From the Max Planck Institute of Psychiatry, Kraepelinstrasse 2-10, 80804 Munich, Germany
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ABSTRACT |
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The role of insulin-like growth factor 1 (IGF-1)
for the treatment of neurodegenerative disorders, such as Alzheimer's
disease, has recently gained attention. The present study demonstrates that IGF-1 promotes the survival of rat primary cerebellar neurons and
of immortalized hypothalamic rat GT1-7 cells after challenge with
oxidative stress induced by hydrogen peroxide
(H2O2). Neuroprotective concentrations of
IGF-1 specifically induce the transcriptional activity and the DNA
binding activity of nuclear factor Insulin-like growth factor 1 (IGF-1)1 is a
pleiotropic factor with a wide spectrum of action in the central
nervous system and also in the peripheral nervous system (1, 2). It
belongs to a superfamily of structurally related proteins that includes insulin and IGF-2. The biological functions of the IGFs and insulin are
mediated by specific membrane receptors, designated the IGF-1, IGF-2,
and insulin receptors (2, 3). The IGF-1 receptor is the primary
mediator of the action of IGF-1.
Recently, IGF-1 has gained increasing attention for the treatment of
neurodegenerative disorders, such as the amyotrophic lateral sclerosis,
which is characterized by the progressive loss of motor neurons (4, 5).
Moreover, with respect to the central nervous system, IGF-1 has been
found to protect hippocampal neurons against the toxicity of the
Alzheimer's disease-associated amyloid The molecular signaling pathways by which IGF-1 promotes survival, in
particular survival of neurons of the central nervous system, are not
well characterized. Dudek et al. (9) revealed a critical
function of the serine-threonine protein kinase Akt to mediate the
protective effects of IGF-1 on the survival of cerebellar neurons
against serum deprivation. Moreover, the inhibition of apoptosis by
IGF-1 has been shown to require the activation of signaling molecules
such as the phosphatidylinositol (PI) 3-kinase (9, 10).
The nuclear factor As for IGF-1, an important role for NF- Reagents--
All media, sera, and media supplements were from
Life Technologies, Inc. IGF-1 was purchased from Promega (Heidelberg,
Germany) and Sigma. IGF-1 stock solutions were dissolved in 0.1 M acetic acid at a concentration of 250 µg/ml and,
subsequently, dissolved in water. The inhibitors wortmannin and
LY294002 were from Calbiochem (Bad Soden, Germany). All other chemicals
were from Sigma unless otherwise stated.
Plasmids--
NF- Cell Cultures--
Primary cultures of rat cerebellar granule
neurons were prepared from 8-day-old Sprague-Dawley (Charles River,
Sulzfeld, Germany) rat pups, as described previously (26). Neurons
dissociated from cerebellar were plated at different densities (see
below) on plastic dishes coated with poly-L-lysine (10 µg/ml) and grown in basal modified Eagle's medium containing 10%
heat-inactivated fetal calf serum, 25 mM KCl, 2 mM glutamine, and 100 µg/ml gentamicin. Cultures were
placed in a humidified incubator at 37 °C under 95% air/5%
CO2 atmosphere. Experiments were performed after 5-8 days
in vitro as indicated. Glial proliferation was prevented by
addition of 10 µM cytosine arabinofuranoside, an
inhibitor of cell proliferation, 16 h after plating. GT1-7 cells
(kindly provided by Dr. R. Weiner, University of California, San
Francisco, CA) were cultured in Dulbecco's modified Eagle's medium,
supplemented with 10% fetal calf serum under standard culture conditions.
Cell Survival Analysis--
Cell viability was assessed using a
modified 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
(MTT) assay, which is a sensitive first indicator of mitochondrial
damage induced by oxidative stressors (7, 27). Briefly, 8000-10,000
GT1-7 cells or 12,000 cerebellar granule neurons were plated in
96-well microtiter dishes with 100 µl of medium. The next day, medium was exchanged to medium without serum, the cells were preincubated for
24 h with different concentrations of IGF-1 and were challenged for additional 24 h with the indicated concentration of
H2O2. Then, 10 µl MTT (5 mg/ml stock in
phosphate-buffered saline) were added to each well, and the incubation
was continued for 4 h. Finally, 100 µl of solubilization
solution (50% dimethyl formamide, 20% SDS, pH 4.8) were added, and
adsorption readings were performed at 570 nm not earlier than 16 h
thereafter. In a set of control experiments, IGF-1 was also added to
wells containing medium alone to exclude possible interferences of
IGF-1 with the colorimetric MTT assay. All MTT assays were at least
repeated four times in triplicate. When inhibitors were used in
survival assays, cells were pretreated with the inhibitors for 45 min.
Cells that did not receive inhibitors received control vehicle
(dimethyl sulfoxide for LY294002, ethanol for wortmannin). For the
survival assays performed after transient transfection of cells, the
MTT assays were adapted to the 500 µM final volume of the
24-well plates.
As additional cell survival test, a trypan blue exclusion assay was
performed in combination with cell countings using morphological criteria for cell death as described previously (7). For this assay,
cells were plated in 60-mm dishes; after the experimental treatment, at
least five optical fields >200 cells were observed, and survival rates
were determined by an investigator blinded to the experimental conditions.
Transfections--
For transient transfection, GT1-7 cells and
cerebellar neurons were seeded at 120,000 and 50,000 cells per well in
24-well tissue culture dishes, respectively, and were transfected with polyethylenimine (PEI) after 1 day in vitro for GT1-7 cells
or 5 days in vitro for cerebellar neurons as described
previously (28). PEI was used at 10 equivalents (10 amino groups per
phosphate group; 0.3 µl of a 10 mM PEI aqueous solution)
per µg of plasmid. Practically, DNA (2 µg/well) and PEI (25 kDa,
Aldrich) were first diluted in 20 µl/well of 0.9% NaCl. Thereafter,
PEI/DNA particles were obtained by gently mixing the two solutions, and
after 10 min, the transfectant solution mixture was diluted into 500 µl of Dulbecco's modified Eagle's medium without serum and applied to the cells for 2 h. The cells were rinsed and cultured in fresh medium without serum, and IGF-1 was applied for 12-15 h. When inhibitors were used following transfections, cells were pretreated with the inhibitors for 45 min. Cells that did not receive inhibitors received control vehicle (dimethyl sulfoxide for LY294002, ethanol for
wortmannin). Luciferase activity was monitored as previously reported
(29). Each transfection experiment was done in triplicate, repeated at
least four times, and normalized for identical amounts of protein using
the Bio-Rad protein reagent to determine protein concentrations of the
samples (Bio-Rad).
Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts for the EMSAs were prepared by a mini-extraction protocol
(30). The NF- Western Blotting--
Western blotting was performed as
described previously (31, 22) with minor modifications. Briefly, medium
of subconfluent GT1-7 cells was exchanged to serum-free medium, the
cells were treated with IGF-1 as indicated, and cytoplasmic and nuclear
extracts were prepared by a mini-extraction protocol (30). Protein
concentrations were determined using the Bio-Rad protein reagent
(Bio-Rad). Aliquots (10 µg) were mixed with SDS sample buffer
containing 4% Statistical Analysis--
An unpaired Student's t
test was used to calculate differences between means; differences were
considered significant at p < 0.05.
IGF-1 Protects Primary Cerebellar Neurons and GT1-7 Cells against
Oxidative Stress Induced by H2O2--
The
treatment of primary postmitotic cerebellar granular neurons and GT1-7
cells with H2O2 (60 µM), a
mediator of the oxidative toxicity of A IGF-1 Activates NF-
The specificity of NF-
To demonstrate that IGF-1 does not lead to a general nonspecific
increase of various transcription factors, the DNA binding activity of
the transcription factor Oct-1 was investigated as control. No increase
in Oct-1 DNA binding activity could be observed after treatment of
GT1-7 cells with IGF-1 under the same experimental conditions (Fig.
3D).
NF- IGF-1 Increases the Amount of Nuclear p65 and Decreases the Level
of Inhibitory I Overexpression of c-Rel Protects GT1-7 Cells against
H2O2--
Several previous reports suggest a
role for NF-
To further ascertain the involvement of NF-
These results show that neuroprotection by IGF-1 can be mimicked by
NF- Inhibition of the PI 3-Kinase Does Block IGF-1-mediated
Neuroprotection and NF-
To investigate whether the PI 3-kinase is also involved in the
activation of NF- The aim of the present study was to investigate the ability of
IGF-1 to protect neurons against H2O2-induced
neuronal cell death and to examine a possible involvement of NF- The fact that IGF-1 is a potent neuroprotective agent against oxidative
stress induced by the lipid peroxidizing agent
H2O2 may offer a new therapeutic avenue for the
treatment of neurodegenerative disorders in which free radicals have
been implicated. For instance, IGF-1 has been shown to reduce neuronal
cell loss observed in vivo following ischemic insults and is
beneficial in the treatment of amyotrophic lateral sclerosis (38, 39).
With respect to its potency to rescue neurons against A The PI 3-kinase pathway has been shown to be implicated in the
signaling of IGF-1-mediated cell survival, including survival of
neurons (9, 40). PI 3-kinase phosphorylates inositol lipids that act as
second messengers for several targets, such as the serine-threonine
kinase Akt (41), which has been shown accordingly to be involved in
anti-apoptotic signaling (42-44). Experiments with pharmacological
inhibitors demonstrated that IGF-1-mediated neuroprotection against
serum deprivation in neuronal cells is dependent on PI 3-kinase
activity (9, 10).
The data obtained in the present study extend this observation showing
that the PI 3-kinase pathway is also involved in the protective effect
of IGF-1 against H2O2-induced cell death. Two potent PI 3-kinase inhibitors, LY294002 (35) and wortmannin (36), were
able to block the cellular protection afforded by IGF-1 against
oxidative challenge in GT1-7 cells. The data from Parizzas et
al. (10) additionally suggest a role of the mitogen-activated protein kinase pathway in IGF-1-mediated neuroprotection because the
anti-apoptotic function of IGF-1 could not completely be blocked by
inhibition of the PI 3-kinase pathway in the PC12 cells employed in
their study. In our cellular system, the neuroprotective effect of
IGF-1 was entirely prevented by blocking PI 3-kinase, arguing against
the involvement of other signal transduction pathways in IGF-1-mediated neuroprotection.
Because the transcription factor NF- To confirm the role of NF- In view of the idea that the activation of NF- The mechanisms by which NF-B (NF-
B), a transcription
factor that has been suggested to play a neuroprotective role. This
induction is associated with increased nuclear translocation of the p65
subunit of NF-
B and with degradation of the NF-
B inhibitory
protein I
B
. IGF-1-mediated protection of GT1-7 cells against
oxidative challenges was mimicked by overexpression of the NF-
B
subunit c-Rel. Partial inhibition of NF-
B baseline activity by
overexpression of a dominant-negative I
B
mutant enhanced the
toxicity of H2O2 in GT1-7 cells. The pathway
by which IGF-1 promotes neuronal survival and activation of NF-
B
involves the phosphoinositol (PI) 3-kinase, because both effects of
IGF-1 are blocked by LY294002 and wortmannin, two specific PI 3-kinase inhibitors. Taken together, our results provide evidence for a novel molecular link between IGF-1-mediated neuroprotection and induction of NF-
B that is dependent on the PI 3-kinase pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
protein (A
) (6). A
is
the main component of the senile plaques in the brain of Alzheimer's
disease patients and its cytotoxic action on neurons results from
oxidative damage to susceptible cells (7). Specifically, it has been
shown that hydrogen peroxide (H2O2) is an
important intermediate in A
neurotoxicity (7). In accordance with a
neuroprotective role of IGF-1 against oxidative injury, this growth
factor is also effective in protecting neurons against glutathione
depletion, suggesting a more general protective potency of IGF-1
against oxidative stress (8).
B (NF-
B) is composed of homo- and heterodimers
of members of the Rel family of related transcription factors that are
well characterized for controlling the expression of numerous immune
and inflammatory response genes (11, 12). Frequently, NF-
B is
present as a heterodimer comprising a 50-kDa (p50) and a 60-kDa (p65)
subunit that is sequestered in the cytoplasm by an inhibitory protein
of the I
B family, with I
B
being the best characterized member
of this family (13, 14). NF-
B-inducing agents, such as cytokines,
viruses, phorbol esters, and UV light, result in the phosphorylation
and degradation of the I
B inhibitory protein (15, 16) allowing free
NF-
B to enter the nucleus, to bind to its cognate DNA sequences, and
to induce target gene transcription.
B during cell death has been
suggested (17). Several reports have shown that NF-
B counteracts the
induction of apoptosis by the cytokine tumor necrosis factor-
(18-20), by ionizing radiation, and by the cancer chemotherapeutic agent daunorubicin (21). Recently, we have demonstrated that constitutively increased NF-
B activity mediates the protection of
neuronal cells against oxidative stress (22). This prompted us to
investigate whether IGF-1 offers neuroprotection against oxidative
insult by affecting the activity of NF-
B as downstream target in
neurons. For this study, two IGF-1 receptor-expressing neuronal cell
systems were employed, the gonadotropin-releasing hormone-secreting
neuronal cell line GT1-7 (23) and primary cultures of rat cerebellar
granule neurons (24, 25). We demonstrate that IGF-1 exerts a
neuroprotective effect against oxidative stress, mediated by a PI
3-kinase-dependent pathway that finally leads to the
activation of NF-
B.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-Luc was a plasmid construct containing six
NF-
B-binding DNA consensus sites linked to a luciferase reporter
gene. Tk-Luc was an empty cassette construct used as a negative control
for NF-
B-Luc. Both plasmids were provided by Dr. P. Baeuerle
(Tularik Inc., San Francisco, CA). The c-Rel-expression plasmid was a
kind gift from Dr. P. Jalinot (CNRS UMR 49, Lyon, France), and the dominant-negative I
B
-expression plasmid was from Dr. D. W.
Ballard (Vanderbilt University, Nashville, TN).
B double-stranded oligonucleotide corresponding to the
NF-
B consensus sequence in the
light chain enhancer in B cells
(5'-AGT TGA GGG GAC TTT CCC AGG C-3'), and the
oligonucleotide for Oct-1 were from Promega (Heidelberg, Germany) and
were end-labeled with [
-32P]ATP (3000 Ci/mmol,
Amersham, Braunschweig, Germany), and T4 polynucleotide kinase
(Promega). EMSA was performed as described previously (22). Briefly,
nuclear extracts (8-12 µg) were incubated for 20 min at room
temperature with 20 µl of 2 µg of poly(dI·dC) (Pharmacia,
Freiburg, Germany), 10% glycerol, 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 15,000-25,000
counts/min of 32P-oligonucleotides. For competition
studies, prior to the addition of NF-
B labeled probe, nuclear
extracts were preincubated for 10 min at room temperature with a
100-fold excess of unlabeled (cold) oligonucleotide. For the reaction
with specific antibodies, the nuclear extracts were incubated for
16 h at 4 °C with 2 µl of either anti-p50 (sc-114X) or
anti-p65 (sc-109X) antibody stocks (Santa Cruz Biotechnology, Santa
Cruz, CA) prior to the binding reaction. DNA-protein complexes were
resolved on a 6% nondenaturing polyacrylamide gel at 20 mA for 3 h in 0.5 X TBE (45 mM Tris borate, 1 mM EDTA).
Gels were vacuum-dried and exposed to Fuji x-ray films at
80 °C
for 12-24 h.
-mercaptoethanol and resolved on a 10%
SDS-polyacrylamide gel. Proteins were transferred onto a polyvinylidene
difluoride membrane (Millipore, Bedford, United Kingdom) and reacted
with an anti-I
B
antibody (C-21, 1:500 dilution, Santa Cruz
Biotechnology) or an anti-p65 antibody (C-20, 1:200 dilution, Santa
Cruz Biotechnology). The primary antibody was detected by
counterstaining with a horseradish peroxidase-linked antibody and
visualized by the ECL detection kit (Amersham). A part of the SDS gel
was stained with Coomassie Blue to verify whether equal amounts of
proteins had been used. Densitometer reading of the autoradiograph of
the Western blot was performed using a Beckmann photometer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and other neurotoxins,
reduced neuronal viability as assessed by trypan blue exclusion/cell
countings using phase-contrast microscopy and by MTT assays (Figs.
1B and
2). When the cells were preincubated for
24 h with different concentrations of IGF-1 (1-50 ng/ml) before
the addition of the oxidative stressor H2O2, the cell viability was dose-dependently increased in both
neuronal systems (Fig. 2). This was also evident in the maintenance of morphology and integrity of GT1-7 cells after incubation with 50 ng/ml
IGF-1 (Fig. 1C). Higher concentrations of IGF-1 did not further increase cell survival (data not shown). These data demonstrate the ability of IGF-1 to exert a neuroprotective function against oxidative stress.
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Fig. 1.
Morphological changes of GT1-7 cells in
response to H2O2 and neuroprotective effect of
IGF-1. Phase contrast photomicrographs of GT1-7 cells treated
with vehicle only (A), treated for 24 h with 60 µM H2O2 (B), or
treated for 24 h with 60 µM
H2O2 after a 24-h preincubation with IGF-1 (50 ng/ml) (C). D, subsequently to these experimental
treatments, 0.12% trypan blue was added to the cell cultures, and the
viable cells were determined per low magnification field in five
optical fields. The data are expressed as the mean ± S.E. of
viable cells relative to control cultures (defined as 100%). (*,
p < 0.001 compared with cells not treated with IGF-1.)
Scale bar = 50 µm.
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Fig. 2.
IGF-1 protects cerebellar granule neurons and
GT1-7 cells against H2O2 in a
dose-dependent manner. Cerebellar granule neurons
(A) and GT1-7 cells (B) were preincubated for
24 h with different concentrations of IGF-1 (1-50 ng/ml) and were
challenged for another 24 h with 60 µM
H2O2. Thereafter, MTT assays were performed,
and MTT reduction was expressed as percentage of cell survival compared
with control cells that received no H2O2. The
viability of control cells was defined as 100%. Experiments were
repeated four times in triplicate with comparable results. The
presented data are means ± S.E. of one representative triplicate
determination. (*, p < 0.05 and **, p < 0.001 compared with cells that received no IGF-1).
B DNA Binding Activity in Neuronal
Cells--
To assess whether IGF-1 has any effect on the activity of
NF-
B in neuronal cells, we first analyzed the influence of IGF-1 on
the DNA binding activity of this transcription factor. We carried out
EMSAs with nuclear extracts from primary cerebellar neurons and GT1-7
cells, employing a DNA probe that represents the NF-
B DNA-binding
motif. Because IGF-1 produced a considerable neuroprotective effect at
a concentration of 50 ng/ml in our neuronal systems (Fig. 2), we used
this concentration to study its influence on NF-
B DNA binding. Upon
treatment with IGF-1, DNA binding activity of NF-
B was increased in
cerebellar neurons after 1, 4, and 6 h and in GT1-7 cells after 4 and 6 h (Fig. 3, A
(lanes 2-4) and B (lanes 3 and
4)). A longer treatment of the cells with IGF-1 did not
further increase the binding of NF-
B to DNA (data not shown).
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Fig. 3.
IGF-1 increases the DNA binding activity of
NF- B in cerebellar granule neurons and in
GT1-7 cells. Nuclear extracts from cerebellar granule neurons
cultured for 5 days in vitro (A) and from GT1-7
cells (B-D) were analyzed by EMSAs. Cell cultures were left
untreated (control (CT)) or were treated for the indicated
time points with 50 ng/ml IGF-1. The EMSAs were performed with labeled
oligonucleotides representing an NF-
B (A-C) or Oct-1
(D) DNA-binding site. Binding of nuclear extracts after
reaction with a 100-fold excess of unlabeled probe is also shown
(competitor). In C, lanes 3 and 4, nuclear extracts from GT1-7 cells treated for 6 h with IGF-1 (50 ng/ml) were preincubated for 16 h at 4 °C with 2 µl of either
anti-p65 (sc-109X) or anti-p50 (sc-114X) antibody (Santa Cruz
Biotechnology) prior to the binding reaction. Filled
arrowheads represent the position of specific NF-
B/DNA bound
complexes, circles depict the position of nonspecific
complexes, and open arrowheads indicate the position of the
free DNA probe. The arrow in C indicates the
position of a supershift after anti-p50 pretreatment (lane
4).
B DNA binding was demonstrated by the addition
of a 100-fold excess of unlabeled NF-
B oligonucleotide probe that
acts as a competitor for the binding of NF-
B (Fig. 3, A
(lane 5) and B (lane 5)). To further
characterize the DNA-protein complexes, nuclear extracts from
IGF-1-treated cells (6 h, 50 ng/ml) were incubated with antibodies
against the p65 and p50 subunits of NF-
B in a separate set of
experiments. Both antibodies markedly reduced the specific DNA-protein
complexes, with the anti-p50 antibody creating a supershift as shown
for GT1-7 cells (Fig. 3C, lanes 3 and 4). This
suggests the presence of both p65 and p50 in the protein complexes that
bind to the NF-
B oligonucleotide. These results are consistent with
previous observations in other cellular systems (22, 32, 33).
B-dependent Gene Transcription Is Increased by
IGF-1--
To determine whether IGF-1 induced DNA binding of NF-
B
results in activation of NF-
B-mediated gene transcription,
cerebellar granule neurons and GT1-7 cells were transiently
transfected with a plasmid construct containing a promoter composed of
six NF-
B-binding DNA consensus sites linked to a luciferase reporter
gene (NF-
B-Luc). Upon stimulation with neuroprotective doses of
IGF-1 (25 and 50 ng/ml) for 12-14 h, luciferase activities were
significantly increased in cerebellar neurons and in GT1-7 cells (Fig.
4). Lowering the IGF-1 concentration to
10 ng/ml could not significantly enhance NF-
B-dependent
reporter-gene activity. Transcription of the plasmid construct Tk-Luc,
which served as a control, was not altered by the growth factor neither
in cerebellar neurons nor in GT1-7 cells (Fig. 4), indicating the
specificity of NF-
B activation by IGF-1.
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Fig. 4.
IGF-1 increases the transcriptional activity
of NF- B in cerebellar granule neurons and in
GT1-7 cells. Cerebellar granule neurons cultured for 5 days
in vitro (A) and GT1-7 cells (B) were
transfected with either 2 µg of the indicator plasmid NF-
B-Luc
(gray bars) or 2 µg of Tk-Luc control vector (black
bars) and were incubated for 16 h with increasing
concentrations of IGF-1 (1-50 ng/ml). Thereafter cells were harvested
and luciferase activity was determined. Results are expressed in
arbitrary units of luciferase activity (relative luciferase activity)
corrected for identical amounts of protein. Experiments were repeated
four times in triplicate. The presented data are the means ± S.E.
of one representative triplicate determination. (*, p < 0.05 and **, p < 0.001 compared with control
values).
B
Protein--
Because it is known that NF-
B
inducing stimuli result in degradation of the inhibitory protein
I
B
and subsequent nuclear translocation of p65, we investigated
whether neuroprotective doses of IGF-1 have the same effect. In nuclear
extracts from GT1-7 cells treated with 50 ng/ml IGF-1 for 1-6 h, p65
expression was already increased after 1 h. This increase
correlated with the rapid degradation of I
B
in the cytoplasmic
portion of these cells as shown by Western blotting (Fig.
5). After 6 h of IGF-1 treatment,
the amount of translocated nuclear p65 was increased 2.3-fold, whereas
the level of I
B
protein was reduced approximately 60% as
compared with extracts from untreated cells (Fig. 5, A and
B, compare the left lane with the 6 h
lane in each). Taken together, the above results indicate that
following degradation of the inhibitory protein I
B
and nuclear
translocation of p65, neuroprotective concentrations of IGF-1 can
specifically induce the DNA binding activity and the transcriptional
activity of NF-
B in neuronal cells.
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Fig. 5.
IGF-1 increases the amount of
nuclear p65 and decreases cytoplasmic
I B
protein levels in
GT1-7 cells. GT1-7 cells were treated with vehicle only or with
50 ng/ml IGF-1 for the indicated time. Cytoplasmic and nuclear extracts
were prepared, and equal amounts of protein were analyzed for the
presence of nuclear p65 (A) and cytoplasmic I
B
(B) by Western blotting. Intensity of the signals was
analyzed by densitometer reading of the autoradiographs of the Western
blots and is presented as relative protein expression. The expression
in untreated cells was defined arbitrarily as 1.
B in cell survival. In view of these findings, we
investigated whether the activation of this transcription factor is
directly involved in IGF-1-mediated neuroprotection. Therefore, GT1-7
cells were transiently transfected with an expression plasmid coding
for c-Rel, a subunit of NF-
B, the expression of which leads to the
induction of an NF-
B-dependent luciferase reporter gene
(Fig. 6B). Whereas cells transfected with an empty control vector showed a decreased survival upon challenge with H2O2, c-Rel-overexpressing
cells were completely protected against the oxidative challenge as
determined by the MTT assay performed subsequently to transfection
(Fig. 6A).
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Fig. 6.
Effect of c-Rel and dominant-negative
I B
expression on cell
survival and basal NF-
B transcriptional
activity. A, GT1-7 cells were transfected with 2 µg
of either an expression plasmid for c-Rel or for mutant I
B
or
with an empty control plasmid. 5 h after transfection cells were
challenged with 60 µM H2O2 for
24 h and MTT assay was performed subsequently. MTT reduction was
expressed as percentage of cell survival compared with cells that
received no H2O2 (defined as 100%).
Experiments were repeated three times in triplicate with comparable
results. The presented data are means ± S.E. of one
representative triplicate determination. (*p < 0.05 compared with cells transfected with the empty expression vector.)
B, GT1-7 cells were cotransfected with 1 µg of the
indicator plasmid NF-
B-Luc together with 1 µg of either an empty
expression vector (control), an expression plasmid for c-Rel, or an
expression plasmid for mutant I
B
. After 16 h, cells were
harvested, and luciferase activity was determined. Results are
expressed in arbitrary units of luciferase activity (relative
luciferase activity) corrected for identical amounts of protein.
Experiments were repeated three times in triplicate. The presented data
are the means ± S.E. of one representative triplicate
determination. (*, p < 0.01 compared with control
values.)
B in protection of
neuronal cells, we transiently transfected GT1-7 cells with an
expression plasmid coding for a mutant form of I
B
, which is
resistant to both phosphorylation and proteolytic degradation and
therefore prevents nuclear translocation of NF-
B (34). This
dominant-negative I
B
, the overexpression of which significantly reduced basal NF-
B-mediated reporter gene activity (Fig.
6B), indeed decreased cell survival after oxidative
challenge when compared with cells expressing an empty control vector
(Fig. 6A).
B activation, and cell viability is decreased by NF-
B inhibition, indicating that NF-
B might at least in part be involved in the mediation of the neuroprotective function of IGF-1.
B Activation--
Recent data from various
groups suggested that the molecular signaling pathway by which IGF-1
exerts its neuroprotective function might involve PI 3-kinase (9, 10).
This is confirmed in our cellular system, because in MTT assays, the
potent PI 3-kinase inhibitors LY294002 (35) and wortmannin (36) did
indeed block IGF-1-mediated protection of GT1-7 cells against hydrogen
peroxide induced cell death (Fig. 7).
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Fig. 7.
LY294002 and wortmannin inhibit
IGF-1-mediated cell survival. GT1-7 cells were left untreated or
were incubated with 50 ng/ml IGF-1 in the presence or absence of 10 µM LY294002 (A) or 100 nM
wortmannin (B). After 24 h, cells were challenged for
another 24 h with 120 µM
H2O2. Thereafter, MTT assays were performed and
MTT reduction was expressed as percentage of cell survival compared
with control cells that received no H2O2. The
viability of control cells was defined as 100%. Experiments were
repeated three times in triplicate with comparable results. The
presented data are means ± S.E. of one representative triplicate
determination. (**, p < 0.001 and ***,
p < 0.0005; IGF-1 stimulated cells compared with cells
that were treated with inhibitor in addition to IGF-1.)
B by IGF-1, we examined the influence of these
inhibitors on IGF-1 induced transcription of an
NF-
B-dependent luciferase gene in transiently
transfected GT1-7 cells. In this assay, the inhibition of PI 3-kinase
by LY294002 (10 µM) and wortmannin (100 nM)
completely abolished the stimulation of NF-
B transcriptional activity by IGF-1 (Fig. 8), consistent
with their ability to block IGF-1-mediated neuroprotection (Fig. 7).
These results strongly support a novel pathway for NF-
B activation
exerted by IGF-1 that involves the PI 3-kinase and mediates IGF-1
neuroprotection.
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Fig. 8.
LY294002 and wortmannin inhibit IGF-1 induced
increase in NF- B transcriptional
activity. GT1-7 cells were transfected with 2 µg of the
indicator plasmid NF-
B-Luc and were left unstimulated or were
incubated with 50 ng/ml IGF-1 in the presence or absence of 10 µM LY294002 (A) or 100 nM
wortmannin (B). After 16 h, cells were harvested, and
luciferase activity was determined. Results are expressed in arbitrary
units of luciferase activity (relative luciferase activity) corrected
for identical amounts of protein. Experiments were repeated three times
in triplicate. The presented data are the means ± S.E. of one
representative triplicate determination. (***, p < 0.005; IGF-1 stimulated cells compared with cells that were treated
with inhibitor in addition to IGF-1.)
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B in
this function. We employed GT1-7 hypothalamic neuronal cells and
mature primary cerebellar granule neurons that have been shown to
express functional IGF-1 receptors (9, 37). As previously reported for
other oxidative stressors, such as the Alzheimer's disease-associated A
and the glutathione depleting agent buthionine sulfoximine (6, 8),
we found that IGF-1 is effective in protecting neuronal cells against
oxidative injury induced by H2O2. A 24-h
pretreatment with 50 ng/ml of IGF-1 caused an enhanced cell survival of
about 30% after H2O2 challenge in GT1-7 cells
and primary mature postmitotic neurons of 5 days in vitro.
IGF-1 did not significantly affect GT1-7 cell growth in serum-free
conditions (data not shown), confirming that the increased cell
survival is not due to a proliferative effect of IGF-1 in this neuronal system.
toxicity
(38), one can likewise imagine that the development of IGF-1-related
compounds could be a promising strategy toward the treatment of
Alzheimer's disease.
B can modulate neuronal
vulnerability and has recently gained great interest for its potential
role in neuroprotection (45-47), we tested the ability of IGF-1 to
induce NF-
B activation. We found that increasing concentrations of
IGF-1 in GT1-7 cells stimulate the NF-
B transcriptional activity
with a substantial effect at 50 ng/ml. These results were also
confirmed in postmitotic cerebellar granule neurons, indicating that
the enhancement of NF-
B activity by IGF-1 is not dependent on the
cell cycle. Consistent with this increase in NF-
B transcriptional
activity, NF-
B DNA binding activity could also be enhanced upon
stimulation with 50 ng/ml IGF-1 in both neuronal systems. In agreement
with these findings, we observed increased nuclear translocation of the
p65-subunit of NF-
B and a parallel decrease in the amount of the
NF-
B cytoplasmic inhibitory protein, I
B
. Thus, the induction
of NF-
B-driven protective transcriptional programs by IGF-1 could be
one way for the neurons to protect themselves against exogenous
insults. In support of this hypothesis, we have recently shown that in
a clone of the sympathetic precursor-like cell line PC12 selected for
its resistance against A
and H2O2,
constitutively increased NF-
B activity mediates this resistance. The
suppression of NF-
B activation in these cells reverses the oxidative
stress resistance phenotype (22). Consistently, the inhibition of
NF-
B activation results in apoptosis in PC12 cells, and nerve growth
factor did not protect from apoptosis when NF-
B activation is
blocked (32).
B in IGF-1-mediated neuroprotection, we
(i) induced an activation of NF-
B by transient expression of the
c-Rel subunit of this transcription factor, and (ii) inhibited NF-
B
activity by expression of a dominant-negative form of I
B
. The
expression of c-Rel in GT1-7 cells, which mimicked IGF-1 induction of
NF-
B activity, protected these cells completely against oxidative stress, consistent with findings from other investigators showing that
the c-Rel expression is able to reduce apoptosis in nonneuronal cells
(17, 19). On the other hand, the dominant-negative I
B
decreased
the intrinsic survival of GT1-7 cells against oxidative insult, most
likely by reducing the high basal NF-
B activity in these cells.
However, this decrease in survival was not significant but rather
indicative, which might be due to the fact that either the
dominant-negative I
B
was not able to completely abrogate NF-
B
activity (supported by the luciferase assay done in parallel) or
additional factors, other than NF-
B, are involved in the protection of neuronal cells against oxidative stress. Together, these results suggest that the protection of neuronal cells by IGF-1 may be mediated,
at least in part, through the activation of NF-
B.
B represents an
intermediary step in IGF-1-mediated neuroprotection, one would expect
that both processes are mediated by a common pathway. Upon investigating the effect of LY294002 and wortmannin on the
IGF-1-induced transcriptional activity of NF-
B, it indeed turned out
that these PI 3-kinase inhibitors completely blocked the IGF-1-induced
increase in NF-
B mediated luciferase activity, indicating that the
IGF-1 activation of NF-
B involves the PI 3-kinase pathway. In
contrast to these findings, Bertrand et al. (48) reported
that an insulin activation of NF-
B in Chinese hamster ovary cells
does not involve the PI 3-kinase, but rather occurs via the
Raf-1-mediated signal transduction pathway. However, these authors
argued that the anti-apoptotic role of insulin in Chinese hamster ovary
cells, besides NF-
B activation, additionally requires independent
processes that involve PI 3-kinase activity (48). The reasons of these
apparent discrepancies remain to be investigated, but might be due to
the different cell type and the specific stimuli applied to the cells.
So far, besides Raf-1 (33), various kinases have been reported to be
involved in the pathway leading to activation of NF-
B, such as
mitogen-activated protein kinase/extracellular signal-regulated protein
kinase (ERK) kinase kinase 1 (49) and the kinase finally
phosphorylating I
B
, conserved helix-loop-helix ubiquitous kinase
(50), or I
B
kinase (51). Here we report for the first time that
also the PI 3-kinase can participate in the induction of NF-
B
activity, suggesting that dependent on the stimulus multiple NF-
B
activation pathways may exist.
B may prevent neuronal cell death upon
stimulation with IGF-1 remain unknown. The activity of this
transcription factor may drive defense programs that afford the
protection against oxidative insult. Therefore, in future, it will be
important to identify neuroprotective target genes of NF-
B and to
investigate their inducibility by exogenous factors, such as IGF-1, to
increase the survival of neurons under oxidative stress.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. R. Weiner for providing the
GT1-7 cell line, P. Baeuerle for the NF-B-luciferase and
TK-luciferase constructs, P. Jalinot for the c-Rel construct, and
D. W. Ballard for the mutant I
B
construct.
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FOOTNOTES |
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* This work was supported in part by a grant from the Deutsche Hirnliga e.V. (to C. B.).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 authors contributed equally to this work.
§ Present address: INSERM U 446, Faculty of Pharmacy, 92296 Chatenay-Malabry, France.
¶ To whom correspondence should be addressed. Tel.: 49-89-30622-246; Fax.: 49-89-30622-642; E-mail: chris{at}mpipsykl.mpg.de.
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ABBREVIATIONS |
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The abbreviations used are:
IGF-1, insulin-like
growth factor;
A, amyloid
protein;
PI 3-kinase, phosphatidylinositol 3-kinase;
NF-
B, nuclear factor
B;
I
B, inhibitor of
B;
Luc, luciferase;
Tk, thymidine kinase;
MTT, 3-(4,
5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide;
PEI, polyethylenimine;
EMSA, electrophoretic mobility shift assay.
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REFERENCES |
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