From the Neuroanatomy and Interdisciplinary Center for Neurosciences (IZN), University of Heidelberg, Im Neuenheimer Feld 307, D-69120 Heidelberg, Germany
Received for publication, October 1, 2002, and in revised form, December 16, 2002
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ABSTRACT |
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Growth differentiation factor-15 (GDF-15) is a
novel member of the transforming growth factor- Transforming growth factor- All TGF- We and others (12, 13) have recently cloned a novel member of the
TGF- Cultured cerebellar granule neurons (CGN) from postnatal rat represent
a highly homogeneous neuron population that provides an excellent
system to study neuronal apoptosis. CGN survive for weeks in
vitro and develop characteristics of mature CGN in vivo when maintained in depolarizing concentrations of K+ (25 mM), but undergo apoptosis when cultured in physiological low K+ (5 mM) conditions (17-19). Although
mechanisms underlying CGN apoptosis are not clear as yet, a requirement
of RNA/protein synthesis, generation of reactive oxygen species (ROS),
activation of caspases, and phosphorylation of c-Jun have been
implicated in this apoptotic model (18, 20, 21).
Our previous observation that GDF-15 is specifically up-regulated in
lesioned neurons prompted us to investigate pro- or anti-apoptotic effects of GDF-15 and underlying mechanisms in CGN. Here we demonstrate that (i) GDF-15 prevents death of CGN in low K+ by
activating Akt and down-regulating ERK, (ii) low K+-induced
ERK activation, in turn, induces c-Jun, which can be inhibited by
GDF-15, and (iii) GDF-15 mediated survival is accompanied by prevention
of low K+-induced ROS generation in CGN.
Materials--
Insulin-like growth factor-1 (IGF-1) was
purchased from Ciba Geigy (Basel, Switzerland). The Eagle's basal
medium with Earle's salts, glutamine, pencillin, streptomycin,
trypsin, phosphate-buffered saline, and Hanks' balanced salt solution
were from Invitrogen (Karlsruhe, Germany). Culture plates were
from Falcon. Fetal calf serum was obtained from Seromed (Berlin,
Germany). LY294002, wortmannin, rabbit polyclonal antibodies
(anti-phospho-Akt (Thr-308), anti-Akt, anti-phospho-p44/42 (ERK1/2),
mitogen-activated protein kinase (Thr-202/Tyr-204), anti-ERK1/2,
anti-phopho-c-Jun (Ser-63), anti-phospho-MEK1/2 (Ser-217/221),
anti-phospho-p-38 (Thr-180/Tyr-182), anti-phopho-JNK (Thr-183/Tyr-185)), and the anti-rabbit IgG-HRP conjugated antibody were from New England Biolabs Gmbh (Frankfurt, Germany). The anti-c-Jun (Santa Cruz Biotechnology, Inc., Heidelberg, Germany) antibody was a
gift from Prof. Kersten Krieglstein (Göttingen, Germany). The U0126, PD98059, dead end colorimetric apoptotic assay kit, and Cyto
Tox 96 assay for lactate dehydrogenase (LDH) measurements were from
Promega (Madison, WI). Polyvinylidene difluoride membrane and the ECL
chemiluminescence kit were from Amersham Biosciences Europe GmbH
(Freiburg, Germany). The 2',7'-dichlorodihydrofluorescein (DCF-H2) was from Molecular Probes. All other chemicals
were purchased from Sigma.
Expression of Recombinant Human GDF-15--
Full-length GDF-15
cDNA was cloned and sequenced as described previously (13).
Recombinant human GDF-15 protein was expressed in baculovirus-infected
insect SF9 cells and purified from supernatants as described previously
(15). Protein extracts of uninfected cells were treated under the same
conditions and used in parallel for controls.
Cell Cultures--
Cerebellar granule neurons were isolated and
cultured from 8 day old Wistar rats as described previously (22) with
slight modifications. Briefly, the freshly dissected cerebellum was
trypsinized and triturated in ice cold Ca2+- and
Mg2+-free Hanks' balanced salt solution. The cells were
resuspended in high K+ medium (Eagle's basal medium
containing 10% fetal calf serum, 25 mM KCl (Sigma), 2 mM glutamine, and 0.5% (v/v) pencillin/streptomycin). Cells were seeded in poly-L-lysine (100 µg/ml, Sigma)
precoated wells in 12-96-well plates at an average density of 1,500 cells/mm2. Cultures were incubated at 37 °C with 5%
CO2 in a humidified chamber. Cytosine arabinoside (10 µM, Sigma) was added after 24 h in vitro
to prevent proliferation of non-neuronal cells. On day 4 in
vitro the culture dishes were switched from high K+ to
low K+ medium (Eagle's basal medium, 5 mM KCl,
2 mM glutamine, and 0.5% pencillin/streptomycin;
"5K"). Depending on the experiment at the time of medium change
the cells in 5K were treated in the presence or absence of stimulants
for indicated time points and were processed for the assays described
below. All the cell death analyses were performed after 24 h of treatment.
LDH Measurements--
LDH activity was assayed using an LDH
assay kit (Promega). Total LDH release (percent) was calculated from
maximum release, defined as the amount of LDH obtained after exposure
of 25 mM K+ cultures to Triton X-100, 0.1% for
10 min at 37 °C.
Propidium Iodide Staining--
Propidium iodide (PI) was used to
determine numbers of dead cells. PI (4.6 µg/ml) was added directly to
the culture medium for 3 min at room temperature, and the cells
were counted after fixation with 4% paraformaldehyde. Typically around
70-300 PI-stained cells were counted in four randomly selected fields
per dish. Numbers of PI-stained cells in low K+ medium were
normalized to 100%. Cell counts were presented as percentages of low
K+-induced cell death.
TUNEL (Terminal Deoxyuridynyltransferase Nick End Labeling)
Assay--
The TUNEL assay was performed according to the
manufacturer's manual (Promega). Around 500 cells in four different
fields consisting of ~20-180 TUNEL-positive cells were counted per
coverslip under phase contrast microscope. Cell numbers were converted
into percentage of apoptotic cells calculated from total cell numbers.
Reactive Oxygen Species (ROS) Measurement--
ROS measurements
were essentially carried out as described in Ref. 20. The culture
plates were read on Fluostar OPTIMA plate reader (BMG Labtechnologies,
Offenburg, Germany) at 480 nm excitation and 520 nm emmision. Neurons
grown in high K+ showed negligible fluorescence and were
used for background fluorescence.
Western Blot Analysis--
Neurons cultured in 12-well plates
were harvested in 1× SDS lysis buffer containing 62.5 mM
Tris-HCl (pH 6.8), 2% w/v SDS, 10% glycerol, 50 mM
dithiothreitol, and 0.01% w/v bromphenol blue. Cell lysates
containing equal amounts of protein (25 µg/lane) were loaded on 10 or
12% SDS-polyacrylamide gels. The separated proteins were transfered
onto a polyvinylidene difluoride membrane using a wet transfer system
(Amersham Biosciences). The membranes were blocked with blocking
buffer containing 5% dry milk in Tris-buffered saline with Tween 20 (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% Tween 20) and incubated overnight with different primary antibodies. Incubations with secondary antibodies were performed for 1 h, and
the bound antibodies were detected with the enhanced chemiluminescence (ECL, Amersham Biosciences) reagents in accordance to the
manufacturer's protocol. Densitometric quantification of Western blots
was done using NIH image (version 1.61) software.
Statistical Analysis--
Data were expressed as means ± S.E. (n = 3). All experiments were performed in
triplicates or duplicates and repeated at least three times.
Statistical significance was analyzed by Student's t test
(Statview 4.01). p values are *, p < 0.05;
**, p < 0.01; ***, p < 0.001.
GDF-15 Prevents Cell Death of CGN Induced by Low
K+--
We first assayed the effect of GDF-15 on CGN
viability by monitoring the release of LDH into the culture medium.
Fig. 1A (left panel) shows that GDF-15 significantly decreased release of LDH from CGN induced by low K+ (5K) at an optimal concentration
of 10 ng/ml. To further substantiate the protective effect of GDF-15,
we quantified neuron death by counting PI-stained (dead) cells. Fig.
1A (right panel) documents that GDF-15 maximally
reduced numbers of PI positive neurons at a concentration of 10 ng/ml.
TUNEL stainings corroborated these findings by showing (Fig.
1B) an increase in the number of apoptotic cells with DNA
fragmentation in low K+, which was prominently decreased by
GDF-15. Quantitative evaluations revealed a more than 30% increase in
numbers of TUNEL positive cells after 24 h under low
K+ conditions. However, the cultures maintained in
serum-free high potassium showed only ~4% TUNEL-positive cells. In
the presence of IGF-1, an established anti-apoptotic molecule for CGN
following K+ withdrawal (18, 23), cell death was reduced to
10%. GDF-15 reduced numbers of apoptotic neurons in low K+
to 15%. Together, these data indicate that GDF-15 is a potent neuroprotective factor for CGN that matches the effect of IGF-1, the
best established protective factor in this culture system.
Effects of PI3K and MEK1/2 Inhibitors on GDF-15-induced
Neuroprotection--
To begin to analyze the signaling cascades
activated by GDF-15, we chose specific inhibitors that inactivate core
units of different signaling pathways. Inhibition of PI3K, which
directs activation of Akt, has been shown to abolish protective effects of growth factors in CGN (24, 25). We used two selective inhibitors of
PI3-kinase, wortmannin and LY294002, to block the activity of the
enzyme in CGN. To determine whether GDF-15-induced survival is impaired
by PI3K inhibitors, we first performed LDH measurements (Fig.
2A, left panel) and
counted PI-stained cells (Fig. 2A, right panel).
The protective effect of GDF-15 was significantly reduced in wortmannin
(100 nM)- or LY294002 (50 µM)-pretreated
cultures, indicating that PI3K activation is important for GDF-15
signaling. To examine the effect of inhibitors on DNA fragmentation, we
performed TUNEL stainings. Fig. 2B shows an increased number
of TUNEL-positive neurons in the presence of GDF-15 plus wortmannin
compared with GDF-15 alone. However, GDF-15 protection was only partly
(about 50%) attenuated by inactivation of the PI3K, suggesting that
GDF-15 may employ additional mechanisms to protect CGN.
Activation of extracellular signal-regulated protein kinases (ERKs) has
been reported to contribute to neuronal cell survival in certain models
of neurotoxicity (26-28). As our data indicated that GDF-15 is a
potent protective factor for CGN, we hypothesized that regulation of
ERK phosphorylation might be involved in GDF-15-induced survival.
ERK1/2 activity is exclusively regulated through MEK1/2 phosphorylation, an immediate upstream dual-specificity kinase (29). We
used U0126 (20 µM), which blocks MEK1/2 (25), and PD98059
(60 µM), a specific blocker of MEK1 (30), to inhibit ERK
phosphorylation. Fig. 2, A and B, show that
inactivation of ERKs did not reverse the survival promoting effect of
GDF-15. On the contrary, neuronal protection with GDF-15 was even more pronounced in the presence of U0126 (20 µM) or PD98059
(60 µM) as measured by LDH release and numbers of
PI-stained cells. To address the question whether MEK inhibition alone
is sufficient to promote CGN survival in low K+, we applied
U0126 or PD98059 without GDF-15. Fig. 2A provides evidence
that both inhibitors promoted survival, although U0126 showed a
somewhat higher protection compared with PD98059. To examine whether
the MEK1/2 inhibitor increased cell viability by inhibiting DNA
fragmentation, we performed TUNEL stainings and quantified the data. As
shown in Fig. 2B, the MEK1/2 inhibitor U0126 significantly
decreased numbers of TUNEL positive cells. These results suggest that
low K+-induced apoptosis in CGN activates MEK1/2 during
neuron death and that inhibition of this pathway clearly attenuates
cell death.
GDF-15 Induces Akt and GSK-3 Phosphorylation--
The
serine/threonine protein kinase Akt is a downstream effector of PI3K
and has been reported to play a critical role in promoting the survival
of a variety of different cell types (for reviews, see Refs. 31 and
32). As the PI3K inhibitor blocked GDF-15-mediated CGN survival we
investigated whether GDF-15 induces Akt phosphorylation in CGN cultured
in low K+ (5K) conditions. As shown in Fig.
3A, GDF-15 significantly
increased levels of phosphorylated Akt within 5 min after factor
administration. This effect was still detectable after 24 h and
decreased to basal level by 48 h (Fig. 3A). GSK-3 is a
critical downstream element of the PI3K/Akt cell survival pathway, and
its activity can be inhibited by Akt-mediated phosphorylation at Ser-21
of GSK-3 GDF-15 Blocks MEK/ERK Signaling in Low K+
CGN Cultures--
The neuroprotective effect of the MEK inhibitors
U0126 and PD98059 on CGN maintained in low K+ alone
suggests that ERKs may be activated in this system at a certain time
point during apoptosis. To investigate this hypothesis, lysates
obtained at various time points from low K+ CGN culture
were subjected to Western blot analysis to detect phosphorylated
(activated) ERK using an anti-phospho-ERK1/2 antibody. The same blot
was stripped and reprobed to detect total ERK level. As shown in Fig.
4A, activation of ERK1/2
became apparent at about 6 h of K+ deprivation, and
the signal was maintained for 24 h, declined at 48 h, and
remained elevated compared with 0 or 4 h (not shown). Although
timing of robust ERK activation varied slightly between different
culture batches, it was consistently observed between 4 and 6 h
after K+ deprivation. There was no change in the level of
total ERK. The MEK1/2, upstream activators of ERK, were also
phosphorylated with the same kinetics as seen for ERK (Fig.
4A). ERK phosphorylation was completely blocked when
cultures were preincubated with the MEK inhibitor U0126 (20 µM) or PD98059 (60 µM) without affecting total ERK levels (Fig. 4B). To test whether the cell
death-preventing effect of MEK inhibition is
dose-dependent, we analyzed cell death and ERK inhibition
using various concentrations of the potent MEK1/2 inhibitor U0126.
U0126 inhibited cell death as well as ERK phosphorylation in a
dose-dependent manner. As shown in Fig. 5A, the protective effect was
maximal between 5 and 20 µM, the concentrations at which
ERK phosphorylation was strongly inhibited without affecting total ERK
levels (Fig. 5B). Higher concentrations of U0126 were toxic
to CGN.
To address the question whether GDF-15 may prevent cell death by
down-regulating phosphorylation of ERKs, we performed Western blot
analysis using cell lysates prepared after various time points of
GDF-15 (20 ng/ml) administration. As shown in Fig.
6A, sustained ERK activation
observed in control cultures (5K + BC) was markedly suppressed by GDF-15 without affecting total ERK levels.
Phosphorylation of MEK1/2 was also affected by GDF-15 (Fig.
6A). However, GDF-15 did not completely inhibit ERK
activation, while U0126 did (cf. Fig. 4B). In the
presence of both GDF-15 and U0126 suppression of cell death was more
pronounced than by individual treatments (Fig. 6B). We
hypothesized that this could be due to a complete suppression of the
ERK cell death pathway by U0126 and an activation of the Akt survival
pathway by GDF-15. To challenge this hypothesis we performed Western
blot analysis using cell lysates isolated 24 h after GDF-15 (20 ng/ml) treatment in the presence and absence of U0126 (20 µM). Fig. 6B shows a complete suppression of
ERK phosphorylation, while Akt remained phosphorylated in the presence of both GDF-15 and U0126.
MEK1/2 Inhibitor and GDF-15 Block Phosphorylation of
c-Jun--
The c-Jun protein and its phosphorylation have been
shown to be involved in low K+-induced cell death (19, 21).
Given that ERK can phosphorylate c-Jun in vitro (34),
together with our observation that there is a slow and sustained ERK
activation in low K+, we hypothesized that ERK could be a
potential upstream regulator of c-jun. We performed Western
blots using cell lysates obtained from various time points after
K+ deprivation and probed them with phospho-specific c-Jun
and total c-Jun antibodies. As shown in Fig.
7A, c-Jun phosphorylation
increased slightly at 3 h after K+ deprivation, yet
both protein and phosphorylation increased robustly and in parallel to
ERK activation at 6 h and remained high for 24 h. Evidence
that ERK regulates c-Jun protein and phosphorylation in low
K+-induced apoptosis was obtained from ERK inhibition
studies. The potent inhibitor of MEK/ERK signaling, U0126, was added to
low K+ (5K) cultures for 9, 12, and 24 h, and lysates
were subjected to Western blot analysis. Fig. 7A shows that
in the presence of U0126 (20 µM) c-Jun phosphorylation
was dramatically reduced, and the protein level was markedly decreased.
Interestingly, GDF-15, which attenuated ERK phosphorylation, also
markedly reduced c-Jun phosphorylation and protein levels. (Fig.
7A).
The stress-activated kinases, JNK and p38, are known to regulate c-Jun
gene expression and phosphorylation (35-37). In accordance with
previous studies (21, 38), we observed a high activation of JNK and a
low activation of p38 in low K+ that were not affected by
U0126 or GDF-15 treatments (Fig. 7B). Thus, activation of
ERK regulates c-Jun protein synthesis and phosphorylation in low
K+ CGN cultures. Treatment with either U0126 or GDF-15
abolishes this signaling and protects neurons from cell death.
The Anti-apoptotic Effect of GDF-15 Inhibits ROS
Formation--
ROS generated by oxidative stress are known to be
important cell death effector molecules in low K+ CGN
cultures (20, 39). We first studied the time course of ROS generation
in low K+ cultures using the fluorescent ROS indicator dye
DCF-H2. DCF-H2 gets oxidized to DCF by ROS,
which, upon excitation, emits fluorescence (40, 41). Fig.
8A shows two peaks in ROS
formation at 6 and 12 h and return to base-line levels at 24 h. We next investigated ROS generation after 6 h in low
K+ in the presence of GDF-15 (20 ng/ml). As shown in Fig.
8B, GDF-15 effectively prevented ROS generation. The MEK
inhibitors (PD 98059 (60 µM) and U0126 (20 µM)) did not affect DCF fluorescence, corroborating the
notion that ROS generation is upstream of ERK activation.
GDF-15 is a novel divergent member of the TGF- We next analyzed whether the MEK/ERK pathway may be involved in GDF-15
dependent neuron rescue. Surprisingly, we found that GDF-15-induced
protection in CGN was even more pronounced when ERKs were inactivated
via selective inhibition of MEK. Moreover, MEK inactivation alone led
to a potent protection against low K+-induced apoptosis of
CGN. We observed slow and sustained ERK activation in CGN undergoing
low K+-induced cell death. Down-regulation of ERK by MEK
inhibitors led to a prevention of low K+-induced apoptosis.
These findings corroborate several recent reports showing a
pro-apoptotic involvement of ERK in neuronal cell death (58, 59).
Moreover, recent studies also demonstrate that
L-2-chloropropionate- and kainate-mediated cell death in CGN (60, 61) is attenuated by MEK inhibition. Most importantly, GDF-15
clearly suppressed the persistent ERK activity in CGN. Prevention of
cell death by MEK inhibitors is unexpected, because typically the ERK
pathway has been credited with proliferative and cell survival
responses (62, 63). However, our results together with the above
studies expand the functional role of ERKs and suggest that the
duration of ERK activation might determine pro- or anti-apoptotic
decision of the cell.
The AP-1 transcription factor family members c-fos
and c-jun have been implicated with pro-apoptotic functions
(see Ref. 64 for a review). The role of c-Fos in low
K+-induced CGN cell death is not known. A requirement of
c-Jun activation in mediating low K+-induced CGN apoptosis
has previously been demonstrated by Watson et al. (21). The
present study has provided evidence that c-Jun phosphorylation and
protein levels increase in parallel to the increase in ERK activation.
Notably, MEK1/2 inhibition or treatment with GDF-15, which both inhibit
ERK activation, also inhibited c-Jun protein up-regulation and
phosphorylation. Although the in vivo role of ERK in
regulating c-Jun has yet to be determined (65), evidence that c-Jun can
be phosphorylated by ERK in vitro (34) and our finding that
MEK1/2 inhibition attenuates the increase in c-Jun protein and
phosphorylation argue in favor of the possibility that ERK may be
directly involved in regulating c-jun in low K+
CGN culture. It is also worthwhile noting that the MEK1/2 inhibitor U0126 was originally characterized based on its ability to inhibit AP-1
transcription elements c-jun and c-fos via the
inhibition of MEK/ERK signaling cascade (29). Furthermore, the
demonstration that kainate-induced toxicity involves c-Jun activation
(66), as well as the ability of MEK inhibitors to prevent kainate
toxicity in CGN (61), may further strengthen the possible role of ERK activation in modulating c-Jun activity. GDF-15-induced inhibition of
c-Jun indicates that the factor prevents cell death by down-regulating a key signal in the mediation of cell death.
Whether GDF-15-mediated inhibition of c-Jun transmits to other
important regulators of cell death remains open. Possible target molecules with roles in cell cycle control and apoptosis are cell cycle-regulated protein kinases (67). Several studies have demonstrated a MEK/ERK-mediated regulation of the cyclin-dependent
kinases (CDKs) cyclin D1, cyclin E, and cyclin kinase inhibitors p21
and p27. Activation of CDKs and cyclin kinase inhibitors can result in
cell cycle arrest or apoptosis (68-70). In the nervous system CDKs
have been shown to be involved in the death of PC12 cells and
postmitotic neurons (71-74). For example, serum-deprived neuronal PC12
cells and sympathetic neurons deprived of trophic support contain
inappropriate amounts of cyclin B and cyclin D transcripts prior to
death (75, 76). Most importantly, Padmanabhan et al. (77)
documented the requirement of CDK proteins in low
K+-induced CGN cell death. They demonstrated an
up-regulation of cyclin D1 and cyclin E after 6 h in low
K+. This is consistent with the late and persistent ERK
induction observed in our studies. Although the mechanisms underlying
CDK-activated neuronal cell death are still unknown, non-neuronal cells
have provided model systems for the control of cell death via the
MEK/ERK signaling pathway. The ability of MEK inhibitors to regulate
CDKs and cyclin kinase inhibitor proteins (70, 78, 79), the presence of
a consensus AP-1 promoter sequence in cyclin D1, and its direct induction by c-Jun (67) suggest that the strong MEK/ERK/c-Jun signals
observed in this study may direct the regulation of cell cycle protein
kinases for the orchestration of neuronal cell death.
To elucidate the GDF-15-mediated mechanisms that suppress ERK
activation in CGN, we continued by documenting that the protective effect of GDF-15 is connected to the regulation of ROS. It has been
reported that increased generation of ROS is closely associated with
low K+-induced cell death (20, 39), and several studies
have provided evidence that ROS induces ERK activation (80-82). We now
show for the first time that GDF-15 inhibits the formation of ROS.
Furthermore, the inability of both MEK inhibitors to prevent ROS
generation suggests that ROS generation is upstream of ERK activation
in low K+ CGN. It should be noted, however, that in other
systems, as superior cervical ganglionic neurons, nerve growth
factor can prevent ROS generation by activating the ERK pathway
(83), suggesting that ROS formation in this system is downstream of
ERK.
The present in vitro work has provided clues for the
possible signaling pathways that are activated by GDF-15 for executing its functions. Activation of Akt is known to play a critical role in
controlling the balance between survival and apoptosis (84, 85). The
discovery that ERK inhibition promotes survival was surprising but
needs to be discussed in the context of the putative in vivo
relevance of this finding. Recent in vivo studies have revealed that inhibition of ERK can reduce infarct volume after focal
cerebral ischemia, suggesting a deleterious effect of ERK activation
(86, 87). In addition, an abnormal punctate staining pattern of
activated ERK has been described in the brains of patients with
Alzheimer's disease (88). While the status of ERK activation in
patients with Parkinson's disease is currently unknown, the involvement of ERK in 6-hydroxydopamine toxicity (89), and our previous in vivo studies, in which we demonstrated potent
protective effects of GDF-15 in 6-hydroxydopamine-lesioned rat
brains (15), suggest that the GDF-15 may exert its protective effect in
neurons through an ERK-inactivating mechanism.
In conclusion, we have demonstrated that GDF-15 prevents cell death in
low K+ CGN cultures by two different modes of action.
First, GDF-15 induces survival by activating Akt and GSK via the PI3K.
Second, GDF-15 down-regulates the MEK/ERK/c-Jun cell death signaling
pathway. Moreover, GDF-15 also attenuates ROS formation, an inducer of ERK, thereby preventing cell death. Putative cross-talks between these
pathways need to be addressed by future experiments. An important issue
also to be resolved in the future concerns the identity of GDF-15
receptors. Finally, the question whether GDF-15 may act as a regulator
of apoptosis for CGN in vivo remains to be answered. Ongoing
studies with a GDF-15-deficient lacZ knock-in transgenic mouse, recently developed in our laboratory, may help to
address this question.
superfamily
and has been shown to be induced in neurons subsequent to lesions. We
have therefore begun to study its putative role in the regulation of
neuron survival and apoptosis. Cultured cerebellar granule neurons
(CGN) survive when maintained in high K+ (25 mM) but undergo apoptosis when switched to low
K+ (5 mM). GDF-15 prevented death of CGN in low
K+. This effect could be blocked by phosphatidylinositol
3-kinase/Akt pathway inhibitors LY294002 or wortmannin. In contrast,
mitogen-activated protein kinase (MEK)/extracellular-signal-regulated
kinase (ERK) pathway inhibitors U0126 and PD98059 potentiated GDF-15
mediated survival and prevented cell death in low K+ even
without factor treatment. Immunoblots revealed GDF-15-induced phosphorylation of Akt and glycogen synthase kinase-3
. This
activation was suppressed by phosphatidylinositol 3-kinase inhibitors.
Low K+ induced delayed and persistent ERK activation, which
was blocked by MEK inhibitors or GDF-15. ERK activation induced
c-Jun, a member of the AP-1 transcription factor family. GDF-15
or U0126 prevented c-Jun activation. Furthermore, we show that GDF-15
prevented generation of reactive oxygen species, a known activator of
ERK. Together, our data suggest that GDF-15 prevents apoptosis in CGN
by activating Akt and inhibiting endogenously active ERK.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
s
(TGF-
s)1 comprise a
superfamily of contextually acting cytokines with a broad array of
biological activities. Depending on the developmental stage and cell
type TGF-
s regulate diverse processes including development, cell proliferation, differentiation, survival, and death (1-4). Members of
the TGF-
superfamily can be subdivided into subfamilies of structurally closer related proteins, including the TGF-
s 1-3, bone
morphogenetic proteins, the growth/differentiation factors (GDFs), activins, and the glial cell line-derived neurotrophic factor
(GDNF) family (5-7).
famliy members, except members of the GDNF subfamily,
signal through heteromeric complexes of type I and type II serine/threonine kinases receptors. Within the type I and type II
receptor families there are several subgroups, e.g. bone
morphogenetic protein receptors, depending on their respective ligands
(for reviews, see Refs. 7 and 8). GDNF, neurturin, persephin, and
artemin signal through a heteromeric receptor complex consisting of the receptor tyrosine kinase Ret (9, 10) and
glycosylphosphatidylinositol-linked
-receptors (11).
superfamily, GDF-15/macrophage inhibitory cytokine-1 (MIC-1).
The protein does not belong to one of the known TGF-
subfamilies and
represents a divergent member of the TGF-
superfamily. GDF-15 is
widely synthesized in the central nervous system, most strongly
expressed in the choroid plexus, and secreted into the cerebrospinal
fluid, from where it may penetrate into the brain parenchyma (14-16).
We have previously shown survival promoting effects of GDF-15 on
unlesioned and intoxicated midbrain dopaminergic neurons in
vitro and in vivo (15). Serotonergic neurons of the embryonic rat raphe were one additional neuron population that responded to GDF-15 by an increase in transmitter synthesis and uptake.
However, motoneurons of the spinal cord and sensory dorsal root
ganglionic neurons did not respond at all or only very moderately to
GDF-15. At this point, it is not clear whether other types of central
nervous system neurons are affected by GDF-15. In a previous study, we
had also shown that GDF-15 protein is up-regulated in cortical neurons
subsequent to a cold lesion (16). This may indicate functions of the
protein in neurons affected by injury and suggest a role of GDF-15 in
the execution of either survival or cell death programs.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
GDF-15 prevents low K+
(5K)-induced cell death.
A, GDF-15 significantly decreases LDH release (left
panel) and the number of propidium iodide-stained (right
panel) CGN induced by 5K in a concentration-dependent
manner. B, GDF-15 decreases 5K-induced apoptosis. TUNEL
quantification data (left panel) and representative
photomicrographs are shown (right panel). Cultures were
treated, and measurements were performed as described under
"Experimental Procedures." Significance was calculated at
p < 0.05 (*), p < 0.01 (**), and
p < 0.001 (***) compared with 5K cultures.
BC, purified supernatant of uninfected Sf9 cells.
Control cultures were treated with IGF-1 (25 ng/ml).
View larger version (55K):
[in a new window]
Fig. 2.
Effect of PI3K and MEK inhibitors on
GDF-15-induced neuronal protection. A,
GDF-15-dependent effects on LDH release (left
panel) and propidium iodide staining (right panel) in
low K+ cultures (5K) are attenuated in the
presence of the PI3K inhibitors wortmannin (W, 100 nM) and LY294002 (LY, 10 µM). MEK
inhibitors U0126 (20 µM) or PD98059 (PD, 60 µM) alone significantly decrease LDH release (left
panel) and the percentage of propidium iodide-stained cells
(right panel). Protection is even more pronounced in the
presence of both MEK inhibitors and GDF-15 (left and
right panels). B, effect of inhibitors on
GDF-15-mediated survival. The left panel shows the TUNEL
quantification data for the mentioned groups. The right
panel shows the photomicrographs of TUNEL-stained cells.
Measurements and cell counts were performed as described under
"Experimental Procedures." , no inhibitor; DMSO
(dimethyl sulfoxide), vehicle control. *, significance compared with 5K
cultures. °, significance compared with 5K + GDF-15 cultures.
Significance was calculated at p < 0.05 (*/°),
p < 0.01 (**/°°), and p < 0.001 (***).
and Ser-9 of GSK-3
(33). We used an
anti-phospho-GSK-3
(Ser-9) antibody to detect phosphorylated
GSK-3
. As shown in Fig. 3A, GDF-15-induced
phosphorylation of GSK-3
was paralleled by an activation of Akt.
Moreover, preincubation of the cultures with the PI3K inhibitors
wortmannin (100 nM) or LY294002 (10 µM)
markedly blocked the GDF-15-induced Akt and GSK3 phosphorylation (Fig. 3B). Total Akt levels were not changed (Fig. 3, A
and B). These data corroborate our findings that GDF-15
rescues CGN from cell death via the PI3K/Akt survival pathway.
Moreover, the rapid phosphorylation of Akt/GSK within 5 min after
factor administration points to a direct effect of GDF-15.
View larger version (46K):
[in a new window]
Fig. 3.
GDF-15 induces phosphorylation of Akt and
GSK-3 in low K+
(5K) cultures. A, after treating the
cells with GDF-15 (20 ng/ml) for the indicated time points the lysates
were prepared and processed for immunoblot using antibodies against
phosphorylated Akt (p-Akt) or GSK-3
(p-GSK).
The same blot was stripped and reprobed to detect total Akt
(Akt). B, GDF-15 induces phosphorylation of Akt
and GSK via PI3K. Cells were pretreated with PI3K inhibitors wortmannin
(W, 100 nM) or LY294002 (LY, 10 µM) for 30 min before stimulating with GDF-15 (for 1 h). PI3K inhibitors attenuate GDF-15-induced Akt and GSK
phosphorylation. The same blot was stripped and reprobed for total Akt.
BC, control lysate of cultures treated with purified
supernatant of uninfected Sf9 cells. The quantification of the
intensity was depicted below the respective blots.
View larger version (39K):
[in a new window]
Fig. 4.
Low K+ (5K)
induces slow and sustained MEK/ERK activation. A, after
switching the medium to 5K for the indicated time points the cells
lysates were prepared and processed to detect phosphorylation of ERK1/2
(p-ERK) by Western blotting. The same blot was stripped for
the detection of total ERK (ERK). Similarly, the
representative lysates were processed for Western blot analysis to
detect phosphorylation of MEK1/2 (p-MEK). B, MEK
inhibitors attenuate ERK activation in 5K cultures. Immediately after
changing the medium to 5K the cells were treated with 20 µM U0126 or 60 µM PD98059 for the indicated
period of time. The lysates were processed for the detection of
phosphorylated ERK, and the same blot was stripped for the detection of
ERK. DMSO (dimethyl sulfoxide), vehicle control for
inhibitors. The quantification of the intensity was depicted below the
respective blots.
View larger version (22K):
[in a new window]
Fig. 5.
MEK1/2 inhibitor U0126 decreases cell death
and ERK activation in a dose-dependant manner. A,
different concentrations of U0126 were added to the culture immediately
after switching the medium to low K+ (5K). Cell
death was measured after 24 h by staining with propidium iodide.
Significance was calculated at p < 0.05 (*) or
p < 0.01 (**) compared with untreated cells.
B, U0126 inhibits ERK phosphorylation in a
dose-dependent manner. U0126 was added to the 5K cultures
immediately after switching to 5K medium. After the above mentioned
time points cell lysates were assessed for phosphorylation of ERK
(p-ERK), and the same blot was stripped and reprobed for
total ERK (ERK). DMSO (dimethyl sulfoxide),
vehicle control for inhibitors. The quantification of the intensity was
depicted below the respective blots.
View larger version (42K):
[in a new window]
Fig. 6.
A, GDF-15 blocks low K+
(5K)-induced sustained ERK activation. GDF-15 was added to
5K cultures for the indicated time points, and the cells were lysed and
processed for Western blot analysis to detect phosphorylated ERK
(p-ERK). The same blot was stripped to detect total ERK
(ERK). Similarly, the representative lysates were processed
to detect phosphorylation of MEK1/2 (p-MEK). B,
GDF-15- and U0126-treated low K+ (5K) cultures show active
Akt. The cells in 5K were treated with GDF-15 (20 ng/ml) and/or U0126
(20 µM) for 24 h. The cell lysates were processed
for Western blotting to detect phosphorylated ERK and ERK. The
representative samples were processed to detect phosphorylation of Akt
(p-Akt). , no treatment; BC, control lysate of
cultures treated with purified supernatant of uninfected Sf9
cells. The quantification of the intensity was depicted below the
respective blots.
View larger version (41K):
[in a new window]
Fig. 7.
GDF-15 and U0126 inhibit c-Jun protein
expression and phosphorylation. After switching to low
K+ (5K) medium the cells were treated with U0126
(20 µM) or GDF-15 (20 ng/ml) for the above mentioned time
points. The cells were lysed and processed for Western blotting.
A shows the Western blot analysis using antibodies against
phosphorylated c-Jun (p-c-Jun), total Jun
(c-Jun), and phosphorylated ERK (p-ERK) and the
densitometric quantification of p-c-Jun and c-Jun levels for 9, 12, and
24 h. B shows Western blot analysis using antibodies
against phosphorylated JNK (p-JNK) and phosphorylated p38
(p-p38) and the densitometric quantification of pJNK
levels for 9, 12, and 24 h.
View larger version (18K):
[in a new window]
Fig. 8.
A, low K+ (5K)
CGN cultures generate ROS. The time course of ROS production
in low K+ as detected by DCF fluorescence was measured as
described under "Experimental Procedures." B, effect
of GDF-15 and MEK inhibitors on ROS generation. After switching the
culture to low K+ (5K) medium, GDF-15 (20 ng/ml), U0126 (20 µM), or PD98059 (60 µM)
were added and DCF fluorescence was measured after 6 h as
described under "Experimental Procedures." *, p < 0.05 compared with 5K cultures.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
superfamily with
largely unknown functions. Recent studies demonstrated that the
molecule is expressed in the central nervous system, and the first
functional characterization of the factor revealed pronounced neurotrophic effects on midbrain dopaminergic and raphe
serotonergic neurons in vitro and in vivo (15).
However, it is not clear whether other central nervous system neuron
populations are also promoted by GDF-15. TGF-
s can affect neurons in
both anti- and pro-apoptotic fashions. Thus, elimination of TGF-
in
chick embryos has been shown to abolish ontogenetic neuron death (36,
42) acting as a master molecule in the regulation of pro-apoptotic signaling cascades. In contrast, TGF-
can also synergize with neurotrophic factors, e.g. neurotrophins, fibroblast growth
factor-2, and GDNF, in the promotion of neuron survival (43-47). With
regard to GDF-15, a pro-apoptotic role in several cell lines has been suggested (48-51). Moreover, our observation that GDF-15 is robustly induced in lesioned cortical neurons (16) can be interpreted in terms
of both an anti- or pro-apoptotic role. To clarify this, we started to
analyze the effect of GDF-15 and underlying mechanisms on neuronal
apoptosis using the well established CGN model (18). Reverse
transcriptase-PCR analysis showed that cultured granule neurons
express GDF-15, but expression is down-regulated upon withdrawal of
survival promoting signals (not shown). The present study demonstrates
that GDF-15, in contrast to TGF-
2, which has been shown to
accelerate apoptosis in CGN (52), prevents primary CGN from low
K+-induced apoptosis. Concerning underlying mechanisms, we
first showed that the protective effect of GDF-15 was attenuated by inhibitors of the Akt-activating PI3K. Western blot analyses clearly confirmed the functional results and demonstrated a
GDF-15-dependent activation of Akt and its downstream
target GSK-3
. Consistent with our data is the observation that other
TGF-
members, including GDNF, neurturin, persephin, and TGF-
1,
promote cell survival via the PI3K/Akt pathway in various cell types
(53-57).
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ACKNOWLEDGEMENTS |
---|
We thank Jutta Fey, Gerald Bendner, and Marion Schmitt for excellent technical assistance; Thomas Fath and Dr. Neelam Shahani from the department of Neurobiology, University of Heidelberg, for providing the Fluostar OPTIMA plate reader. We also thank Alan Summerfield for assisting in graphics and Dr. Heike Peterziel for critically reading the manuscript.
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FOOTNOTES |
---|
* This work was supported by grants from Deutsche Forschungsgemeinschaft (STR 616/1-2) and Bundesministerium für Bildung und Forschung.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.
These two authors contributed equally to this work.
§ To whom correspondence should be addressed: Neuroanatomy, IZN, University of Heidelberg, Im Neuenheimer Feld 307, 2. OG, D-69120 Heidelberg, Germany. Tel.: 49-6221-54-8227; Fax: 49-6221-54-5604; E-mail: Jens.Strelau@urz.uni-heidelberg.de.
Published, JBC Papers in Press, January 3, 2003, DOI 10.1074/jbc.M210037200
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ABBREVIATIONS |
---|
The abbreviations used are:
TGF, transforming growth factor;
GDF-15, growth differentiation factor-15;
CGN, cerebellar granule neuron;
PI3K, phosphatidylinositol 3-kinase;
MEK, mitogen-activated protein kinase kinase;
ERK, extracellular
signal-regulated kinase;
GSK-3, glycogen synthase kinase-3
;
LDH, lactate dehydrogenase;
ROS, reactive oxygen species;
DCF-H2, 2',7'-dichlorodihydrofluorescein;
GDNF, glial cell
line-derived neurotrophic factor;
CDK, cyclin-dependent
kinase;
IGF, insulin-like growth factor;
JNK, c-Jun
NH2-terminal kinase;
PI, propidium iodide;
TUNEL, terminal
deoxyuridynyltransferase nick end labeling.
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