproliferative inflammatory renal diseases.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-53715.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. Kester, The Pennsylvania State Univ., The Milton S. Hershey Medical Center, Dept. of Pharmacology, PO Box 850, Hershey, PA 17033 (E-mail: mxk38{at}psu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 August 2000; accepted in final form 22 December 2000.
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