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INTRODUCTION |
Apoptosis is a fundamental biological process used to eliminate
unwanted, superfluous, or potentially harmful cells. In the developing
nervous system, about half of all neurons that are produced die by
apoptosis around the time of birth (1). Cultured rat cerebellar granule
cells are widely used as a model system for studying neuronal
apoptosis. These neurons are usually cultured and matured in medium
containing 26 mM potassium (high K+;
HK).1 After maturation,
changing to medium containing 5 mM potassium (low
K+; LK) induces neuronal cell death in which the cells show
apoptotic features. In addition, this culture system provides a large
homogeneous neuronal population. Therefore, these neurons are widely
used as a primary cell culture system to investigate the biochemical and molecular mechanisms underlying neuronal apoptosis in the central
nervous system.
Mitogen-activated protein (MAP) kinases are serine/threonine kinases
that play an important role in signal transduction from the cell
surface to the nucleus. The mammalian MAP kinases can be subdivided
into extracellular signal-regulated kinases, c-Jun N-terminal kinases
(JNK), and p38 MAP kinases (p38). p38 is activated by phosphorylation
on Thr-180 and Tyr-182 in response to environmental stress (2, 3).
Recently, p38 activation has been suggested to be involved in mediating
apoptosis in various cell types (4-6). SB203580, a specific inhibitor
of p38, prevents several types of cell death, including
glutamate-induced apoptosis of cultured cerebellar granule neurons (7,
8).
c-Jun is a member of the activator protein-1 family of transcription
factors possessing leucine zippers. c-Jun is phosphorylated on Ser-63
and Ser-73 within its N-terminal region by JNK (9). Phosphorylation of
c-Jun is necessary for apoptosis of superior cervical ganglion and
cerebellar granule neurons (10, 11). However, the mechanism of c-Jun
phosphorylation remains unclear.
In the present study, we investigated whether p38 is necessary for
LK-induced apoptosis and whether p38 is involved in the phosphorylation
of c-Jun in cerebellar granule neurons. Our results suggest that p38
was activated after lowering potassium concentration, and active p38
phosphorylates c-Jun directly. We found the p38-c-Jun pathway is very
important for LK-induced apoptosis in cerebellar granule neurons.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Primary cultures of dissociated cerebellar
granule neurons were prepared from the cerebella of postnatal day 9 rats (Wistar ST, both sexes) as described previously (12-16). Briefly,
cells were gently dissociated with a plastic pipette after digestion with papain (90 units/ml, Worthington) at 37 °C. The cells were then
cultured in medium consisting of 5% precolostrum newborn calf serum
(Mitsubishi Kasei), 5% heat-inactivated horse serum (55 °C, 30 min;
Life Technologies, Inc.), and 90% 1:1 mixture of Dulbecco's modified
Eagle's medium and Ham's F-12 medium containing 15 mM
HEPES buffer (pH 7.4), 30 nM selenium, and 1.9 mg/ml sodium bicarbonate, at a final cell density of 5 × 105
cells/cm2 on a polyethyleneimine-coated surface in
6-cm-diameter dishes (21 cm2 of culture surface area,
Sumitomo Bakelite). After culture for 1 day in a humidified
CO2 (5%) incubator, the medium was changed to 26 mM potassium-containing (HK) minimum essential medium (MEM) supplemented with 5% heat-inactivated horse serum and 1 µM cytosine arabinoside. MEM was supplemented with 2.2 mg/ml glucose and 2.2 mg/ml sodium bicarbonate. HK-MEM was prepared by
increasing the KHCO3 concentration from the normal low
value of 5.4 to 26 mM, with the omission of the
corresponding concentration NaHCO3. After 4 days in culture
in a 10% CO2 incubator, the medium was changed to
serum-free 5.4 mM potassium-containing (LK) MEM or HK-MEM. The assays described below were then performed.
Assay of Neuronal Survival--
Neuronal survival was determined
by MTT assay, according to the original procedure (17) with some
modifications (18). Briefly, the tetrazolium salt MTT
(3-(4,5-dimethyl-2-thizolyl)-2,5-diphenyl-2H-tetrazolium bromide) was added to cultures to a final concentration of 1 mg/ml. After incubation for 2 h at 37 °C, the assay was stopped by
adding 80% volume of lysis buffer (10% SDS in 50%
N,N-dimethyl formamide, pH 4.7). The absorbance was measured
spectrophotometrically at 570 nm, following overnight incubation at
37 °C. The percent survival was defined as
(A(experimental
blank)/A(control
blank)) × 100; the blank
value was taken from wells without cells.
Immunocytochemistry--
To stain neurons, the cultured cells
were fixed with 4% paraformaldehyde for 20 min and incubated with
anti-MAP2 antiserum (1:1000; a gift from Dr. H. Murofushi) overnight at
4 °C. Cells were visualized with a Vectastain ABC kit (Vector
Laboratories), followed by exposure to 0.02% 3-3'-diaminobenzidine
4-HCl, 0.3% H2O2 and 0.1%
(NH4)2Ni(SO4)2.
Immunoblotting--
Cells were lysed in SDS lysis buffer
containing 1% SDS, 20 mM Tris-HCl (pH 7.4), 5 mM EDTA (pH 8.0), 10 mM NaF, 2 mM
Na3VO4, 0.5 mM phenylarsine oxide,
10 mM Na4P2O4, and 1 mM phenylmethylsulfonyl fluoride. The lysates were boiled
for 3 min and then clarified by ultracentrifugation at 60,000 × g at 8 °C for 30 min. The protein concentration was
determined using a BCA protein assay kit (Pierce), and then aliquots of
10 µg of protein were resolved by electrophoresis on 10%
SDS-polyacrylamide gels. Proteins were transferred onto polyvinylidene
fluoride membranes (Millipore Corp.) in 0.1 M Tris base,
0.192 M glycine, and 20% methanol using a semi-dry
electrophoretic transfer system. The membranes were blocked with 0.1%
Tween 20, Tris-buffered saline (T-TBS) containing 5% nonfat dried milk
at room temperature for 1 h. Membranes were probed with 1:200
anti-phospho-JNK antibody, 1:200 anti-JNK antibody, 1:200
anti-phospho-p38 antibody, 1:1000 anti-p38 antibody, 1:200
anti-phospho-c-Jun antibody, or 1:1000 anti-c-Jun antibody in T-TBS
containing 1 or 5% nonfat dried milk at room temperature for 1 h.
After washing three times with T-TBS, the membranes were incubated with
horseradish peroxidase-conjugated goat anti-rabbit IgG or donkey
anti-mouse IgG secondary antibody (Zymed Laboratories
Inc. or Jackson ImmunoResearch Laboratories, Inc.) diluted 1:1000
with T-TBS at room temperature for 1 h. The membranes were then
washed at least 4 times with T-TBS and were visualized using the ECL
chemiluminescence system (Amersham Pharmacia Biotech) or Immunostar (Wako).
In Vitro Kinase Assay--
The cell lysates were prepared at 0, 3, 6, and 9 h after changing to LK medium using Triton lysis
buffer containing 1% Triton X-100, 20 mM Tris-HCl (pH
7.4), 150 mM NaCl, 5 mM EDTA (pH 8.0), 10 mM NaF, 2 mM Na3VO4,
0.5 mM phenylarsine oxide, 10 mM
Na4P2O4, 2 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride. Then 1 µg of anti-p38
antibody was added to the lysates followed by incubation at 4 °C for
at least 3 h. Protein G-Sepharose (10-µl gel) was then added and
rotated at 4 °C for 1 h. The immune complexes were pelleted by
centrifugation at 10,000 × g at 4 °C for 1 min and
then washed twice with Triton lysis buffer and twice with kinase buffer
containing 40 mM HEPES (pH 7.4), 10 mM
MgCl2, 3 mM MnCl2. p38 kinase
reaction was carried out for 20 min at 25 °C with 5 µg of GST-ATF2
(rat, residues 1-109) or GST-c-Jun (human, residues 1-125) as a
substrate and the immunoprecipitate in kinase buffer containing 10 µCi of [
-32P]ATP, 20 µM ATP. After
SDS-polyacrylamide gel electrophoresis, the incorporation of
32P into GST-ATF2-(1-109) or GST-c-Jun-(1-125) was
visualized using a Fuji BAS2000 image analyzer.
Antibodies and Reagents--
Anti-MAP2 antibody was kindly
provided by Dr. H. Murofushi. Anti-phospho-p38, anti-phospho-JNK, and
anti-JNK antibodies were purchased from New England Biolabs Inc., and
anti-p38, anti-phospho-c-Jun and anti-c-Jun antibodies were from Santa
Cruz Biotechnology Inc. SB203580 was obtained from Calbiochem and
dissolved at 10 mM in dimethyl sulfoxide
(Me2SO) as a stock solution.
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RESULTS |
LK-induced Apoptosis of Cerebellar Granule Neurons--
We
utilized primary cultures of cerebellar granule cells from neonatal
rats as a model system to investigate the intracellular signaling in
LK-induced cell death. This cell death shows characteristic features of
apoptosis (14, 15). After maturation for 4 days in HK medium, the
medium was switched to serum-free LK medium. At 24 h after
reduction of potassium concentration, cells were stained with anti-MAP2
antibody (Fig. 1A), and cell
survival was determined by MTT assay (Fig. 1B). As a result,
51.7% of granule neurons died in LK medium.

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Fig. 1.
LK-induced apoptosis of cerebellar granule
neurons. After maturation for 4 days, the medium was switched to
serum-free LK- or HK-MEM. After culture for 24 h, the granule
neurons were stained with anti-MAP2 antibody (A) and cell
survival was quantified by MTT assay (B). *,
p < 0.001
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The Phosphorylation and Expression of c-Jun in LK-induced Cell
Death--
Previous studies showed that the level of c-Jun
expression was increased, and the N-terminal phosphorylation of c-Jun
was necessary for the process of apoptosis (11, 19). Therefore, we
performed Western blotting analysis with monoclonal anti-phosphorylated c-Jun (Ser-63) antibody to detect the phosphorylation of c-Jun (Fig.
2A). Three hours after
changing to LK medium, the level of c-Jun phosphorylation was
increased, and this phosphorylation was maintained for 12 h. This
phosphorylated and activated c-Jun may form homo- or heterodimers with
c-Fos, and the dimerized c-Jun may up-regulate the expression of c-Jun
itself. The c-Jun up-regulation is known to be important in DNA
damage-induced apoptosis (20) and apoptosis induced by the PI3-K
inhibitor, LY294002, in cerebellar granule cells (21). Therefore, we
examined the level of c-Jun protein during LK-induced cell death using
a polyclonal anti-c-Jun antibody (Fig. 2B). Our results
indicated that the expression of c-Jun was markedly increased from 6 to
12 h after potassium deprivation. The delay in the changes in the
protein level compared with the phosphorylation of c-Jun was considered
to be due to the period required for transcription and translation of
c-Jun. In contrast, the phosphorylation and protein levels of c-Jun in serum-free HK-MEM were not changed from basal level.

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Fig. 2.
Phosphorylation and expression of c-Jun
during LK-induced apoptosis. After maturation for 4 days, the
lysates were prepared from granule neurons cultured for 0, 3, 6, 9, and
12 h in serum-free LK- or HK-MEM using SDS lysis buffer. The
lysates were immunoblotted with anti-phospho-c-Jun antibody
(A) or anti-c-Jun antibody (B). IB,
immunoblot.
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p38 Was Activated in LK-induced Apoptosis--
Next we
investigated what kind of kinase phosphorylates c-Jun in cerebellar
granule neurons. The first candidate was c-Jun N-terminal kinase (JNK)
that belongs to the MAP kinase superfamily and is activated by
phosphorylation on Thr-183 and Tyr-185. JNK activation is known to
participate in apoptosis of brain neurons and PC12 cells (22-24). In
cerebellar granule neurons, glutamate-induced exitotoxicity and DNA
damage activated JNK (7, 25). We examined whether JNK was activated to
phosphorylate c-Jun during LK-induced apoptosis. To examine JNK
activation, the lysates from granule cells cultured for 0, 3, 6, 9, and
12 h in serum-free LK or HK medium after maturation were
immunoblotted with anti-phospho-JNK (Fig.
3A). We could not detect any
increase in the JNK phosphorylation after lowering potassium
concentration. This result suggested that JNK is not involved in c-Jun
phosphorylation during LK-induced apoptosis of granule cells,
consistent with the observations of Watson et al. (11).

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Fig. 3.
p38 activation during LK-induced cell
death. After maturation for 4 days, the lysates were prepared from
granule neurons cultured for 0, 3, 6, 9, and 12 h in serum-free
LK- or HK-MEM, using SDS lysis buffer. The lysates were immunoblotted
(IB) with anti-phospho-JNK and anti-JNK antibody
(A) or anti-phospho-p38 and anti-p38 antibody
(B). C, the cell lysates were prepared at 0, 3, 6, and 9 h using Triton X lysis buffer. p38 MAP kinase was
immunoprecipitated with 1 µg of anti-p38 antibody. The kinase
reaction was carried out for 20 min at 25 °C with the p38
immunoprecipitate and 5 µg of GST-ATF2-(1-109) as a substrate in
kinase buffer. After SDS-polyacrylamide gel electrophoresis, the
32P incorporated into GST-ATF2-(1-109) was visualized
using a Fuji BAS2000 image analyzer (upper). The
immunoprecipitates were immunoblotted with anti-p38 antibody
(bottom).
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The next candidate was p38, which also belongs to the MAP kinase
superfamily. To examine whether p38 was activated during potassium
deprivation-induced apoptosis, lysates from granule cells cultured for
0, 3, 6, 9, and 12 h in serum-free LK or HK medium were
immunoblotted with anti-phospho-p38 antibody (Fig. 3B). p38
was markedly phosphorylated within 3 h, and the increased level of
phosphorylation was prolonged to 9 h after potassium deprivation.
To confirm this observation, we further performed in vitro
kinase assay of p38 (Fig. 3C). p38 was immunoprecipitated from the lysates with anti-p38 antibody, and the p38 kinase assay was
carried out with [
-32P]ATP using GST-ATF2-(1-109) as
a substrate. ATF2 was markedly phosphorylated by p38 3 h after
potassium deprivation. These results indicated that p38 is indeed
activated during LK-induced cell death.
Direct Phosphorylation of c-Jun by p38 in Vitro--
It is an
intriguing question whether p38 phosphorylates c-Jun. As shown in Figs.
2A and 3B, the time courses of phosphorylation of
p38 and c-Jun were very similar. To examine whether p38 is involved in
the regulation of c-Jun, we investigated the effects of SB203580, a
specific inhibitor of p38, on the phosphorylation and expression of
c-Jun. The addition of SB203580 to LK medium markedly suppressed the
phosphorylation and up-regulation of c-Jun in a
dose-dependent manner (Fig.
4A). Next, we examined whether p38 directly phosphorylated c-Jun using in vitro kinase
assay. After immunoprecipitation with anti-p38 antibody, the p38 kinase assay was carried out with [
-32P]ATP, using
GST-c-Jun-(1-125) as the substrate. At 3 h after potassium
deprivation, c-Jun was phosphorylated by p38 (Fig. 4B). These results suggested that p38 directly phosphorylates c-Jun during
LK-induced apoptosis of granule cells.

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Fig. 4.
Involvement of p38 in c-Jun phosphorylation
during LK-induced apoptosis. A, after maturation for 4 days, the lysates were prepared from granule neurons cultured for 0, 3, 6 and 9 h in serum-free LK- or HK-MEM with or without SB203580
using the SDS lysis buffer. The lysates were subjected to Western
blotting analysis using anti-phospho-c-Jun antibody (upper)
and anti-c-Jun antibody (bottom). B, the granule
neurons were cultured for 0, 3, 6 and 9 h after changing to
LK-MEM. The cell lysates were prepared using Triton-X lysis buffer. p38
was immunoprecipitated with 1 µg of anti-p38 antibody. The p38 kinase
reaction was carried out for 20 min at 25 °C with the
immunoprecipitate and 5 µg GST-c-Jun-(1-125) as a substrate in
kinase buffer. After SDS-polyacrylamide gel electrophoresis, the
32P incorporated into GST-c-Jun-(1-125) was visualized
using a Fuji BAS2000 image analyzer (upper). The
immunoprecipitates were immunoblotted with anti-p38 antibody
(bottom).
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SB203580 Prevented LK-induced Apoptosis--
To examine whether
the activation of p38 is involved in the process of LK-induced
apoptosis, we utilized SB203580, an inhibitor of p38. When apoptosis
was induced by lowering the potassium concentration, the inhibitor was
added to LK medium (Fig. 5). After
24 h, cell survival was quantified by counting the number of
MAP2-positive cells. Resultingly SB203580 inhibited neuronal apoptosis.
In the presence of 3, 10, and 30 µM SB203580, 59, 74, and
81% of cells survived, respectively. This result indicated that p38 is
involved in the signaling pathway of LK-induced apoptosis.

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Fig. 5.
Prevention of LK-induced cell death by
SB203580. After maturation for 4 days, the medium was switched to
serum-free LK or HK-MEM without (0 µM) or with 3, 10 or
30 µM SB203580. After culturing for 24 h, granule
neurons were stained with anti-MAP2 antibody (A) and cell
survival was quantified by counting MAP2-positive neurons
(B). *p < 0.005, **p < 0.001
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DISCUSSION |
Several recent studies have suggested that p38 is required for
some apoptotic processes (5, 6). For example, during glutamate-evoked
apoptosis of cerebellar granule neurons, p38 was transiently activated,
and this apoptosis was prevented by SB203580 (7). However, function of
p38 in apoptotic process remains unclear. In this study, we
investigated the role of p38 in LK-induced apoptosis of cultured
cerebellar granule neurons. Our results indicated that p38 was
phosphorylated and activated during LK-induced apoptosis (Fig. 3,
B and C). In Fig. 3B, control neurons
in HK medium also showed a transient and slight activation of p38. The
transient activation of p38 was considered to be due to changing from
serum-containing to serum-free HK-MEM.
Brain-derived neurotrophic factor (BDNF) and insulin-like growth
factor-1 (IGF-1) are known to rescue granule neurons from LK-induced
apoptosis (14, 26-27). We observed that BDNF and IGF-1 rescued about
60 and 80%, respectively, of neurons from cell death (data not shown).
Furthermore, the addition of BDNF and IGF-1 to LK medium suppressed the
activation of p38 similarly to control neurons in HK medium (data not shown).
The mRNA and protein levels of c-Jun were selectively increased
after NGF withdrawal in sympathetic neurons (10, 28). In cerebellar
granule neurons, Watson et al. (11) showed that dominant
negative c-JunAla, which cannot be phosphorylated,
inhibited LK-induced apoptosis. We observed marked changes in the
levels of phosphorylation and expression of c-Jun during this apoptotic
process. Although the importance of c-Jun is well known, its kinase has
remained unclear. The first candidate was JNK, because the
phosphorylation of c-Jun by JNK was well known in sympathetic neurons
(29). However, in cerebellar granule neurons, the JNK activity was high
under basal conditions and was not activated further during apoptosis. The next candidate was p38, because the time courses of the
phosphorylation of p38 and c-Jun during apoptosis were very similar. To
investigate the role of p38 in the phosphorylation of c-Jun, we
utilized SB203580. The addition of this inhibitor completely prevented
both phosphorylation and up-regulation of c-Jun (Fig. 4A).
In addition, in vitro kinase assay of p38 showed that p38
could directly phosphorylate c-Jun (Fig. 4B). Furthermore,
we found that SB203580 prevented LK-induced apoptosis in a
dose-dependent manner (Fig. 5). This prevention of
apoptosis seemed to be due to the inhibition of phosphorylation of
c-Jun. It has been reported that SB203580 can block some JNKs at high
doses (30-32). Since we could not detect low potassium-induced activation of JNKs, its contribution to the present findings is likely
to be limited but warrants further investigation. SB202190 is also
utilized as a specific inhibitor of p38 (33). We observed a weak but
significant inhibitory effect of SB202190 on the apoptosis (data not shown).
ATF2 is a member of the ATF/CREB family of basic region leucine zipper
(bZIP) DNA-binding proteins. The N-terminal transactivation domain of
ATF2 is phosphorylated by both p38 and JNK (34-36). Therefore, we
investigated whether ATF2 was also phosphorylated by p38 during LK-induced apoptosis. The phosphorylation of ATF2 was detected by
Western blotting analysis using anti-phospho-ATF2 antibody. ATF2 was
markedly phosphorylated at 3 h (data not shown). The time course
of ATF2 phosphorylation corresponded to those of phosphorylation of p38
and c-Jun. These observations suggested that p38 is involved in
phosphorylation of not only c-Jun but also of ATF2. We observed that
p38 showed greater affinity for ATF2 than c-Jun as a substrate in the
kinase assay utilizing the same amounts of GST-ATF2 and GST-c-Jun (data
not shown). In addition, we investigated whether SB203580 inhibited
phosphorylation of ATF2 as well as that of c-Jun. SB203580 indeed
inhibited ATF2 phosphorylation (data not shown). This result confirmed
that SB203580 suppresses the p38 pathway.
The results presented here demonstrate that p38 can directly
phosphorylate c-Jun during LK-induced apoptosis in cultured cerebellar granule neurons. However, the mechanisms of c-Jun-mediated apoptosis remain unclear. Therefore, further studies should be performed to
identify c-jun target genes and to determine how
c-Jun regulates apoptosis.