From the Signal Transduction Laboratories, the
§ Lipid and Lipoprotein Research Group, and the Department
of Biochemistry, University of Alberta,
Edmonton, Alberta T6G 2S2, Canada
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ABSTRACT |
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Treatment of confluent rat2 fibroblasts with
C2-ceramide (N-acetylsphingosine),
sphingomyelinase, or tumor necrosis factor- Ceramides are important lipid second messengers that are generated
through sphingomyelin hydrolysis by sphingomyelinases (1). Agonists
such as There are several proposed downstream targets for ceramide action
including ceramide-activated protein phosphatase (18), ceramide-activated protein kinase (19), and protein kinase C- PI 3-kinase phosphorylates the 3-position of the inositol ring to
produce a family of 3-phosphoinositides that play important roles in
cell signaling. In various cell types, PI 3-kinase is implicated in
regulating cell growth and inhibiting apoptosis (27, 28), intracellular
vesicle trafficking and secretion (29-31), and cytoskeletal
organization (32-34). PI 3-kinase exists as a heterodimer consisting
of a p110 catalytic subunit and a p85 regulatory subunit. The p85
subunit contains two SH2 domains, one SH3 domain, a Bcr homology
domain, and proline-rich sequences (for review see Refs. 35-38), which
suggests that PI 3-kinase is regulated by multiple mechanisms. Tyrosine
phosphorylated proteins such as the EGF and PDGF receptors IRS-1 and
CD28 bind the SH2 domains of the p85 subunit (35-38). This binding
increases PI 3-kinase activity and, in some situations, causes
translocation of PI 3-kinase to plasma membranes to bring it into
proximity with its lipid substrates (39). Other possible mechanisms
regulating PI 3-kinase activation include binding of the SH3 domains of
the Src family of tyrosine kinases to the proline-rich sequences on the
p85 subunit (40-42), interaction of Cdc42 or Rac with the Bcr homology
domain (43, 44), tyrosine phosphorylation of p85, and
autophosphorylation of p85 by the p110 kinase at serine 608 (45). In
addition, binding of Ras-GTP to the catalytic domain p110 increases PI
3-kinase activity (46-48).
In this work we investigated whether ceramides are able to stimulate PI
3-kinase in rat2 fibroblasts. We demonstrated that C2-ceramide but not dihydro-C2-ceramide
activates PI 3-kinase transiently after 5-20 min through a pathway
that involves tyrosine kinase activity and the activation of Ras.
Treatment of the fibroblasts with TNF Materials--
DMEM, penicillin, streptomycin, and fetal bovine
serum were purchased from Life Technologies, Inc.
C2-ceramide
(N-acetyl-D-erythro-sphingosine), dihydro-C2-ceramide, and Ly 294002 were obtained from
Biomol. Bovine serum albumin, N-oleoylethanolamine,
aprotinin, leupeptin, phosphatidylinositol, human TNF Cell Culture--
Rat2 fibroblasts (50) were normally plated at
10,000 cells/cm2 in 10-cm culture dishes. Fibroblasts were
then cultured as described previously (51) for 4-5 days until
confluence. Cells were maintained in DMEM supplemented with 10% fetal
bovine serum, 100 units/ml penicillin, and 100 µg of streptomycin/ml
in a humidified atmosphere of 5% CO2, 95% air at
37 °C. Rat2 fibroblasts stably expressing wild type Ha-Ras and the
dominant-negative mutant Ha-Ras (N17 Ras) were generated by infection
with retroviruses containing the respective cDNA sequences and
appropriate drug selection using 0.6 mg of Geneticin (G418)/ml or 2.5 µg of puromycin/ml, respectively. The N17 Ras containing vector was
obtained from Dr. S. E. Egan, and it was subjected to DNA sequence
analysis to verify the presence of the N17 mutation (52). N17 Ha-Ras is
thought to form a stable inactive complex with Ras exchange factors and
thereby prevents activation of endogenous Ras (53). The N17-expressing
fibroblasts contained about 28-fold more total Ras than did the Rat2
cells (Fig. 1A). It should be
noted that we purposely loaded five times more lysate protein for the
parental rat2 and vector control fibroblasts to visualize the
relatively low levels of expression of endogenous Ras. The increase in
the number of fibroblasts expressing N17 after the third day in culture
was about 30% lower than for vector control or parental rat2
fibroblasts (Fig. 1B). The same growth trend was observed
for DNA, measured by a fluorescent assay (54), and analyzed for protein
content (results not shown). In these experiments the initial seeding
density was about 7,000 cells/cm2, and confluence was
reached after about 6 days.
Preparation of Cell Membranes--
Confluent cells were cultured
overnight in DMEM containing 15 µM lipid-free bovine
serum albumin followed by the addition of ceramide or
dihydro-C2-ceramide in Me2SO (final
concentration, 0.1%) as indicated. The final concentration of
Me2SO was 0.08%. Cells were then washed twice with ice-cold
phosphate-buffered saline, harvested by centrifugation, and resuspended
in buffer A, which contained 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4,
1.5 mM KH2PO4, 2.5 mM
EDTA, 1 mM dithiothreitol, and 0.1 mM
phenylmethylsulfonyl fluoride. Cells were then sonicated twice for
10 s each and then centrifuged for 5 min at 800 × g. After discarding the nuclei and unbroken cells, membranes
were prepared by ultracentrifugation at 250,000 × g
for 60 min. The pellets (membranes) were washed and resuspended in
buffer A.
Immunoprecipitation and Immunoblotting--
PI 3-kinase was
immunoprecipitated from cell lysates (300 µg of protein) by adding 2 µg of anti-p85 PI 3-Kinase Assay--
PI 3-kinase activity was determined by
measuring the formation PI 3-[32P]phosphate (56).
Confluent cells were washed twice with buffer containing 50 mM HEPES, 137 mM NaCl, 1 mM
MgCl2, 1 mM CaCl2, and 100 µM Na3VO4 and then harvested in
lysis buffer (1% Nonidet P-40, 10% glycerol, 50 mM HEPES,
137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 2 mM
Na3VO4, 10 mM
Na4P2O7, 100 mM NaF, 5 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and
1 µg/ml leupeptin). PI 3-kinase was immunoprecipitated by incubating
the cell lysates with 2 µg of anti-p85 Activation of Ras--
Ras-GTP was measured by using a
nonisotopic method (57). Briefly, fibroblasts overexpressing wild type
Ha-Ras were treated with different agents and then lysed in buffer
containing 25 mM HEPES, pH 7.5, 150 mM NaCl,
1% Nonidet P-40, 0.25% deoxycholate, 10% glycerol, 25 mM
NaF, 10 mM MgCl2, and 1 mM EDTA.
Cleared lysates were incubated for 30 min at 4 °C with 20-30 µg
of glutathione S-transferase-Ras binding domain (amino acids
1-149 of cRaf1 fused to glutathione S-transferase) that was
bound to glutathione-Sepharose beads. After three washes with lysis
buffer, bound proteins were resolved by SDS-polyacrylamide gel
electrophoresis on 11% gels, and Ras was detected by immunoblotting
with a pan Ras polyclonal antibody. Control experiments confirmed that
Ras-GTP does not bind to glutathione-Sepharose beads alone.
Determination of DNA Synthesis and Cell
Proliferation--
Because rat2 cells are thymidine kinase negative,
we first generated a population of rat2 derivatives by infection with a retrovirus vector expressing herpes simplex virus type I thymidine kinase (21). Confluent rat2 fibroblasts containing nuclear thymidine kinase were cultured in DMEM containing 15 µM lipid-free
bovine serum albumin and 0.1% fetal bovine serum for 24 h. Cells
were then incubated for 24 h with TNF Measurement of Ceramidase Activity and Protein
Concentration--
Ceramidase activity was measured by incubating
fibroblasts for 30 min with 40 µM
N-[14C]acetylsphingosine in DMEM. The cells
were washed with ice-cold incubation buffer, and the combined media and
resuspended cells were extracted separately with
chloroform/methanol/water (final volumes, 1:1:0.9). The distribution of
[14C]ceramide and [14C]acetate in the
organic and aqueous phases, respectively, were determined by
scintillation counting. Protein concentrations were measured by the
Bio-Rad or BCA method using bovine serum albumin as a standard (58).
DNA concentrations were determined by a fluorescent assay using calf
thymus DNA as a standard (54).
C2-ceramide Activates PI 3-Kinase in Rat2
Fibroblasts--
PI 3-kinase activity in cell lysates was assessed
using an immune complex kinase assay that employed an anti-p85 antibody to precipitate the enzyme and using PI as a substrate. Treatment of
rat2 fibroblasts with 40 µM C2-ceramide
stimulated PI 3-kinase activity by 4-6-fold after 10-20 min, and the
activity then decreased to the basal level by 60 min (Fig.
2A). The stimulation at 10 min
was evident between 10 and 80 µM C2-ceramide
with a peak at about 40 µM (Fig. 2B). It
should be noted that these incubations contained 15 µM
albumin, which increases the amount of ceramide required to produce a
biological effect. By comparison, the optimum increase in PI 3-kinase
activity obtained after treating cells with 5 ng of PDGF/ml for 10 min
was 6-8-fold (results not shown). PI 3-kinase was also assessed using
anti-phosphotyrosine antibodies for precipitation. Under the latter
conditions, PI 3-kinase activity in the immunoprecipitate increased by
about 4.3-fold after 10 min following treatment with 40 µM C2-ceramide (Fig. 2A). By
comparison, the use of an optimum dose of PDGF (5 ng/ml) produced about
an 8-fold increase after 10 min using this procedure (results not shown). Control experiments showed that similar amounts of precipitated PI 3-kinase protein were used to measure PI 3-kinase activity (Fig.
2C).
We also tested whether PI 3-kinase activation by ceramide is correlated
with translocation of the protein to cell membranes. Treatment with
ceramide caused an approximately 2-3-fold increase in the amount of
the p85 subunit detected in the particulate fraction, indicating
translocation from the cytosol (Fig.
3A). Furthermore, when cell
lysates were immunoprecipitated with anti-p85 and analyzed by Western
blotting with anti-phosphotyrosine antibody, C2-ceramide treatment increased the tyrosine phosphorylation of p85
To verify that the activation of PI 3-kinase was caused by
C2-ceramide itself as opposed to its conversion to
sphingosine, we incubated rat2 fibroblasts with
[14C]acetylsphingosine and measured its recovery and the
production of [14C]acetate. Only 0.047 ± 0.019%
(mean ± S.E. from three independent experiments) of the total
C2-ceramide was converted to [14C]acetate
after a 30-min incubation. Rat2 fibroblasts were also pretreated for 20 min with 5 µM D-MAPP or 500 µM
N-oleoylethanolamine to inhibit alkaline and acid
ceramidases, respectively (59). The two inhibitors decreased the
formation of [14C]acetate by 66-70% when added
separately and by about 80% when added together, demonstrating their
efficacies in these experiments. Treatment of rat2 fibroblasts with
ceramidases inhibitors alone caused a marginal increase (1.2-1.6-fold)
of PI 3-kinase activity, which is compatible with the expected increase
in endogenous ceramide concentrations. The ceramidase inhibitors did
not significantly affect the activation of PI 3-kinase by
C2-ceramide after a 10-min incubation (Fig.
4). Taken together, our results
demonstrate that the activation of PI 3-kinase is caused by ceramide
and not its metabolites.
Activation of PI 3-Kinase by C2-ceramide Involves the
Stimulation of Ras--
p21ras can activate PI 3-kinase
through binding to the p110 catalytic subunit (46-48). We therefore
investigated whether C2-ceramide stimulates PI 3-kinase
through Ras by using fibroblasts expressing N17 Ras, which interferes
with activation of endogenous Ras by exchange factors (53).
C2-ceramide (40 µM) did not increase PI
3-kinase activity significantly after a 10-min incubation in cells
expressing N17 Ras compared with about a 4-fold increase of PI 3-kinase
activity in the vector control cells (Fig.
5A). We also tested the
effects N17 Ras on the activation of PI 3-kinase by EGF and PDGF. The
expression of N17 Ras blocked the activation of PI 3-kinase by 5 ng/ml
PDGF by about 80%. By contrast, there was no significant effect of N17
Ras expression on PI 3-kinase activation that was produced by 100 ng/ml
of EGF (Fig. 5A). Our results therefore implicate Ras-GTP in
the ceramide-induced activation of PI 3-kinase. This conclusion was
confirmed in experiments that used rat2 cells that overexpressed wild
type Ha-Ras. C2-ceramide increased the amount of Ras-GTP in
a time-dependent manner with a peak (2-3-fold) after a
10-min incubation (Fig. 5B). Treatment of cells with the
relatively inactive dihydro-C2-ceramide (40 µM) for 10 min did not significantly increase Ras-GTP
levels (Fig. 5B). As a positive control, treatment of cells
with 100 ng of EGF/ml for 5 min caused a 10.3 ± 1.9-fold increase
in Ras-GTP levels.
We also tested whether C2-ceramide would increase the
amount of p21ras physically associated with PI 3-kinase in rat2
fibroblasts stably expressing wild type Ha-Ras. PI 3-kinase was
immunoprecipitated from cell lysates with anti-p85 antibodies, and the
precipitate was resolved by SDS-polyacrylamide gel electrophoresis and
then immunoblotted with anti-pan Ras antibodies. Treatment with
C2-ceramide but not dihydro-C2-ceramide
increased the co-precipitation of Ras with PI 3-kinase by about 8-fold
after 5 min (Fig. 5C). By comparison, treatment with 100 ng
of EGF/ml for 5 min caused a 27-fold increase in this assay (results
not shown). These results prompted us to assay the PI 3-kinase activity
in anti-pan Ras immunoprecipitates. Treatment of rat2 fibroblasts
overexpressing wild type Ha-Ras with 40 µM
C2-ceramide increased the PI 3-kinase activity that
co-precipitated with Ras by about 3-fold at 20 min (Fig.
5D). The relatively inactive analog,
dihydro-C2-ceramide (40 µM), had no
significant effect on the association of Ras with PI 3-kinase (results
not shown). In control experiments, no PI 3-kinase activity was
associated with beads in the absence of anti-Ras antibody.
Activation of PI 3-Kinase by C2-ceramide Involves the
Stimulation of Tyrosine Kinases--
Protein tyrosine phosphorylation
could be involved at a number of levels in ceramide-induced activation
of PI 3-kinase For example, PI 3-kinase is activated through the
binding of SH2 domains on the p85 subunit to tyrosine phosphorylated
receptor or nonreceptor proteins (35-38). Therefore, we tested the
effect of tyrosine kinase inhibitors on the activation of PI 3-kinase
by C2-ceramide. Rat2 fibroblasts were pretreated with 50 µM genistein or 1 µM PP1 for 1 h
before adding 40 µM C2-ceramide for 10 min.
These tyrosine kinase inhibitors blocked PI 3-kinase activation by
C2-ceramide (Fig.
6A). C2-ceramide
treatment also increased protein tyrosine phosphorylation as indicated
in Fig. 6B including a protein of approximately 120 kDa. We
therefore determined whether this protein was FAK because this has been
implicated in activation of PI 3-kinase (60). C2-ceramide-
and TNF Treatment of Fibroblasts with TNF TNF PI 3-kinase can be activated in different cell types by
protein-tyrosine kinase- and Ras-dependent pathways (for
review see Refs. 35-38). These two mechanisms can activate PI 3-kinase
synergistically (47). The present work establishes that TNF The activation of PI 3-kinase observed after incubating rat2
fibroblasts for 10 min with 5-80 µM
C2-ceramide was demonstrated by assaying the activity of
the enzyme after immunoprecipitation with anti-p85 or
anti-phosphotyrosine antibodies. Furthermore, ceramide caused p85 to
translocate to membranes where its associated p110 subunit would have
access to its lipid substrates. C2-ceramide also increased
the tyrosine phosphorylation of p85. The activation of PI 3-kinase was
ascribed to C2-ceramide itself rather than metabolism to
sphingosine because this rate of conversion was very low (estimated as
<0.02% over the 10 min incubations), and the ceramide effect was not
diminished by inhibitors of ceramidases. The specificity of the
ceramide effect was confirmed because dihydro-C2-ceramide had no significant effect on PI 3-kinase activity.
Tyrosine kinases could contribute to the activation of PI 3-kinase by
several mechanisms. For example, binding the p85 SH2 domains to
tyrosine phosphorylated receptors or nonreceptor proteins increases PI
3-kinase activity. This binding also localizes PI 3-kinase to the
membrane bringing it into proximity with its lipid substrates thus
contributing to the increased PI 3-kinase activity in vivo
(39). Alternatively, or additionally, tyrosine phosphorylation events
can cause the Ras guanyl nucleotide exchange protein, Sos, to be
recruited to the plasma membrane where it can activate Ras and
facilitate binding of Ras to p110. Tyrosine phosphorylation of p85
might represent yet another mechanism of PI 3-kinase activation by
protein-tyrosine kinases, but this remains controversial. The role of
tyrosine kinases in the C2-ceramide-induced activation of
PI 3-kinase was confirmed by the inhibition of the ceramide-induced activation by genistein and PP1 and also our observation of increased tyrosine phosphorylation of the p85 subunit of PI 3-kinase (Fig. 3B), FAK, and various proteins in cell lysates after
treatment with C2-ceramide (Fig. 6, B and
C).
There is growing evidence linking PI 3-kinase to Ras-mediated
signaling. Ras interacts with the p110 catalytic subunit in a
GTP-dependent manner (46-48), and PI 3-kinase
co-immunoprecipitates with Ras (62, 63). The involvement of Ras in the
activation of PI 3-kinase by C2-ceramide is established by:
1) the lack of PI 3-kinase activation in cells expressing
dominant-negative (N17) Ras, 2) the ceramide-induced increase in
Ras-GTP in rat2 fibroblasts stably overexpressing wild type Ha-Ras, and
3) the ceramide-induced increase in the physical association of Ras
with PI 3-kinase. This last point was demonstrated both by
co-precipitation of Ras with PI 3-kinase using the anti-p85 antibody
and also by the increase in PI 3-kinase activity found in anti-Ras
immunoprecipitates. The idea that PI 3-kinase activation by ceramide
involves an association with both tyrosine phosphorylated cellular
proteins and Ras-GTP is significant. Our results show that
C2-ceramide causes maximal activation of Ras after 5 min.
However, the maximum PI 3-kinase activity found in anti-Ras
immunoprecipitates occurs after 20 min. This could be because Ras
activation is necessary, but alone is not enough to cause a detectable
increase in PI 3-kinase activity. It may require concomitant tyrosine
phosphorylation of proteins, such as FAK, which is maximal after a
20-min incubation with C2-ceramide (Fig. 6C).
Such a dual regulatory mechanism in other systems causes the
synergistic stimulation of PI 3-kinase activity (47).
Our results are not limited to the effects of exogenously added
ceramide but probably reflect the normal biological signaling events
downstream of cell activation by TNF The complete inhibition of the ceramide-induced activation of PI
3-kinase in rat2 cells expressing N17 Ras is striking. Warner et
al. (64) showed that stable expression of N17 Ras in rat1 fibroblasts using the same vector as described here causes a
significant but partial block to MAP kinase activation in response to
EGF treatment. However, in these cells, N17 Ras expression completely blocked activation of phospholipase A2 by EGF. By contrast,
Burgering et al. (65) found that N17 Ras expressed by means
of a vaccinia vector in rat1 fibroblasts did not block EGF stimulation
of MAP kinase. Burgering et al. argued that rat fibroblasts
possess both Ras-dependent and Ras-independent pathways
that function in EGF signaling to MAP kinase. Likewise, we found that
N17 Ras expression does not block PI 3-kinase activation by EGF,
although there was a 80% inhibition of the PDGF activation of PI
3-kinase (Fig. 5A). Also N17 Ras expression in rat2 cells
decreased the stimulation of MAP kinase by 0.1-100 ng/ml EGF by only
about 8%.2 In contrast,
stimulation of MAP kinase activity by 5 ng/ml PDGF was decreased by
33-43% in rat2 fibroblasts expressing N17 Ras (results not shown).
The existence of Ras-independent pathways of MAP kinase activation
might explain why expression of N17 Ras had only a modest effect on the
growth rate of rat2 cells in our studies. In any case, our results with
N17 Ras indicate that sphingomyelinase and C2-ceramide
activate PI 3-kinase by a pathway that depends absolutely on Ras-GTP in
rat2 fibroblasts. This conclusion is supported by our observation that
ceramide treatment leads to activation of wild type Ras.
The activation of PI 3-kinase by ceramides and TNF The stimulation of fibroblast proliferation by TNF (TNF
) increased
phosphatidylinositol (PI) 3-kinase activity by 3-6-fold after 10 min.
This effect of C2-ceramide depended on tyrosine kinase
activity and an increase in Ras-GTP levels. Increased PI 3-kinase
activity was also accompanied by its translocation to the membrane
fraction, increases in tyrosine phosphorylation of the p85 subunit, and
physical association with Ras. Activation of PI 3-kinase by TNF
,
sphingomyelinase, and C2-ceramide was inhibited by tyrosine
kinase inhibitors (genistein and PP1). The stimulation of PI 3-kinase
by sphingomyelinase and C2-ceramide was not observed in
fibroblasts expressing dominant-negative Ras (N17) and the stimulation
by TNF
was decreased by 70%. PI 3-kinase activation by
C2-ceramide was not modified by inhibitors of acidic and
neutral ceramidases, and it was not observed with the relatively inactive analog, dihydro-C2-ceramide. It is proposed that
activation of Ras and PI 3-kinase by ceramide can contribute to
signaling effects of TNF
that occur downstream of sphingomyelinase
activation and result in increased fibroblasts proliferation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-interferon, TNF
(2),1 interleukin-1 (3), Fas
ligand (4), and nerve growth factor (5) activate sphingomyelinases
leading to ceramide accumulation. The ability of ceramides to initiate
apoptosis in myeloid and lymphoid tumor cell lines as well as in normal
lymphocytes is well known (6, 7). Depending upon the cell type,
however, ceramides can display other effects. Ceramides play an
important role in the differentiation of HL-60 cells induced by vitamin D3 (8), TNF
, and
-interferon (2). Furthermore, TNF
and ceramides can cause cell proliferation depending on the target cells (9-13). For example, ceramides stimulate cell division in confluent quiescent Swiss 3T3 fibroblasts (12, 13). TNF
-induced proliferation of fibroblasts has been implicated in the pathogenesis of
diseases such as rheumatoid arthritis (14), neuroma formation after
peripheral nerve damage (15), pulmonary fibrosis (16), and chronic
intestinal inflammatory disorders such as ulcerative colitis and
Crohn's disease (17).
(20).
Additionally, we and others found that ceramides inhibit the
agonist-induced activation of phospholipase D (21, 22). We recently
showed that treatment of 3T3-L1 adipocytes for 12 h with
C2-ceramide increased the PI 3-kinase activity that was physically associated with IRS-1, and this increased glucose uptake in
the absence of insulin (23). These effects of ceramides mimic those of
TNF
, which can increase the tyrosine phosphorylation of IRS-1, its
binding of PI 3-kinase (24), the synthesis of GLUT1 (23, 25), and the
basal uptake of glucose by cells (26).
or sphingomyelinase for 20 min
also stimulated PI 3-kinase activity through activation of tyrosine
kinase activity and Ras. This work therefore identifies a novel TNF
and ceramide signaling pathway that could contribute to cellular
responses such as proliferation of fibroblasts.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, EGF, PI, calf
thymus DNA, and genistein were purchased from Sigma.
4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo{3,4-d}pyrimidine (PP1) was obtained from Calbiochem-Novabiochem Corporation, and PDGF
was from Intergen.
(1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-MAPP) was a generous gift from Drs. Y. A. Hannun
and A. Bielawska. Rabbit polyclonal anti-p85
(sc-423), anti-pan Ras
(sc-32, for immunoprecipitation), and anti-focal adhesion kinase were
obtained from Santa Cruz Biotechnology, anti-pan Ras (R02120, for
Western analysis) was from Transduction Laboratory, monoclonal
anti-phosphotyrosine (05-321) was from Upstate Biotechnology Inc., and
thin layer chromatography plates of Silica Gel 60 were from British
Drug Houses. [
-32P]ATP, [1-14C]acetic
anhydride, anti-rabbit IgG linked to horseradish peroxidase, and
enhanced chemiluminescence kit (ECL) were purchased from Amersham Life
Science. Sphingomyelinase and [3H]thymidine were from ICN
Biomedicals. N-[14C]Acetylsphingosine was
synthesized essentially by the method of Ohta et al. (49)
and then purified by thin layer chromatography on plates of silica gel
G using chloroform/methanol/NH4OH/water (80:20:0.5:0.5
v/v/v/v) for development.
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Fig. 1.
Expression of N17 Ras and effects on the
growth of rat2 fibroblasts. In Panel A, lysates from
rat2 fibroblasts (200 µg of protein), vector control cells (200 µg
of protein), rat2 fibroblasts expressing N17 Ras (40 µg of protein),
and rat2 fibroblasts overexpressing wild type Ha-Ras (40 µg of
protein) were analyzed by Western blotting using a pan Ras antibody.
Panel B shows the growth rates of rat2 fibroblasts ( ),
vector control cells (
), and rat2 fibroblasts overexpressing N17 Ras
(
). The initial plating density of the cells in the dishes was about
7000 cells/cm2. The values are mean cell numbers in
millions ± ranges of two independent experiments.
or 5 µg of anti-phosphotyrosine antibodies and
incubating for 6 h at 4 °C with constant gentle rocking
followed by adding 40 µl of 50% protein A-Sepharose in phosphate-buffered saline. The mixtures were incubated for overnight at
4 °C. The immunoprecipitates were then washed three times with 50 mM HEPES, 150 mM NaCl, 100 mM NaF,
1 mM phenylmethylsulfonyl fluoride, 2 mM
Na3VO4, 1% Triton X-100, and 0.1% SDS.
Identical amounts of proteins were separated by SDS-polyacrylamide gel
electrophoresis as described by Laemmli (55) and were transferred to
nitrocellulose membranes. The membranes were blocked overnight with
Tris-buffered saline (pH 7.5) containing 1% bovine serum albumin and
1% dry skim milk at 4 °C and then incubated with the primary
antibody at room temperature. The immunoreactive proteins were detected using anti-rabbit IgG linked to horseradish peroxidase and enhanced chemiluminescence. Western blots were exposed for various length of
times to ensure the proportionality of the response and then analyzed densitometrically.
, 5 µg of
anti-phosphotyrosine, or 5 µg of anti-pan Ras antibodies for 6 h
at 4 °C with constant gentle rocking followed by adding 40 µl of
50% protein A-Sepharose in phosphate-buffered saline. The mixtures
were incubated overnight at 4 °C with constant gentle rocking.
Immunoprecipitates were washed three times with buffer I
(phosphate-buffered saline containing 1% Nonidet P-40 and 100 µM Na3VO4) and then three times
with buffer II (100 mM Tris-HCl, pH 7.5, 500 mM
LiCl, and 100 µM Na3VO4) followed
by three times with buffer III (Tris-HCl, pH 7.5, 100 mM
NaCl, 1 mM EDTA, and 100 µM
Na3VO4). Immunoprecipitates were then
resuspended in 50 µl of buffer III. After adding 0.5 mM
PI, the samples were incubated for 10 min at room temperature with
constant shaking. Then 10 µl of 100 mM MgCl2
and 10 µl of 440 µM ATP containing 30 µCi of
[
-32P]ATP were added, and the incubation was continued
for 10 min with constant shaking. Reactions were stopped with 20 µl
of 8 M HCl and 160 µl of chloroform/methanol (1:1).
Lipids were separated on thin layer silica gel plates (pretreated with
10% (w/v) potassium oxalate) by development with
chloroform/methanol/water/NH4OH (60:47:11:2.2 v/v/v/v).
Incorporation of 32P into PI 3-phosphate was detected by
autoradiography, and the activity was measured by scraping the
corresponding region from the plates followed by scintillation counting
(23).
or
C2-ceramide, where indicated, and
[3H]thymidine (1 µCi/ml) was present in the last 6 h of the incubation. Cells were then washed, and the incorporation of
[3H]thymidine into DNA was measured (21). In a separate
cell proliferation assay, fibroblasts were treated with TNF
or
C2-ceramide for 48 h and then released from the dishes
by trypsinization, and cell number was determined using a Coulter counter.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Activation of PI 3-kinase by
C2-ceramide. Confluent rat2 fibroblasts were incubated
with 40 µM C2-ceramide for the time indicated
(A) or for 10 min with different concentrations of
C2-ceramide (B). PI 3-kinase was
immunoprecipitated with anti-p85 (
) or antiphosphotyrosine
antibodies (
), and activity was measured by the phosphorylation of
PI. Error bars illustrate S.E. from three to seven
independent experiments. Panel C shows a representative
Western blot performed with anti-p85
of the precipitate obtained
with the same antibody. This shows that approximately the same amount
PI 3-kinase was used in the assays.
subunit of
PI 3-kinase in a time-dependent manner (Fig.
3B). To demonstrate that these effects were specific, we
treated rat2 fibroblasts with dihydro-C2-ceramide, which is
a relatively inactive analog of C2-ceramide.
Dihydro-C2-ceramide did not cause a significant increase in
PI 3-kinase activity (0.7 ± 0.2-fold, mean ± S.E. from six
independent experiments), translocation of PI 3-kinase to membranes
(Fig. 3A), or tyrosine phosphorylation of PI 3-kinase.
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Fig. 3.
Translocation and tyrosine phosphorylation of
PI 3-kinase by C2-ceramide. Panel A shows the
effect of treating confluent fibroblasts for 15 min with 40 µM C2-ceramide and
dihydro-C2-ceramide on the relative concentration of PI
3-kinase in the membrane fraction as measured by Western blotting with
anti-p85 antibody. Panel B demonstrates the increase in
tyrosine phosphorylation of p85
after treatment with 40 µM C2-ceramide as measured after
precipitation with anti-p85 antibody followed by Western blotting with
anti-phosphotyrosine antibody. Error bars illustrate S.E.
from three independent experiments. The lower blot in panel
B is a representative Western blot of anti-PI 3-kinase in the
immunoprecipitate to show that the same amount of PI 3-kinase protein
was used for measuring the tyrosine phosphorylation of PI 3-kinase.
DMSO, dimethyl sulfoxide.
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Fig. 4.
Lack of effect of ceramidase inhibitors on
the activation PI 3-kinase by C2-ceramide. Confluent
rat2 fibroblasts were pretreated for 20 min with 5 µM
D-MAPP or 500 µM
N-oleoylethanolamine (NOETH) to inhibit alkaline
and acid ceramidases, respectively. The figure shows the PI 3-kinase
relative to that in untreated fibroblasts. The PI 3-kinase activity in
confluent rat2 fibroblasts treated with D-MAPP or NOETH
alone was not significantly different (p > 0.05) from
the basal PI 3-kinase activity. Results are shown as the means ± S.E. from three independent experiments.
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Fig. 5.
Activation of PI 3-kinase by
C2-ceramide requires activated Ras. Panel A
shows the effects of 40 µM C2-ceramide (15 min), 5 ng/ml PDGF (10 min), and 100 ng/ml EGF (5 min) on PI 3-kinase
activity in fibroblasts expressing dominant-negative (N17) Ras compared
with vector control cells. Panel B illustrates the
concentration of Ras-GTP, measured as described under "Experimental
Procedures," following stimulation of rat2 fibroblasts overexpressing
wild type Ha-Ras for 0-20 min with 40 µM
C2-ceramide or with dihydro-C2-ceramide for 10 min. The representative Western blot illustrates Ras-GTP that
precipitated with cRaf1-gluthione S-transferase. Panel
C is a representative Western blot performed with anti-pan Ras
antibody on immunoprecipitates obtained with anti-p85 antibody to
show the relative concentration of Ras that was co-precipitated by the
anti-p85
antibody. In these experiments, rat2 fibroblasts expressing
wild type Ha-Ras were treated with 40 µM
C2-ceramide for 1 or 5 min or with 40 µM
dihydro-C2-ceramide for 5 min. Panel D
demonstrates the time course for the relative increase in PI 3-kinase
activity in anti-pan Ras precipitates using rat2 fibroblasts
overexpressing wild type Ha-Ras. Results are shown as the means ± S.E. from three independent experiments. DMSO, dimethyl
sulfoxide.
-induced tyrosine phosphorylation of FAK in a
time-dependent manner. Dihydro-C2-ceramide
produced no significant increases in the tyrosine phosphorylation of
FAK (Fig. 6C).
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Fig. 6.
Activation of PI 3-kinase by
C2-ceramide involves activated tyrosine kinase activities.
Panel A demonstrates that the activation of PI 3-kinase
obtained after treating confluent rat2 fibroblasts with 40 µM C2-ceramide for 10 min is inhibited by the
tyrosine kinase inhibitors, 50 µM genistein, and 1 µM PP1 and that 40 µM
dihydro-C2-ceramide does not activate PI 3-kinase activity
significantly. Results are shown as the means ± S.E. from three
independent experiments. Panel B shows an
anti-phosphotyrosine Western blot analysis of total cell lysates (50 µg of protein) obtained from cells that were treated with
Me2SO (lane 1), 40 µM
dihydro-C2-ceramide for 15 min (lane 2), 5 ng/ml
PDGF for 5 min (lane 3), 40 µM C2-ceramide
for 5, 10, 15, 20, and 30 min, respectively (lanes 4-8),
and 80 µM C2-ceramide for 15 min (lane
9). Panel C shows the tyrosine phosphorylation of FAK
obtained with anti-phosphotyrosine precipitation of 400 µg of lysate
protein for each lane followed by immunoblotting with anti-FAK. Results
in panels B and C were reproduced in a further independent
experiment. DMSO, dimethyl sulfoxide.
or Sphingomyelinase Activates
PI 3-Kinase--
Many signaling responses of TNF
can be mimicked by
treating cells with cell-permeable ceramides or sphingomyelinase, which generates long chain ceramides (61). We therefore tested whether TNF
and sphingomyelinase would also activate PI 3-kinase in rat2 fibroblasts. Treatment of rat2 fibroblasts with 10 ng of TNF
/ml or
with 0.1 units of sphingomyelinase/ml increased PI 3-kinase activity
after about 20 min (Fig. 7A).
Pretreatment of rat2 fibroblasts with the tyrosine kinase inhibitors
genistein (50 µM) or PP1 (1 µM) decreased
the stimulation of PI 3-kinase by TNF
and sphingomyelinase by 88 and
82%, respectively (Fig. 7B). This indicates that PI 3-kinase activation by TNF
and sphingomyelinase requires a tyrosine kinase activity, as does the effect of C2-ceramide.
Similarly, the activations of PI 3-kinase by TNF
and
sphingomyelinase, respectively, were inhibited by about 70 and 99%, in
fibroblasts expressing N17 Ras compared with control cells (Fig.
7C). This indicates the involvement of Ras-GTP in PI
3-kinase activation by these agonists.
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Fig. 7.
Activation of PI 3-kinase by tumor necrosis
factor- and sphingomyelinase. Confluent
rat2 fibroblasts treated with 10 ng of TNF
/ml or 0.1 units of
sphingomyelinase/ml and the time course for the activation of PI
3-kinase are shown in Panel A. In Panel B, rat2
fibroblasts were pretreated for 1 h with 1 µM PP1 or
50 µM genistein to inhibit tyrosine kinase activity.
Panel C shows the inability of TNF
or sphingomyelinase to
stimulate PI 3-kinase activity in fibroblasts expressing
dominant/negative (N17) Ras after a 20-min incubation. Results are
shown as the means ± S.E. from three independent
experiments.
and C2-ceramide Induce Cell Proliferation in
Confluent Rat2 Fibroblasts--
PI 3-kinase activation is often
implicated in the control of cell proliferation. We therefore
investigated whether this is the case for the TNF
- and
C2-ceramide-mediated increases in PI 3-kinase. TNF
and
C2-ceramide caused about a 2-fold increase in the
incorporation of [3H]thymidine into DNA after 24 h,
and this effect was completely blocked by PI 3-kinase inhibitor, Ly
294002 (Fig. 8A). Furthermore, incubation of fibroblasts with 10 ng/ml TNF
or 40 µM
C2-ceramide for 48 h caused 1.68 ± 0.05- and
1.28 ± 0.1-fold increases in cell number, respectively (Fig.
8B).
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Fig. 8.
Tumor necrosis factor-
and C2-ceramide stimulate PI
3-kinase-dependent proliferation of rat2 fibroblasts.
Panel A demonstrates that 10 ng/ml TNF
and 40 µM C2-ceramide caused about a 2-fold increase
in the incorporation of [3H]thymidine into DNA after
24 h, and this effect was completely blocked by PI 3-kinase
inhibitor, Ly 294002 (20 µM). Panel B shows
the changes in cell number that occurred after 48 h as a result of
these treatments. Results are shown as the mean ± ranges from two
independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
sphingomyelinase, and C2-ceramide all activate PI 3-kinase
in rat2 fibroblasts. This process is dependent on tyrosine kinase
activity, and it involves an increase in the amount of Ras-GTP. The
novelty of the present study is that TNF
, sphingomyelinase, and
ceramides should initiate such a signaling pathway.
. This cytokine exerts its
effects, in part, by stimulating sphingomyelinase with subsequent accumulation of ceramides (2). TNF
and sphingomyelinase stimulated PI 3-kinase, and these effects were blocked by the tyrosine kinase inhibitors, genistein and PP1. In addition, activation of PI 3-kinase by sphingomyelinase was completely abolished in fibroblasts that expressed N17 Ras, as was the case for C2-ceramide.
However, the activation of PI 3-kinase by TNF
in the same cell line
was inhibited by only 70%. This indicates that a maximum of about 30%
of the TNF
-induced increase in PI 3-kinase is probably mediated by a sphingomyelinase-independent pathway and the formation of ceramides and
Ras-GTP. Taken together, our results demonstrate that tyrosine kinase
stimulation and formation of Ras-GTP is upstream of PI 3-kinase
activation by TNF
as well as by C2-ceramide and long chain ceramide.
could play an
important role in regulating several cellular functions. For example,
we demonstrated that cell-permeable ceramides increase PI 3-kinase
activity associated with IRS-1 in 3T3-L1 adipocytes (23). We have also
shown that treatment of rat2 cells for 20 min with either
C2-ceramide or TNF
leads to activation of MAP kinase.2 These effects are substantially blocked by Ly
294002 and by expression of N17 Ras. Other workers demonstrated that
ceramides can activate MAP kinase in some cell types through the
stimulation of a ceramide-dependent kinase that activates
Raf (66). Our studies provide an alternative pathway for the
ceramide-induced activation of MAP kinase that involves the stimulation
of tyrosine kinases, Ras and PI 3-kinase. The conclusion that ceramides
can activate mitogenic enzymes such as PI 3-kinase and MAP kinase in
confluent rat2 fibroblasts may appear counter-intuitive because
ceramides are often associated with producing apoptosis. In an attempt
to explain this contradiction, Kolesnick and Fuks (67) suggested that
the cellular responses to ceramides depend on the genetic component of
cells as well as the microenvironment in which the signal is generated.
plays an
important role in the pathogenesis of many autoimmune and chronic inflammatory diseases (14-17). TNF
-induced fibroblast proliferation has been reported to be dependent on PDGF secretion (11), stimulation of c-raf-1 kinase (68), and MAP kinase activation (15). Our results
provide another mechanism for the TNF
-induced proliferation of
fibroblasts, which is dependent on PI 3-kinase activation. PI 3-kinase
plays a key role in many cell processes such as growth (27, 28),
intracellular vesicle trafficking, secretion (29-31), and regulation
of the cytoskeleton (32-34). The demonstration that PI 3-kinase is
activated by TNF
and ceramides in a tyrosine kinase- and
Ras-GTP-dependent manner identifies a pathway that may
contribute to signal transduction by cytokines and other agonists that
stimulate sphingomyelinase activities.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Y. Hannun and A. Bielawska for their gift of D-MAPP and Dr. S. E. Egan for the vector containing N17 Ras. We are also grateful to Dr. S. Bourgoin for helpful advice and David Li for experimental assistance.
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FOOTNOTES |
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* This work was supported by grants from the Medical Research Council of Canada, the Canadian Diabetes Foundation (in honor of Helen Margaret Clery), and the Heart and Stroke Foundation of Alberta (to D. N. B.) and a grant from the National Cancer Institute of Canada (to J. C. S.).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.
¶ Recipient of a Research Fellowship from the Alberta Heritage Foundation for Medical Research.
Recipient of a Medical Scientist Award from the Alberta
Heritage Foundation for Medical Research. To whom correspondence should be addressed: Signal Transduction Laboratories, Lipid and Lipoprotein Research Group, and Dept. of Biochemistry, University of Alberta, 357 Heritage Medical Research Centre, Edmonton, Alberta T6G 2S2, Canada. Tel.: 403-492-2078; Fax: 403-492-3383.
2 A. N. Hanna and D. N. Brindley, unpublished work.
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ABBREVIATIONS |
---|
The abbreviations used are:
TNF, tumor
necrosis factor-
;
DMEM, Dulbecco's minimum essential medium;
EGF, epidermal growth factor;
FAK, focal adhesion kinase;
IRS-1, insulin
receptor substrate-1;
MAP, mitogen-activated protein (Erk);
PI, phosphatidylinositol;
PDGF, platelet-derived growth factor;
PP1, 4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo{3,4-d}pyrimidine;
SH, Src homology domain;
D-MAPP, (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol.
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