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Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain
1Correspondence: Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain. E-mail: mgp{at}solea.quim.ucm.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: cell death • sphingolipids • mitogen-activated protein kinases • neural cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. Likewise, exposure of neural
cells to physical (e.g., ultraviolet radiation), chemical (e.g., tumor
necrosis factor, TNF), bacterial (e.g., lipopolysaccharide), or viral
(e.g., human immunodeficiency virus 1) stimuli may increase
intracellular ceramide levels and therefore evoke changes in the cell
survival/death decision (4
, 5)
. Moreover, changes in
ceramide metabolism exert important regulatory effects on neuronal
growth and development (6)
.
In ceramide signaling pathways leading to apoptosis, ceramide
generation through sphingomyelin hydrolysis by neutral and/or acid
sphingomyelinase is usually considered the norm. The link between
receptor activation, sphingomyelinase activation, and ceramide
generation is mostly supported by comprehensive studies of the p55 TNF
receptor, the p75 neurotrophin receptor, and CD95/Fas (4
, 5)
. However, the de novo synthesis pathway has been
gaining recognition as an alternative means of generating a signaling
pool of ceramide. Thus, compounds such as L-cycloserine, an inhibitor
of serine palmitoyltransferase, and fumonisin B1, an inhibitor of
ceramide synthase, prevent ceramide accumulation and apoptotic death in
hematopoietic (7)
and pancreatic ß cells
(8)
exposed to long-chain fatty acids, which are
substrates for ceramide synthesis de novo. A significant
contribution of the de novo pathway to ceramide generation
and apoptosis has also been reported in endothelial cells exposed to
TNF, a paradigmatic example of ligands that are believed to generate
ceramide solely through sphingomyelin breakdown (9)
, and
in PC12 pheochromocytoma cells exposed to angiotensin II
(10)
. In addition, the chemotherapeutic drug daunorubicin
may induce apoptosis by enhancing ceramide synthesis de novo
(11)
as well as by inducing sphingomyelin breakdown
(12
, 13)
.
Despite these recent observations supporting the importance of ceramide synthesis de novo in the induction of apoptosis, the characterization of the downstream targets linking de novo-synthesized ceramide to apoptosis remains elusive. Moreover, although ceramide may significantly contribute to cell death in neurological disorders, the possible involvement of ceramide synthesis de novo in neural cell death is as yet unknown. The present study was therefore undertaken to address two questions: 1) Does de novo-synthesized ceramide induce apoptosis of neural cells? 2) If so, which may be downstream targets of de novo-synthesized ceramide leading to apoptosis?
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. For all the experimental determinations performed,
the serum-containing medium was removed and cells were transferred to a
chemically defined, serum-free medium consisting of DMEM/Ham’s F12
(1:1, v/v) supplemented with 5 µg/ml insulin, 50 µg/ml transferrin,
20 nM progesterone, 50 µM putrescine, 30 nM sodium selenite, and
1.0% (w/v) defatted and dialyzed bovine serum albumin. Cortical
neurons from 24 h rats were cultured exactly as described before
(14)
.
Cell death
Cell viability was determined by trypan blue exclusion.
Oligonucleosomal DNA fragmentation, a characteristic biochemical
feature of apoptotic cell death, was measured using a nucleosome DNA
enzyme-linked immunoabsorbent assay (Boehringer, Mannheim, Germany),
which quantitatively records histone-associated DNA fragments.
Ceramide and sphingomyelin syntheses
Cells were transferred to chemically defined medium. After
24 h, reactions were started by the addition of 1 µCi of
L-[U-14C]serine per well together with the
different modulators. Reactions were terminated at the times indicated
by aspiration of the medium and addition of 1 ml methanol. Lipids were
extracted and saponified, and ceramide and sphingomyelin were resolved
by thin-layer chromatography in parallel with standards on silica-gel
G60 plates with chloroform:methanol:water (100:42:6, v/v/v) as the
developing system until the front had reached two-thirds of the plate.
The solvent was then evaporated and plates were subsequently run with
chloroform:methanol:acetic acid (94:1:5, v/v/v) until the front had
reached the top of the plate (15)
.
Fatty acid uptake and metabolism
Cells were transferred to chemically defined medium. After
24 h, reactions were started by the addition of 0.2 mM (1 µCi)
albumin-bound [9,10-3H]palmitate together with
the different modulators. At the times indicated, the medium was
separated from the cells, and lipids were extracted from the two
compartments and subsequently resolved by thin-layer chromatography
together with standards. Fatty acid uptake was calculated as the
disappearance of [3H]palmitate from the
extracellular medium. Nonesterified fatty acids and triacylglycerols
were separated on silica-gel G60 plates with chloroform/diethyl
ether/acetic acid (70:30:1, v/v/v) as the developing system.
Phosphatidylcholine was resolved on silica-gel H60 plates with
chloroform/methanol/acetic acid/water (50/25/8/1, v/v/v/v) as
developing system. Ceramide and sphingomyelin were resolved as
described above.
Mitogen- and stress-activated protein kinase activities
Cells were washed and lysed, and supernatants were obtained as
described before (16)
. Extracellular signal-regulated
kinase (ERK) activity was determined as the incorporation of
[
-32P]ATP into a specific peptide substrate
(16)
. The activity of c-Jun amino-terminal kinase (JNK)
and p38 mitogen-activated protein kinase (MAPK) was monitored as the
incorporation of [-32P]ATP into specific
substrates (c-Jun 1–169 and MAPKAP kinase-2 46–600, respectively)
after sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), autoradiography, and radioactive counting of the excised
substrate bands according to manufacturer’s instructions (Upstate
Biotechnology, Lake Placid, N.Y.) (17)
.
Raf-1 activity
Raf-1 was immunoprecipitated from cell lysates as described
before (16)
. The kinase reaction was carried out for 30
min at 30°C with 0.7 µg kinase-negative MEK1[97A] (Upstate
Biotechnology) and 2 µCi [-32P]ATP as
substrates in assay buffer containing 25 mM Tris-HCl, pH 7.4, 10 mM
MgCl2, 0.5 mM EDTA, 5 mM NaF, 1 mM
NaVO4, 1 mM 4-nitrophenylphosphate, and
proteinase inhibitors (17
, 18)
. Reactions were stopped
with SDS sample buffer, and substrate phosphorylation was determined in
the excised bands after SDS-PAGE and autoradiography.
Kinase suppressor of Ras (KSR) activity
KSR was immunoprecipitated from cell lysates with an anti-KSR
antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) bound to
protein G-Sepharose. The kinase reaction was carried out for 30 min at
30°C, with 0.3 mM synthetic Raf-1 peptide (17
, 18)
and 2
µCi [-32P]ATP as substrates in the assay
buffer described above for Raf-1. Phosphorylated peptide was resolved
by P81 phosphocellulose paper.
Protein kinase B (PKB) activity
PKB was immunoprecipitated from cell lysates with 2 µg of
anti-PKB
antibody bound to protein G-Sepharose (19)
.
PKB activity was determined as the incorporation of
[-32P]ATP into a specific peptide substrate.
Phosphorylated peptide was resolved by P81 phosphocellulose paper
(19)
.
Statistical analysis
Results shown represent the means ± SD of the
number of experiments indicated in every case. Five to six different
replicates of the various conditions included in each experiment were
routinely performed. Statistical analysis was performed by analysis of
variance. A post hoc analysis was made by the
Student-Neuman-Keuls test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and
cell viability was determined at different times. As shown in
Fig. 1A
, palmitate induced the death of astrocytes in a
time-dependent fashion. This effect of palmitate was more pronounced
when mitochondrial fatty acid oxidation was blocked with
tetradecylglycidic acid (TDGA), a specific inhibitor of carnitine
palmitoyltransferase I (CPT-I), the key regulatory enzyme of long-chain
fatty acid translocation into mitochondria in astrocytes
(14)
. TDGA alone had no significant effect on cell
viability.
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The lipid second messenger ceramide is involved in the induction of
apoptosis in a number of pathophysiological situations (4
, 5)
. Ceramide is mostly generated by degradation of sphingomyelin
or by de novo synthesis. Because long-chain fatty acids are
biosynthetic precursors of ceramide, the possibility that intracellular
ceramide accumulation resulting from enhanced ceramide synthesis
mediates palmitate-induced astrocyte death was tested. Palmitate
notably increased ceramide synthesis in primary astrocytes (Fig. 2A
). By contrast, no significant effect of palmitate on serine
incorporation into sphingomyelin was evident (Fig. 2B
).
Coincubation of the cells with palmitate and TDGA exacerbated the
effect of the fatty acid on ceramide synthesis (Fig. 2A
). A
similar time course was observed in astrocytes for palmitate-induced
death (Fig. 1A
) and palmitate-induced ceramide synthesis
(Fig. 2A
).
|
To obtain additional evidence that enhanced ceramide synthesis in
palmitate-treated astrocytes reflected de novo ceramide
formation, cells were incubated with two inhibitors of ceramide
biosynthesis: 1) L-cycloserine, an inhibitor of serine
palmitoyltransferase, the first committed step of ceramide synthesis
de novo; 2) fumonisin B1, an inhibitor of
ceramide synthase, which catalyzes the condensation of sphinganine and
acyl-CoA to generate dihydroceramide. As shown in Table 1
, both L-cycloserine and fumonisin B1 were able to block both
palmitate-induced ceramide synthesis and palmitate-induced astrocyte
death, even in the presence of TDGA.
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Next, we tested whether astrocyte death occurred by a process of
apoptosis, as expected for ceramide-mediated cell death. As shown in
Fig. 3
, treatment of astrocytes with palmitate led to a significant increase
in oligonucleosomal DNA fragmentation, a hallmark of apoptosis. Again,
the effect of palmitate was more remarkable when TDGA was
simultaneously present in the incubations. Moreover, the apoptotic
effect of palmitate was prevented by L-cycloserine.
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De novo-synthesized ceramide signals apoptosis of
astrocytes via Raf-1/ERK
It is generally accepted that the ERK cascade promotes cell
proliferation. However, recent investigations have begun to define
situations in which sustained ERK activation mediates antiproliferative
effects (21
, 22)
. We therefore studied the possible
involvement of ERK in fatty acid-induced apoptosis of astrocytes.
PD098059, a selective inhibitor of the ERK cascade, prevented the
decrease in astrocyte viability elicited by palmitate, even in the
presence of TDGA. Thus, values of viability of primary astrocytes were
103 ± 10% after 48 h exposure to 25 µM PD098059; 101 ± 11% after 48 h exposure to 0.2 mM palmitate and 25 µM
PD098059; and 99 ± 7% after 48 h exposure to 0.2 mM
palmitate, 20 µM TDGA and 25 µM PD098059 (n=6; 100%:
incubations with no additions). Likewise, palmitate-induced apoptotic
DNA fragmentation in astrocytes was prevented by PD098059 (Fig. 3)
. The
effect of palmitate on ERK activity was subsequently determined. As
shown in Table 2
, palmitate was able to induce a sustained activation of ERK in
astrocytes. The stimulatory effect of palmitate was more remarkable
when TDGA was simultaneously present in the medium. Moreover, the
palmitate-induced stimulation of ERK in astrocytes was prevented by
L-cycloserine and, as expected, by PD098059.
|
It is widely accepted that Raf-1 represents a pivotal element in the
control of cell fate by the ERK cascade (23)
. Although the
molecular link between ceramide accumulation and Raf-1 activation is
not well understood and contradictory data have been reported,
Kolesnick and co-workers have shown that KSR is a ceramide-activated
protein kinase that may phosphorylate and activate Raf-1 (18
, 24)
. The effect of palmitate on Raf-1 and KSR activity in
astrocytes was therefore determined. Thus, cell incubation with
palmitate induced a significant increase of Raf-1 kinase activity
(Fig. 4A
). This effect was exacerbated by coincubation with TDGA.
Palmitate-induced activation of Raf-1 was prevented by L-cycloserine
(Fig. 4A
), pointing to an involvement of de
novo-synthesized ceramide. In contrast to Raf-1, KSR activity was
not significantly affected by palmitate, either alone or in combination
with TDGA (Fig. 4B
).
|
Other protein kinases distinct from ERK—namely, JNK, p38 MAPK, and
PKB—have been proposed as potential targets of ceramide in the control
of cell fate (4
, 5)
. The activity of those kinases was
therefore determined. However, exposure of astrocytes to 0.2 mM
palmitate (with or without 20 µM TDGA) for 48 h did not
significantly affect JNK activity (n=4), p38 MAPK activity
(n=3), and PKB activity (n=4).
Neurons are resistant to the apoptotic action of palmitate
In contrast to what was observed in primary astrocytes, the
viability of cortical neurons in primary culture was not reduced by
palmitate along the 72 h experimental period, even in the presence
of TDGA in the medium (Fig. 1B
). Likewise, palmitate was
unable to stimulate ceramide synthesis in neurons, either alone or in
combination with TDGA. Thus, incorporation of
L-[14C]serine into ceramide in neurons was
97 ± 20% after 48 h exposure to 0.2 mM palmitate and
98 ± 13% after 48 h exposure to 0.2 mM palmitate and 20
µM TDGA (n=4; 100%: incubations with
L-[14C]serine alone).
To test whether neurons, unlike astrocytes, possess the ability to
prevent exogenous fatty acids from entering the ceramide synthesizing
pathway, cells were cultured in the presence of exogenous
[3H]palmitate and the metabolic fate of the
fatty acid was determined. As shown in Table 3
, compared to astrocytes, neurons had a very low capacity to take up
palmitate, and to incorporate the fatty acid into glycerolipids
(phosphatidylcholine and especially triacylglycerols) and sphingolipids
(sphingomyelin and especially ceramide). Furthermore, TDGA
significantly enhanced palmitate uptake and incorporation into ceramide
in astrocytes but not in neurons (Table 3)
.
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If ERK activation mediates apoptosis induced by de novo-synthesized ceramide (see above), then ERK should not be stimulated in palmitate-treated neurons. ERK activity was therefore determined in neurons, and no significant effect of palmitate was observed. Thus, ERK activity in neurons was 112 ± 9% after 48 h exposure to 0.2 mM palmitate and 97 ± 6% after 48 h exposure to 0.2 mM palmitate and 20 µM TDGA (n=4; 100%: incubations with no additions). Likewise, exposure of neurons to 0.2 mM palmitate (with or without 20 µM TDGA) for 48 h did not significantly affect JNK activity (n=4), p38 MAPK activity (n=3), and PKB activity (n=4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 5
, 25)
.
Nevertheless, ample consensus supports the notion that ceramide plays a
pivotal role in the control of neural cell death (1
2
3)
and differentiation (6)
. Ceramide-induced apoptosis is
usually ascribed to ceramide generation through sphingomyelin
hydrolysis by neutral and/or acid sphingomyelinase (4
, 5
, 25)
. By contrast, the present report demonstrates that ceramide
synthesis de novo is important in determining the apoptotic
outcome of neural cells. Work by Bazan in the early 1970s demonstrated
an enhanced breakdown of cellular glycerolipids and a concomitant
accumulation of nonesterified fatty acids, including palmitic acid, in
a number of models of brain trauma/ischemia (26
, 27)
. The
breakdown of membrane phospholipids on trauma/ischemia seems to be the
result of Ca2+-induced stimulation of
phospholipases and of uncoupling of phospholipid resynthesis due to
energy depletion, and may be involved in irreversible damage of
membrane structure and function (28)
. In addition,
nonesterified fatty acids exert various detrimental effects on brain
structure and function such as uncoupling of oxidative phosphorylation,
disruption of plasma membrane and mitochondrial ion fluxes, inhibition
of membrane receptors, enzymes, and ion channels, and elevation of
synaptic glutamate concentration (28)
. Both apoptotic and
necrotic cell death occur in brain trauma/ischemia (1
2
3)
.
Our data indicate that under these situations, ceramide synthesized
de novo from nonesterified fatty acids may contribute to the
apoptotic death of astrocytes.
Selective involvement of ERK in apoptosis of astrocytes induced by
de novo-synthesized ceramide
It is generally accepted that the activation of the ERK cascade
leads to cell proliferation (21
, 22)
. However, recent
investigations have begun to define situations in which ERK mediates
cell cycle arrest (e.g., ref 29
), antiproliferation (e.g.,
ref 30
), as well as apoptotic (e.g., ref 31
)
and nonapoptotic death (e.g., ref 32
) in a number of
cells, including neural cells. Data in the present work show for the
first time that the apoptotic action of de novo-synthesized
ceramide relies selectively on ceramide-induced Raf-1/ERK activation.
This assumption is mostly based on the following observations:
1) PD098059 prevents palmitate-induced ERK activation and
astrocyte death; 2) blockade of ceramide synthesis de
novo with L-cycloserine prevents palmitate-induced ceramide
accumulation and Raf-1/ERK activation; 3) TDGA enhances
palmitate-induced Raf-1/ERK activation and astrocyte death;
4) other potential targets of ceramide in the control of
cell fate were not significantly affected in astrocytes exposed to
palmitate; 5) unlike astrocytes, neurons are reluctant to
palmitate-induced ERK activation and cell death.
Data also show that ceramide-induced activation of Raf-1/ERK in
astrocytes occurs independently of KSR, a protein kinase that has been
suggested to be involved in the stimulation of Raf-1 by the p55 TNF
receptor (18)
and in ceramide-induced apoptosis
(24)
. These authors reported that ceramide selectively
induces the autophosphorylation of KSR, thereby enhancing its capacity
to phosphorylate and activate Raf-1/ERK. Our observations are in line
with those of Huwiler et al. (33)
, however, who have shown
that ceramide directly binds to and activates Raf-1. As a matter of
fact, Raf-1 has a ceramide binding motif (4)
, therefore
linking the ceramide pathway with the ERK cascade in the control of
cell fate. Nevertheless, others have reported that ceramide binding to
Raf-1 does not lead to Raf-1 stimulation (34)
and that the
activation of Raf-1 by KSR is independent of the kinase activity of the
latter (35)
. The precise role of KSR as a modulator of the
ERK cascade is still a matter of debate.
Differential sensitivity of astrocytes and neurons to fatty
acid-induced apoptosis
The importance of ceramide and ERK in fatty acid-induced apoptosis
of astrocytes is supported by the observation that the absence of
palmitate-induced ceramide synthesis and ERK activation in neurons
renders these cells reluctant to apoptosis. Inherent differences in
fatty acid uptake and metabolism are evident between astrocytes and
neurons. Compared to astrocytes, neurons show a very low capacity to
take up palmitate and to divert the fatty acid to glycerolipid and
sphingolipid synthesis. This different behavior of the two cell types
is particularly relevant for triacylglycerol and ceramide biosyntheses,
which were 
25 and 15 times higher in astrocytes than in
neurons, respectively. Triacylglycerols seem to be the major source of
the nonesterified palmitic acid released during ischemia
(36)
.
TDGA, a specific inhibitor of CPT-I, exacerbated the effect of
palmitate on ceramide accumulation, Raf-1/ERK activation and apoptotic
death in astrocytes. Evidence has accumulated during the last two
decades highlighting the physiological importance of CPT-I in the
control of mitochondrial fatty acid oxidation in many cell types,
including astrocytes (14)
. CPT-I has been implicated in
ceramide-mediated apoptosis (7)
. Because palmitate is a
precursor for ceramide synthesis de novo, it is conceivable
that inhibition of CPT-I leads to accumulation of palmitate in the
cytoplasm, increased ceramide synthesis, and apoptosis. This is what
actually occurs in astrocytes treated with TDGA. Likewise, expression
of high CPT-I activity may help cells to withstand palmitate-induced
apoptosis (7
, 37)
. Neural cells have been shown to exhibit
a high activity of sphingosine acylation to generate ceramide
(38)
. In addition, the recently purified neutral
ceramidase might also be involved in ceramide synthesis de
novo owing to its ability to catalyze the reverse amidase
reaction, i.e., the condensation of the fatty acid with sphingosine to
generate ceramide (39)
. However, the situation may be more
complex in that CPT-I in astrocytes is a ceramide-activated enzyme
(14)
, pointing to the existence of a regulatory loop in
which elevated ceramide levels occurring on CPT-I inhibition might be a
signal for the reactivation of the enzyme. The observation that CPT-I
directly interacts with the anti-apoptotic protein Bcl-2 in the
mitochondrial outer membrane (40)
and the well-established
role of mitochondria in the onset of apoptosis (4)
point
to a general role of CPT-I as a regulator of apoptosis
(41)
.
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ACKNOWLEDGMENTS |
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antibody and the PKB substrate
peptide (GRPRTSSFAEG); to Dr. J. M. Lowenstein (Brandeis
University, Waltham, Mass.) for kindly donating the TDGA; and to Dr.
Math J. H. Geelen (Utrecht University, The Netherlands) for
continuous support and expert advice. This work was supported by grants
from Comisión Interministerial de Ciencia y Tecnología
(PM 98/0079) and Comunidad Autónoma de Madrid (CAM 08.5/0017/98). Received for publication February 24, 2000. Accepted for publication May 10, 2000.
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) or presence (•) of 20 µM TDGA, or with 20 µM TDGA alone
(
). Results are expressed as percentage of incubations with no
additions. A) Astrocytes (n=6).
B) Neurons (n=4).
