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INTRODUCTION |
The p53 tumor suppressor gene has recently been shown to play an
important role in regulating apoptosis for neurons and a number of
other cell types (1). Hypoxic insult, as well as excitotoxic
stimulation, involves excessive production of nitric oxide (NO) and has
have been shown to increase p53 production with concomitant increases
in neuronal apoptosis (2). In general, p53-mediated apoptosis requires
a p53 protein that functions to modulate transcription of certain
genes. Important examples of such activated genes include the
apoptosis-inducing Bax gene (3) and the KILLER/DR5 gene (4).
Following exposure to toxic treatments, a number of peptide factors
including neurotrophins and growth factors such as insulin-like growth
factor 1 (IGF-1)1 have been
found to enhance neuronal survival (5). In recent years, considerable
advances have been made in understanding the signal transduction
pathway activated by these growth factors. Among them, the
serine/threonine kinase termed Akt appears to play a central role in
the survival of a number of cell types (6-9). Recently, four Akt
substrates were identified that are also components of the cell's
intrinsic death machinery: the pro-apoptotic Bcl-2 family member known
as Bad (10, 11), the caspase-9 protease (12), I
B kinase
(IKK
)
(13), and a member of the Forkhead family of transcription factors
known as FKHRL1 (14-16). In each case, phosphorylation by Akt kinase
inhibits these protein's pro-apoptotic function, thereby accounting
for at least part of the survival effect imparted by activated Akt kinase.
We now report that activated Akt significantly impairs
p53-dependent neuronal apoptosis and impairs induction of
Bax in response to treatment with hypoxia, NO, or adenovirus-mediated
expression of p53. We further show that activated Akt strongly inhibits
p53-dependent transactivation of target genes with no
apparent effect on p53-protein accumulation, subcellular localization,
and phosphorylation status of p53. These findings suggest a mechanism
where activated Akt kinase inhibits p53-mediated transactivation to
apparently block subsequent apoptosis of the neuron.
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EXPERIMENTAL PROCEDURES |
p53-deficient Mice--
Homozygous p53-deficient mice were
obtained from the Oriental Yeast Co., Ltd. (Tokyo, Japan). Originally
on a mixed C57BL6/CBA strain, p53-deficient mice were backcrossed to
C57BL6 (five times) so that the majority of their genetic background is
C57BL6. Fifth generation heterozygous p53 knockout mice on the C57BL6
background were mated to one another to produce the homozygous p53
knockout mice used in this study. The genotypes of all mice were
confirmed by PCR using DNA extracted from the tails according to the
protocol described previously (17). The primers were mixtures of 1) a genomic sequence in intron 1 and upstream of exon 2 in the p53 gene
(5'-AATTGACAAGTTATGCATCCAACAGTACA-3'), 2) a genomic sequence in exon 4 of the p53 gene (5'-ACTCCTCAACATCCTGGGGCAGCAACAGAT-3'), and 3) a
sequence from the neomycin resistance gene
(5'-GAACCTGCGTGCGTGCAATCCATCCATCTTGTTCAATG-3'). A 500-base pair PCR
product represents the wild-type p53 gene and a 800-base pair PCR
product represents the disrupted p53 gene.
Cell Culture--
Hippocampal neurons were prepared from
postnatal day
1 p53 wild-type and deficient mice and from embryonic
day 18 Harlan Sprague Dawley rat embryos, as described previously (18,
19). Briefly, fetal hippocampi were dissected and digested with 0.25% trypsin for 20 min at room temperature in calcium/magnesium-free Hanks' balanced salt solution (Life Technologies, Inc.). Tissues were
further dissociated by repeated trituration. The cells were seeded at a
density of 1 × 106 cells/cm2 on plates
coated with poly-L-lysine (10 µg/ml) (Falcon Lab and Maintainware, Lincoln Park, NJ) and grown in Dulbecco's modified Eagle's medium supplemented with 10% inactivated fetal calf serum, 30 mM glucose, and 0.5% (v/v) penicillin-streptomycin at
37 °C in a humidified atmosphere of 5% CO2 and 95%
room air. To inhibit growth of glial cells, cytosine arabinoside (10 µM) was added to the cultures 48 h after seeding.
All experiments were performed in 8-10-day-old cultures (8-10
DIV).
Experimental Treatments--
The growth medium was replaced with
a serum-free medium that consisted of Dulbecco's modified Eagle's
medium supplemented with 30 mM glucose and 0.5% (v/v)
penicillin-streptomycin. At 24 h after the replacement, cells were
treated for 6 h with IGF-1 or vehicle (saline) and exposed to
hypoxia or a NO donor, sodium nitroprusside (SNP; Sigma). Human
recombinant IGF-1 was purchased from Sigma. Hypoxia treatment was
performed using an incubator attached to a hypoxia chamber (Coy
Laboratory Products, Ann Arbor, MI) that maintained a humidified
atmosphere with low oxygen tension (pO2; 12-14 Torr) as
described previously (20). In PI 3-kinase inhibition studies, the PI
3-kinase inhibitor wortmannin (Sigma) was added to the cultures 15 min
before treatment with IGF-1.
Recombinant Adenovirus--
The generation of recombinant
adenovirus containing constitutively active Akt (AxCALNmyrAkt) or
kinase-deficient Akt gene (AxCAKDAK179M) has been described previously
(21). In brief, AxCALNmyrAkt encodes constitutively active Akt
(myr-Akt) that lacks a pleckstrin homology domain but contains a Src
myristoylation signal sequence fused to its amino-terminal end. The
myr-Akt gene is subcloned into the Cre-inducible expression cassette,
pAxCALNLw, which is normally not expressed because of the placement of
the neomycin resistance gene. Efficient production of a nuclear
location signal-tagged Cre recombinase under control of the CAG
promoter (22) permits Cre-mediated excision of the neomycin resistance gene from the pAxCALNLw vector allowing expression of the heterologous target gene. The kinase-defective Akt (tagged with hemagglutinin to its
NH2 terminus) was prepared by PCR where Akt1 K179M in
pBT-701 (kindly provided by Dr. Kikkawa, Kobe University, Kobe, Japan), was used as a template. DNA fragments generated by PCR were confirmed by DNA sequence analysis. Then, the kinase-defective Akt gene tagged
with hemagglutinin was cloned into the pAxCAwt expression vector and
was designated as pAxCAAktK179M.
The recombinant adenovirus vectors AxCANCre, which expresses Cre
recombinase; AxCALNLZ, which expresses LacZ when a sufficient amount of
Cre recombinase is expressed; and AxCANLZ, which expresses LacZ under
control of the CAG promoter, were generously provided by Dr. Saito
(University of Tokyo, Tokyo, Japan). Another recombinant adenovirus
vector Ad5CMVp53, which expresses p53 protein under the control of CMV
promoter, was generously provided by Dr. Roth (University of Texas,
Houston, TX) (23). All viruses were grown in 293 cells and
purified by CsCl2 gradient centrifugation. Virus titers
were determined by plaque assay and concentrated virus stored at
80 °C.
Infection was carried out by adding recombinant adenovirus to
serum-containing medium. The cells were incubated with virus-containing media at the indicated multiplicity of infection at 37 °C for 60 min with constant agitation. The medium was changed, and the cells were
incubated at 37 °C for 24 h before exposure to hypoxia or a NO
donor, SNP, or were untreated as stated.
Assessment of Cell Viability--
Neuronal cell viability was
assessed by determining the release of lactate dehydrogenase into the
culture medium thereby indicating a loss of membrane integrity and cell
death. Lactate dehydrogenase activity was measured using a commercial
kit (Kyokuto Chemical Co., Tokyo, Japan) in which a colorimetric assay
measures the pyruvate-mediated conversion of 2,4-dinitrophenylhydrazine
into a visible hydrazone precipitate. Percent neuronal viability was expressed as (1
experimental value/maximum release) × 100, where the maximum release was obtained after exposure of untreated
control cultures to 0.2% Triton X-100 for 15 min at 37 °C.
Levels of cytoplasmic, histone-associated DNA fragments were assayed
using the Cell Death enzyme-linked immunosorbent assay (Roche Molecular
Biochemicals, Mannheim, Germany) according to manufacture's protocol.
In brief, the cytosolic fraction (13,000 × g
supernatant) of ~500 cultured cells was used as an antigen source in
a enzyme-linked immunosorbent sandwich assay with primary anti-histone
antibody coated to the microtiter plate and secondary anti-DNA antibody
coupled to peroxidase. From the absorbance values, the -fold increase
of DNA fragmentation was calculated as (absorbance of treated
cells
absorbance of blank)/(absorbance of control cells
absorbance of blank) where nontreated cells were used as controls.
Caspase-3-like Enzyme Activity--
Caspase-3-like activity was
measured by spectrophotometric assay as described previously (24).
Briefly, neurons were suspended in buffer (50 mM Tris-HCl,
pH 7.4, 1 mM EDTA, and 10 mM EGTA), and then
incubated with 10 µM digitonin (Sigma) at 37 °C for 10 min, followed by centrifugation at 15,000 rpm for 3 min. Protein concentration in the resulting supernatant was measured using a DC
protein assay kit (Bio-Rad). Then, the supernatant containing 30 µg
of protein was incubated with 50 µM enzyme substrate,
7-amino-4-methylcoumarin (AMC)-DEVD, at 37 °C for 1 h. Levels
of released AMC were measured using an excitation wavelength of 380 nm
and an emission wavelength of 460 nm with a spectrofluorometer (Hitachi
F-3000, Hitachi, Tokyo, Japan). One unit was defined as the amount of
enzyme required to release 0.22 nmol of AMC/min at 37 °C.
Caspase-3-like activity of control neurons was 47.1 ± 2.7 units/mg of protein.
Western Blot Analysis--
Cell extracts for Western blot
analysis were prepared by washing the cells three times with PBS and
lysing them in sample buffer (50 mM Tris-HCl, pH 8.0, 20 mM EDTA, 1% SDS, and 100 mM NaCl). The samples
were boiled for 5 min before subjecting 20-µg aliquots to
electrophoresis on 12.5% SDS-PAGE gels. After the proteins were
transferred onto polyvinylidene difluoride membrane (Millipore Corp.,
Bedford, MA), the membrane was incubated in blocking buffer (1× PBS,
5% nonfat dried milk) for 1 h at room temperature and then probed
with a primary antibody in blocking buffer overnight at 4 °C. The
membrane was washed four times in PBS containing 0.3% Tween 20, probed
with the secondary antibody in blocking buffer for 1 h at room
temperature, and washed again in PBS containing 0.05% Tween 20. Detection of signal was performed with an enhanced chemiluminescence
detection kit (Amersham International, Little Chalfont, United
Kingdom). The primary antibodies used were anti-p53 monoclonal antibody
(DO-1; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Bax
polyclonal antibody (P-19: Santa Cruz Biotechnology, Inc.), anti-mouse
Akt polyclonal antibody (New England Biolabs), anti-mouse phospho-Akt
polyclonal antibody that recognizes Akt only when phosphorylated at
Ser-473 (9270, New England Biolabs), and anti-mouse
-actin
monoclonal antibody (Sigma).
RNA Isolation and RT-PCR--
Total RNA derived from 2 × 107 cells was extracted from primary cultures of rat
neurons by the acid guanidium-thiocyanate/phenol chloroform method. For
RT-PCR, 5 µg of total RNA was reversed-transcribed using oligo(dT)
and reverse transcriptase from Moloney murine leukemia virus (Life
Technologies, Inc.) in a volume of 25 µl. For PCR amplification,
specific oligonucleotide primer pairs (10 pmol each) were incubated
with 1 µl of cDNA template in a 20-µl PCR reaction mixture
containing 1.5 mM MgCl2, 25 mM KCl,
10 mM Tris, pH 9.2, 1 µl of deoxynucleotides (1 mM each), and 1 unit of Taq polymerase. The
sequences of primers used in this study were as follows: Bax sense
primer (5'-TGGTTGCCCTTTTCTACTTTG-3') and Bax antisense primer
(5'-GAAGTAGGAAAGGAGGCCATC-3') and as an internal control,
-actin
sense primer (5'-TGCCCATCTATGAGGGTTACG-3') and
-actin antisense
primer (5'-TAGAAGCATTTGCGGTGCACG-3'). Dilutions of cDNA were
amplified for 23-26 cycles at 94 °C for 30 s, 55 °C for
30 s, and 72 °C for 30 s. The amplified PCR products at each cycle number were analyzed by 1% agarose gel electrophoresis and
ethidium bromide staining. The product of constitutively expressed
-actin mRNA served as the internal standard. The amplified PCR products were analyzed by 1% agarose gel electrophoresis and ethidium bromide staining. The product of constitutively expressed
-actin mRNA served as the control. All the products were assayed in the linear response range of the RT-PCR amplification process; the cycle
number used was determined by finding the midpoint of linear amplification on a sigmoid curve for both amplification products with
cycle numbers of 24-30 plotted against band density (25). The identity
of each PCR product was confirmed by subcloning the amplified cDNAs
into the pGEM-T vector (Promega) and sequencing.
Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts were prepared from primary neuronal cultures according to
published method (26) with some modifications. In brief, cells were
plated at a density of 2 × 106 cells/60-mm dish and
were harvested by scraping and washing in 0.5 ml of cold PBS. The cells
were then washed once in 0.1 ml of cold buffer A (10 mM
HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM
KCl, and 0.5 mM dithiothreitol). The washed pellets were
then suspended in 50 µl of buffer A plus 0.1% Nonidet P-40
supplemented with 1 µg/ml leupeptin and aprotinin and were incubated
on ice for 10 min. After incubation, the pellets were mixed briefly by
vortexing and were centrifuged at 10,000 rpm at 4 °C for 5 min in a
microcentrifuge. The supernatant was carefully removed, and the nuclear
pellet was resuspended in 20 µl of cold buffer C (20 mM
HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.15 mM dithiothreitol, and 0.5 mM
phenylmethylsulfonyl fluoride) containing 1 µg/ml leupeptin and
aprotinin and incubated on ice for another 15 min with intermittent
vortexing. The extracts were then centrifuged at 10,000 rpm at 4 °C
for 10 min, and the supernatant was divided into aliquots and frozen at
70 °C. Protein concentrations were determined using Bio-Rad
protein assay kit.
To assay binding activity, a double-strand oligonucleotide containing
the sequence corresponding to the p53 consensus site (5'-AGCTTAGACATGCCTAGACATGCCTA-3') (27) was end-labeled with [
-32P]ATP using T4 kinase (Life Technologies, Inc.).
Unincorporated nucleotides were removed using Sephadex G-50 column
(Amersham Pharmacia Biotech). Binding reactions were carried out in a
final volume of 25 µl consisting of 10 mM HEPES, pH 7.9, 4 mM Tris-HCl, 60 mM KCl, 1 mM
EDTA, 1 mM dithiothreitol, 10% glycerol, 1.5 mg/ml bovine
serum albumin, 2 µg of poly(dI-dC), 2-10 µg of nuclear extract,
and 0.5 ng of 32P-labeled oligonucleotide probe (50,000 cpm). Reactions were incubated for 20 min at room temperature. Binding
reactions were subjected to nondenaturing polyacrylamide
electrophoresis through 4% gels in a 1× Tris borate-EDTA buffer
system. Gels were dried and subjected to autoradiography.
Luciferase Assay--
Primary cultured rat neurons were plated
in six-well tissue culture dishes at 9 × 104
cells/well and used for transient transfection at DIV 7. Cells were
then cotransfected with 0.3 pmol of a p53-Luc plasmid that contains a
firefly luciferase reporter gene driven by a basic promoter element and
a TATA box, which are joined to a tandem repeat of a p53 binding
element (Stratagene), together with 0.3 pmol of pRL-TK plasmid, which
contains an herpes simplex virus thymidine kinase promoter upstream of
the Renilla luciferase gene (Promega). As controls, pGL3
basic vector or pGL3 promoter plasmids that contain an SV40 promoter
upstream of the firefly luciferase gene (Promega) are cotransfected
with pRL-TK plasmid. Transfections are carried out using the modified
calcium phosphate method as described previously (28).
Transfected cells were cultured for 24 h, washed twice with 2 ml
of Ca2+- and Mg2+-free PBS and lysed with
Passive Lysis Buffer (Promega). Firefly luciferase and
Renilla (sea pansy) luciferase activities were measured
sequentially using a dual-luciferase reporter assay system (Promega)
and a Lumat LB9501 luminometer (EG&G, Berthold). After measuring the
firefly luciferase signal (LAF) and the
Renilla luciferase signal (LAR), the
relative luciferase activity (RLA) was calculated as:
RLA = LAF/LAR, where relative
RLA was calculated as a percentage, i.e.
%RLA = RLA/(RLA)max.
In Vivo Kinase Assay--
To examine the effect of Akt
activation on the phosphorylation status of p53, in vivo
kinase assays were performed according to the published method (29).
Neurons derived from rat fetal brains were seeded in 35-mm dishes at
the density of 1-2 × 106 cells/dish. Cells were
incubated in a phosphate-free medium for 30 min and then labeled with
32P for 3 h in a medium containing 0.2 mCi/ml
[32P]orthophosphate. After 32P labeling, they
were lysed in RIPA buffer (0.02 M Tris-HCl, pH 7.4, 0.15 M NaCl, 2 mM EDTA, 1% Nonidet P-40, 1% sodium
deoxycholate, 0.1% SDS) containing protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin) and phosphatase inhibitors (50 mM NaF, 1 mM Na3VO4). p53 proteins were
recovered by immunoprecipitation with anti-p53 antibody and subjected
to SDS-PAGE, which was dried prior to analysis by autoradiography.
Statistical Analysis--
Results are presented as mean ± S.E. Experimental groups were compared by analysis of variance,
followed by Scheffe's post hoc test. p values
less than 0.05 were considered significant.
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RESULTS |
p53 Is Involved in Caspase-3-like Activity and Neuronal Death
Mediated by Hypoxic and Nitric Oxide Treatments--
To address the
possibility that accumulation of p53 plays a role in cell death
following treatment with hypoxia or a NO donor, SNP, we compared
viabilities of primary hippocampal neurons derived from wild-type or
p53-null mice. Treatment with hypoxia or SNP (50 µM)
induced significantly greater degrees of neuronal loss, DNA
fragmentation, and caspase-3-like activation in neurons derived from
wild-type mice as compared with those from p53-null mice (Fig.
1A, a-c).
Administration of the Ac-DEVD-CHO, a peptide inhibitor of
caspase-3-like protease (1 µM), which completely
inhibited casapse-3-like activation (Fig. 1A, c),
reduced the proportion of hypoxia-induced or SNP-induced cell death
(Fig. 1A, b) and DNA fragmentation (Fig.
1A, a), suggesting that increased protease activity is required for cell death. Furthermore, treatment with hypoxia or SNP significantly increased the levels of p53 protein (Fig.
1A, d). These results demonstrated that apoptosis
following hypoxia or SNP treatments is prominently associated with
increased p53 expression.

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Fig. 1.
p53 is involved in hypoxia- and NO-mediated
neuronal death. A, primary cultured p53-null
(p53( / )) or wild-type (p53(+/+)) mouse hippocampal neurons were
exposed to hypoxia or SNP (50 µM) for 24 h, and then
levels of cytoplasmic histone-associated DNA fragments (a),
neuronal viability (b), and caspase-3-like activity
(c) were assayed as described in text. Extracts from cells
were subjected to 10% SDS-PAGE, and blots were probed with antibody to
p53 (d). DEVD, Ac-DEVD-CHO (1 µM).
Data are mean ± S.E. of four independent experiments performed in
triplicates. *, p < 0.05 versus wild-type.
B, primary cultured p53-deficient (p53( / )) mouse
hippocampal neurons were infected or not infected with Ad5CMVp53
(Adp53) or AxCALNLZ (AdLZ) at the indicated
m.o.i. At 24 h after viral infection, extracts from cells were
subjected to 10% SDS-PAGE and blots were probed with antibody to p53
(a). After 24 h of infection, levels of DNA
fragmentation (b), neuronal viability (c), and
caspase-3-like activity (d) were measured. Data are
mean ± S.E. of four independent experiments performed in
triplicates. *, p < 0.05 versus control,
uninfected cells; **, p < 0.05 versus Adp53
(50 m.o.i.).
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To further demonstrate that p53 is involved in neuronal apoptosis, we
used a strategy of adenovirus infection to transduce the expression of
p53 protein in cultures of hippocampal neurons from p53-null mice.
Western blot analysis revealed that cultured neurons deficient in p53
gene and infected with Ad5CMVp53, which expresses human wild-type p53
protein under control of the CMV promoter, displayed increased levels
of p53 protein at 24 h after infection that was dependent on the
multiplicity of infection (m.o.i.) as compared with uninfected cells or
with cells infected with a control virus, AxCANLZ (Fig. 1B,
a). Infection with Ad5CMVp53 (50 and 100 m.o.i.)
also increased neuronal death, DNA fragmentation, and caspase-3-like
activity at 24 h after infection. (Fig. 1B, b-d). Moreover, pretreatment of neurons with the
caspase-protease inhibitor, Ac-DEVD-CHO (1 µM),
significantly inhibited neuronal death induced by infection with
Ad5CMVp53 (Fig. 1B, c). DNA fragmentation, neuronal viability, and caspase-3-like proteolytic activity of AxCANLZ-infected cells were similar to those of control, uninfected neurons (data not shown). These results are consistent with the hypothesis where increased levels of p53 expression are proportionally associated with increased apoptotic cell death.
IGF-1-mediated Signaling Pathways Inhibit p53-induced Neuronal
Death--
Growth factors such as IGF-1 appear to play a
neuroprotective role against ischemic or excitotoxic insult (30). To
assess the role of IGF-1 in the prevention of p53-mediated neuronal
death, we examined the effect of IGF-1 on wild-type or p53-null neurons exposed to hypoxia or SNP treatment. Pretreatment with IGF-1 conferred ~70% protection from hypoxia- or SNP-induced cell death, 60%
reduction in DNA fragmentation, and 70% inhibition of caspase-3-like
activation in wild-type neurons with these effects being blocked by
treatment with wortmannin (20 nM), an inhibitor of PI
3-kinase (Fig. 2, A-C). On
the other hand, IGF-1 treatment in p53-null neurons did not
significantly change the measured parameters of apoptosis observed in
non-IGF-1-treated p53-null neurons (Fig. 2, A-C). These
results clearly suggest that IGF-1 protects neurons from p53-mediated
toxicity through PI 3-kinase activation.

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Fig. 2.
IGF-1 inhibits p53-induced neuronal death
through PI 3-kinase. Neurons derived from wild-type (p53(+/+)) or
p53-null mice (p53( / )) pretreated with IGF-1 (100 ng/ml) or vehicle
(saline) in the presence or absence of wortmannin (W) (20 nM) were exposed to hypoxia or SNP (50 µM)
for 24 h, and then neuronal viability (A), DNA
fragmentation (B), and caspase-3-like activity
(C) were measured as described in text. Data are mean ± S.E. of four independent experiments performed in triplicates. *,
p < 0.05 versus non-IGF-1-treated p53(+/+)
neurons; #, p < 0.05 versus IGF-1-treated
p53(+/+) neurons in the absence of wortmannin.
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IGF-1 Mediates Inhibited DNA Binding and Transcriptional Activity
of p53 with No Effect on p53 Protein Accumulation--
The functional
status of p53 to activate transcription strongly correlates with its
ability to bind specific DNA sequences on target genes (31). To examine
the effect of IGF-1 on p53 accumulation, DNA binding activity, and
transcriptional activity, primary rat hippocampal neurons were
pretreated with or without IGF-1 in the presence or absence of
wortmannin followed by exposure to hypoxia or SNP treatments for up to
24 h. 24 h after exposure to hypoxia or SNP, cells were lysed
and the extracts were subjected to Western blot analysis using anti-p53
monoclonal antibody. As shown in Fig.
3A, IGF-1 treatment with or
without pretreatment of wortmannin had no effect on p53 accumulation in
neurons exposed to hypoxia or SNP treatment. We then examined the
effect of IGF-1 on p53's DNA binding activity by EMSAs. Nuclear
extract were prepared 8 h after treatment with hypoxia or SNP, and
the EMSAs were performed using the p53 consensus response element as a
probe (5'-AGCTTAGACATGCCTAGACATGCCTA-3'). As shown in Fig.
3B, treatment with IGF-1 inhibits hypoxia- or SNP-induced
increases in p53's DNA binding activity. Pretreatment with wortmannin
apparently antagonized the IGF-1 effect and allowed increased p53 DNA
binding activity. Excess unlabeled probe effectively competed the
binding of the labeled probe (Fig. 3B). To examine whether
IGF-1 affects the transactivation potential of p53, we performed
reporter gene assays using reporter plasmids containing a tandem repeat
of a p53 consensus response element placed upstream of a luciferase
cDNA (p53-Luc, Stratagene). 24 h after transient transfection
with the reporter plasmid, cells were exposed to hypoxia or SNP
treatment for 8 h in the presence or absence of IGF-1. Although we
observed 8-9-fold induction of the luciferase activity by hypoxia or
SNP treatment, pretreatment with IGF-1 remarkably reduced the increase
in the transcriptional activation of p53, which was prevented by
wortmannin (Fig. 3C).

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Fig. 3.
Effect of IGF-1 on cellular accumulation, DNA
binding, and transcriptional activity of p53. Cultured rat
hippocampal neurons pretreated with IGF-1 (100 ng/ml) or saline
(vehicle) in the presence or absence of wortmannin (W; 20 nM) were exposed to hypoxia or SNP (50 µM).
A, Western blot analysis. Extracts of neurons were subjected
to 10% SDS-PAGE. Then the blots were probed with antibody to p53.
Visualization of proteins was performed with ECL. B, EMSAs.
Nuclear extracts prepared from neurons exposed to hypoxia or SNP for
8 h were assayed by EMSAs. "Cold" means the addition of excess
of unlabeled oligonucleotide containing p53 consensus binding element
into binding reactions as indicated -fold. The arrowhead
points to the p53/p53 consensus binding element oligonucleotide
complex. C, reporter gene assays. Primary hippocampal
neurons were transiently cotransfected with reporter construct either
p53-Luc plasmid or control plasmid, together with pRL-TK plasmid, and
then neurons were treated with IGF-1 in the presence or absence of
wortmannin. After 24 h of treatments, cultures were exposed for
8 h to hypoxia or SNP (50 µM) and luciferase
activity was assayed. Relative luciferase activity was expressed as
-fold increase, compared with control LacZ-expressing neurons without
any stimuli (Control). Data are mean ± S.E. of four
independent experiments performed in triplicate. *, p < 0.05 versus nontreated neurons.
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Activation of Akt Kinase Inhibits p53-mediated Neuronal
Death--
Akt, a downstream target of PI 3-kinase, has been
implicated in transducing growth factor survival signals in various
cell types including neurons (32). As we found that IGF-1 inhibits p53-mediated neuronal death through activation of PI 3-kinase, we then
assessed a potential function of Akt activation in the prevention of
p53-mediated neuronal death using an adenoviral vector capable of
expressing constitutively active Akt with the Src myristoylation signal
fused in-frame to the c-Akt coding sequence (AxCALNLmyrAkt). As a
control, we employed another vector capable of expressing a
kinase-defective Akt mutant (AxCAAktK179M) in which the lysine of the
adenosine triphosphate-binding site at position 179 was replaced by a
methionine. Primary hippocampal neurons were infected with AxCANCre and
either AxCALNmyrAkt or AxCALNLZ, a control virus carrying LacZ instead
of myrAkt. Other cells were infected with AxCAAktK179M, and whole cell
extracts were prepared for Western blot analysis 24 h after viral
infection. Western blot analysis revealed increased levels of Akt and
phosphorylated Akt in neurons infected simultaneously with AxCANCre (2 m.o.i.) and AxCALNmyrAkt (2 and 10 m.o.i.), in a
m.o.i.-dependent manner (Fig.
4A). As the myrAkt
lacks its pleckstrin homology domain, the size of the protein product
(47.5 kDa) is smaller than endogenous Akt or the product of
kinase-defective Akt (62 kDa). The cells infected with AxCANCre (2 m.o.i.) and AxCALNLZ (10 m.o.i.) did not show increased levels of Akt
protein or phosphorylated Akt protein (Fig. 4A). Infection
with AxCANCre (2 m.o.i.) and Ax CALNLmyrAkt (10 m.o.i.) to express
activated Akt significantly reduced neuronal death and caspase-3-like
activation induced by hypoxia, SNP, and p53 adenovirus infection
compared with infection with AxKDAkt (10 m.o.i.) to express inactive,
kinase-defective Akt protein (Fig. 4, B and C).
Similarly, infection with AxCALnLZ (10 m.o.i.) failed to reduce
neuronal death compared with cells expressing activated-Akt kinase.

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Fig. 4.
Activation of Akt kinase inhibits p53-induced
neuronal death. A, assessment of adenovirus-mediated
expression of active and kinase-defective forms of Akt. Cultured rat
hippocampal neurons were infected with AxCANCre (AxCre) and
AxCALNLZ (AxLacZ) or AxCALNLmyrAkt (AxAkt), or
AxCAAktK179M (AxKDAkt) at the indicated m.o.i. 24 h
after infection, cell extracts were subjected to 10% SDS-PAGE and
blots were probed with antibodies for Akt or phospho-Akt. B
and C, cultures were infected with both AxCANCre
(AxCre; 2 m.o.i.) and AxCALNLZ (AxLacZ;
10 m.o.i.) or AxCALNLmyrAkt (AxAkt; 10 m.o.i.), or
AxCAAktK179M (AxKDAkt; 10 m.o.i.) alone. 24 h
after infection, neurons were exposed for 24 h to hypoxia, 50 µM SNP or infected with Adp53 (50 m.o.i.). Then, the
neuronal viability (B) and caspase-3-like activity
(C) were assessed as described in the text. Data are
mean ± S.E. of four independent experiments performed in
triplicate. *, p < 0.05 versus neurons
infected with both AxCre and AxLacZ in each treatment.
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Effect of Akt Kinase Activation on p53 Accumulation, Nuclear
Translocation, Phosphorylation Status, DNA Binding Activity, and
Transcriptional Activation--
Since we show that IGF-1 reduces the
DNA binding activity and transcriptional activity of p53 without
changing p53 protein accumulation through PI 3-kinase (Fig. 3,
A-C), we further tested whether Akt activation inhibits
changes in p53 accumulation, nuclear translocation, DNA binding
activity, and transcriptional activity that are induced by hypoxia
treatment, SNP treatment, or Adp53 infection. Cells infected with both
AxCANCre (2 m.o.i.) and AxCALNLmyrAkt (10 m.o.i.) or AxCALNLZ (10 m.o.i.) or infected with AxCAAktK179M (10 m.o.i.) were exposed to
hypoxia or SNP (50 µM) or infected with Ad5CMVp53 (50 m.o.i.). Cellular accumulation, nuclear translocation, and DNA binding
activity of p53 were assessed at 24, 12, and 8 h, respectively,
after exposure to hypoxia or SNP, and at 24 h after AdCMVp53
infection. Cellular accumulation and nuclear translocation of p53 were
observed following all the treatments and were not changed by
expression of active Akt, kinase-defective Akt, or LacZ (Fig.
5, A and B).
However, EMSAs using the p53 consensus response element as a probe
showed that expression of active Akt, but not of kinase-defective Akt
or LacZ, did inhibit the hypoxia-, NO-, or Adp53-induced increase in
DNA binding activity of functional p53 to the consensus binding
oligonucleotide (Fig. 5C). Consistent with this finding,
reporter gene assays using reporter plasmids containing tandem repeats
of p53 consensus response element placed upstream of a luciferase
cDNA revealed that expression of activated Akt, but not of
kinase-defective Akt or LacZ, significantly reduced the induction of
the luciferase promoter that is controlled by the p53-responsive
promoter following hypoxia, SNP, or Adp53 infection (Fig.
5D). Next, to examine the possibility that Akt directly or
indirectly phosphorylates p53 and thereby regulates p53's
transcriptional activity, the phosphorylation status of p53 was
assessed by an in vivo kinase assay. Rat primary cultured
neurons were infected with both AxCANCre (2 m.o.i.) and AxCALNLmyrAkt
(10 m.o.i.) or AxCALNLZ (10 m.o.i.) or infected with AxKDAkt (10 m.o.i.) alone. After 24 h of incubation, cells were infected with
Ad5CMVp53 (50 m.o.i.) and incubated for an additional 24 h to
overexpress p53. Then cells were incubated for 3 h in the medium
containing 0.2 mCi/ml [32P]orthophosphate. The
phosphorylation status of p53 was monitored by immunoprecipitating p53
protein and quantifying p53-associated radioactivity by
autoradiography. p53 protein expression was monitored by Western
blotting analysis. As shown in Fig. 5E, our in
vivo kinase assay showed that neither expression of active or
kinase-defective Akt has any effect on phosphorylation status of p53
(Fig. 5E).

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Fig. 5.
Effect of Akt activation on cellular
accumulation, nuclear translocation, DNA binding activity,
transcriptional activation, and phosphorylation status of p53.
A-C, cultured rat hippocampal neurons were infected
AxCANCre (AxCre; 2 m.o.i.) and AxCALNLZ
(AxLacZ; 10 m.o.i.) (Cont. and
lane 1) or AxCALNLmyrAkt (AxAkt;
10 m.o.i.) (lane 2) or AxCAktK179M
(AxKDAkt; 10 m.o.i.) (lane 3). At
24 h of infection, neurons were exposed to hypoxia (a)
or SNP (50 µM) (b), or infected with Ad5CMVp53
(Adp53) (50 m.o.i.) (c). After treatment with
hypoxia, SNP or Ad5CMVp53, p53 protein amount in cell lysate
(A) and subcellular fractions (n, nuclear;
c, cytosolic) (B) was assessed by Western blot.
Immunoreactive -tubulin and cytosolic -actin were used to confirm
the identity of the subcellular fractions and protein loading amount,
respectively. C, EMSAs. After 8 h of treatment with
hypoxia or SNP, or 24 h of infection with Ad5CMVp53, nuclear
extracts prepared from neurons were assayed in EMSAs using p53
consensus response element as the 32P-labeled
oligonucleotide. The arrowhead points to the p53/p53
consensus binding element oligonucleotide complex. Cont,
control LacZ-expressing cells without treatment with hypoxia, SNP, or
Ad5CMVp53. D, reporter gene assays. Primary hippocampal
neurons were transiently cotransfected with reporter construct either
p53-Luc plasmid or control plasmid, together with pRL-TK plasmid, and
then neurons were infected or not infected with AxCANCre (2 m.o.i.) and
AxCALNLZ (10 m.o.i.) or AxCALNLmyrAkt (10 m.o.i.), or AxCAAktK179M (10 m.o.i.) alone. After 24 h of virus infection, cultures were
exposed for 8 h to hypoxia or SNP, or for 24 h infected with
Ad5CMVp53 (50 m.o.i.) and luciferase activity was assayed. Relative
luciferase activity was expressed as -fold increase, compared with
control LacZ-expressing neurons without any stimuli (Cont).
Data are mean ± S.E. of four independent experiments performed in
triplicates. *, p < 0.05 versus control
LacZ-expressing cells in each treatment. E, phosphorylation
status of p53. Cultured rat hippocampus neurons were infected with both
AxCANCre (2 m.o.i.) and AxCALNLZ (10 m.o.i.) or AxCALNLmyrAkt (10 m.o.i.), or infected with AxCAAktK179M (10 m.o.i.) alone. At 24 h
of viral infection, neurons were infected with Ad5CMVp53 (50 m.o.i.)
(lanes 2-5) and incubated for additional 24 h. Neurons were then metabolically labeled in a medium containing 0.2 mCi of [32P]orthophosphate/ml. After 32P
labeling, p53 proteins were recovered by anti-p53 antibody
immunoprecipitation and subjected to SDS-PAGE, which was dried for
analysis by autoradiography. The loading internal control was assessed
by Western blot analysis for p53.
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Akt Activation Inhibits p53-mediated Expression of Bax, a
Proapoptotic Gene--
Previous studies have shown that the Bax genes
contain p53 response elements in their promoter and that their
expression is transcriptionally regulated by functional p53 (3). As a
member of the Bcl-2 family, Bax is required to initiate a
p53-dependent cell death pathway in neurons (33, 34).
Therefore, we examined whether Akt activation inhibits p53-mediated Bax
expression. Cells were infected with both AxCre (2 m.o.i.) and AxAkt
(10 m.o.i.) or AxLacZ (10 m.o.i.) or with AxKDAkt (10 m.o.i.) alone and
allowed to rest for 24 h before treatment with hypoxia, with SNP
(50 µM), or with infection with Adp53 (50 m.o.i.) for
another 24 h. Expression of active Akt, but not the
kinase-defective form of Akt, significantly inhibited up-regulation of
Bax mRNA levels following exposure of cells to hypoxia, to SNP or
to infection with p53 adenovirus, as assessed by RT-PCR (Fig.
6A). Similar to increasing
mRNA levels, Western blot analysis also revealed that expression of
active Akt, but not kinase-defective Akt, blocked the p53-mediated
increase of Bax protein levels (Fig. 6B).

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Fig. 6.
Akt kinase activation inhibits p53-mediated
expression of Bax. Cultured hippocampal neurons were infected with
AxCANCre (2 m.o.i.) and AxCALNLZ (10 m.o.i.; lanes
1 and 2) or AxCALNLmyrAkt (10 m.o.i.;
lane 3), or AxCAAktK179M (10 m.o.i.;
lane 4). 24 h after infection, neurons were
exposed to hypoxia or SNP (50 µM) or infected with
Ad5CMVp53 (50 m.o.i.). Lane 1, control
LacZ-expressing cells without treatment with hypoxia, SNP, or
Ad5CMVp53. A, RT-PCR for bax mRNA. At 24 h after
treatment with hypoxia, SNP (50 µM) or Ad5CMVp53 (50 m.o.i.), total RNA was extracted from neurons and RT-PCR for Bax and
-actin gene was performed using cDNAs made from total RNA as
template. Results were confirmed by three repeated assays.
B, Western blot analysis for Bax. At 24 h after
treatment with hypoxia, SNP, or Ad5CMVp53, extracts of neurons were
subjected to 10% SDS-PAGE and the blots were probed with antibodies to
Bax and -actin. Results were confirmed by three repeated
assays.
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DISCUSSION |
In the present study, we demonstrate that Akt can significantly
inhibit p53-mediated neuronal death by reducing the ability of p53 to
transactivate gene transcription. Previous data showed that p53 is
up-regulated in neuronal cells in response to diverse insults such as
ischemia, and Bax-mediated caspase-3 activation plays an important role
in p53-induced apoptosis of neuronal cells (35). In this study,
treatment of p53-null neurons with hypoxia or a NO donor turned out to
reduce the induction levels of caspase-3-like activity, DNA
fragmentation and neuronal death when compared with those of wild-type
neurons. Exogenous adenovirus-mediated transduction of p53 expression
in p53-deficient neurons up-regulated caspase-3-like activity and
induced caspase-dependent DNA fragmentation. These results
strongly suggest that p53 expression induced by treatments with hypoxia
or a NO donor is responsible for a large portion of caspase-mediated
apoptosis in primary hippocampal neurons.
A variety of mammalian cells are dependent on growth factors for their
survival. Certain growth factors may also confer protection against
apoptosis by stimulating PI 3-kinase and/or a downstream effector, the
Akt kinase, in various cell types including neuronal cells (36). The
present study showed that IGF-1 blocked p53-mediated caspase-3-like
activation and cell death via PI 3-kinase. Adenovirus-mediated expression of active Akt inhibits caspase-3-like proteolytic activity and protects neurons from apoptosis induced by hypoxia, SNP, or p53
adenovirus treatment. These results clearly indicate that activation of
Akt is capable of suppressing the caspase-mediated neuronal death that
is provoked by p53 expression.
How does Akt activation inhibit p53-mediated neuronal death? The final
outcome of p53 activation depends upon many factors including the
downstream targets associated with growth control and cell cycle
checkpoints (p21, GADD45, MDM2), DNA repair (GADD45, p21, PCNA), and
apoptosis (Bax, Bcl-x, Fas, IGF-BP) (37). Sequence-specific transactivation is required for p53-induced apoptosis in specific experimental systems (38). On the other hand, p53-mediated apoptosis does not necessarily require transcriptional activation (39). Here, we
show that the p53 protein induced by treatments with hypoxia, with SNP,
or with p53 adenovirus infection appears to serve as a site-specific
transcription factor that transactivates pro-apoptotic genes such as
Bax. In addition, the increase in p53's transcriptional activity was
inhibited by expression of active Akt, but not of kinase-defective Akt.
This observation was further confirmed by endogenous activation of Akt
induced by IGF-1. Furthermore, p53-mediated Bax expression was also
inhibited by expression of active Akt. Considering the previous
observations that demonstrate a role of Bax in p53-mediated cell death
(33, 34), our results suggest that activation of Akt kinase serves to
protect neurons from apoptosis, at least in part, by inhibiting the
transcriptional activity of p53.
There are several mechanisms that may explain how Akt suppresses
transcriptional activity of p53. Several factors appear to contribute
to the regulation of p53's transcriptional activity including
post-translational protein modification, status of protein conformation, p53 protein levels, and its cellular localization (40).
In our experiments, activated Akt had no effect on the protein
accumulation and nuclear translocation of p53. In addition, Akt
activation has no effect on phosphorylation status of p53. Considering
that essentially all activated Akt translocates into nucleus where it
functions to phosphorylate proteins (41), these data suggest that Akt
phosphorylates coactivators that directly or indirectly modify p53's
transcriptional activity, thereby reducing p53-mediated transcriptional
activity and apoptosis. Additional experiments will be required to
determine the precise mechanism by which activation of Akt kinase
inhibits p53's transcriptional activity.
In summary, we propose a novel mechanism in which activation of Akt
inhibits p53-mediated neuronal death. More specifically, we report that
Akt activation inhibits the transcriptional activity of p53, which we
show leads to a reduction of Bax expression and a reduction in neuronal
apoptosis. Thus, in addition to interfering with apoptotic events
involving Bad and caspase-9, Akt activation appears to be capable of
suppressing p53's function at the level of p53-mediated transcription.