From the Department of Anatomy and Neuroscience and
** Department of Geriatric Medicine, Graduate School of Medicine, Osaka
University, Suita, Osaka 565-0871, Japan, § CREST, Japan
Science and Technology,
Department of Anatomy, Asahikawa Medical
College, Asahikawa, Hokkaido 078-8510, Japan, and the
Division of Neurology, Duke University
Medical Center, Durham, North Carolina 27710
Received for publication, July 12, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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Upon binding to the cAMP-response element of a
gene's promoter, the transcription factor known as cAMP-response
element-binding protein (CREB) facilitates transcription of many
different neuronal genes including those involved with synaptic
function. Based on our previous reports of gene structure
(GenBankTM accession number AF029701), we now
demonstrate that activated CREB binds to the proximal promoter of the
human presenilin-1 (PS-1) gene to activate PS-1 transcription in rat
and in human neuronal cells. Specific stimulation of the
N-methyl-D-aspartate subtype of neuronal
glutamate receptors activates CREB and results in increased PS-1
expression. Similarly, treatment with brain-derived neurotrophic
factor activates CREB and increases PS-1 expression in a
dose-dependent fashion. By using adenovirus vectors
expressing dominant negative forms of CREB, we were able to show that
induction of PS-1 expression requires the activation of CREB.
Conversely, constitutive expression of mitogen-activated protein
kinase/extracellular signal-regulated kinase (MEK) results in
activation of CREB and increased PS-1 expression that can be blocked by
the addition of selective MEK inhibitors. Our findings suggest a
hypothesis where stimulation of
N-methyl-D-aspartate receptors signals CREB activation to enhance PS-1 gene product expression that contributes to
normal neuronal functions.
Alzheimer's disease is a devastating neurological disorder
characterized by progressive memory loss and cognitive deficits. To
date, four genes have been found to be associated with Alzheimer's disease phenotypes including the amyloid precursor protein gene on
chromosome 21 (1-4), the apolipoprotein E gene on chromosome 19 (5-7), the presenilin-1
(PS-1)1 gene on chromosome 14 (8), and the presenilin-2 (PS-2) gene on chromosome 1 (9, 10). The
majority of familial Alzheimer's disease cases are associated with
over 40 independent mutations in the PS-1 genes of unrelated families
that all display an early-age-of-onset Alzheimer's phenotype (8,
11-25). Although the current literature suggests that intracellular
PS-1 is intimately associated with the Human PS-1 is widely expressed in a variety of organs throughout the
body (8). This distribution raises the question as to why mutations in
the PS-1 gene and its products appear to confer a disease state that
selectively affects the brains of patients with familial Alzheimer's
disease without apparent effect on their peripheral organs. In
contrast, rat PS-1 is poorly expressed in liver, kidney, lung, and
heart (27), and mouse PS-1 is poorly expressed in skeletal muscle and
spleen.2 We also reported
that the PS-1 promoter was more active in cells differentiated to
become neurons than in cells differentiated to a muscle phenotype (28).
These differences suggest both cell type-specific and species-specific
distributions of PS-1 expression. The brain, however, is the only organ
where PS-1 is universally well expressed in human, rat, and mouse
species. Using in situ hybridization, PS-1 mRNA is most
highly expressed in neurons and below the limit of detection in other
brain cells (29, 30). These results strongly suggest the existence of a
mechanism where PS-1 transcription is regulated in a neuron-specific
manner in the brain.
To understand presenilin-1 function, one of our approaches is to study
regulatory mechanisms in the brain. PS-1 expression is higher in the
hippocampus and cerebellum than in the cerebral cortex (29-32).
Hippocampal pyramidal neurons, dentate granule neurons, cerebellar
Purkinje neurons, and primary olfactory cortical neurons show higher
PS-1 expression than the neurons in other cortical regions of rat brain
(33, 34). Compared with the adult, the level of PS-1 expression is
significantly higher in the developing rat brain. Peaking at postnatal
day 10, PS-1 expression in the hippocampus and cerebellum is greatest
at a time when proliferation, migration, and synapse formation are
actively being completed (35).
Like PS-1, N-methyl-D-aspartate (NMDA) receptors
are well expressed in hippocampal pyramidal neurons and dentate granule
neurons at this time of development (36-39). Activation of NMDA
receptors and consequent Ca2+ entry into postsynaptic
spines are critical to the developmental maturation of the brain, to
establishment of long term memory, and to the plasticity of synaptic
transmission that are characteristic of functional brain neurons
(40-44). Furthermore, glutamate released from presynaptic terminals
can bind to glutamate receptors at the postsynaptic membrane
stimulating Ca2+ influx, phosphatase, and kinase activities
that serve to modulate several intracellular signaling systems. Of
these systems, phosphorylation of cAMP-response element-binding protein
(CREB) activates a signal transduction cascade that eventually
modulates downstream protein synthesis in postsynaptic neurons
(45-48).
An important relationship between these systems emerged when sequencing
of the PS-1 promoter showed the existence of a CREB binding site near
the major transcription start site. If CREB regulates the expression of
PS-1, then control of CREB activation may be one of the keys to
understanding the differences in PS-1 expression between neurons,
between nonneuronal cells, during the steps of synapse formation, and
during signal transmission at synapses. We now report that activation
of CREB leads to increased PS-1 expression in a
dose-dependent fashion. We also find that PS-1 colocalizes
with neurons expressing NMDA receptors. Thus, we now test the ability
of NMDA receptors to signal CREB activation and specifically stimulate
PS-1 expression. Our findings support a hypothesis where NMDA-mediated
CREB activation regulates PS-1 expression to play a potentially
critical role in physiological and pathological, neuron-specific
functions of presenilin-1.
Reagents--
U0126 and brain-derived neurotrophic factor (BDNF)
were purchased from Promega (Madison, WI). Nifedipine and NMDA were
purchased from Sigma. (+)-MK801 hydrogen malate was purchased from
Research Biochemicals International (Natick, MA). Monoclonal antibody
to the amino-terminal fragment of PS-1 (anti-PS-1-NTF) was purchased from Chemicon International (Temecula, CA). Anti-CREB monoclonal antibody (CREB-1, 24H4B) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Cell Culture--
SK-N-SH human neuroblastoma cells were
purchased from the American Type Culture Collection and were routinely
propagated in Dulbecco's modified Eagle's medium (Sigma) with 10%
fetal calf serum (Life Technologies, Inc.). Cultures of rat primary
hippocampal neurons were prepared from Harlan Sprague-Dawley rat
embryos at embryonic day 18 as described previously (49). All
experiments were performed in 5-8-day-old cultures.
Isolation of Genomic DNA Clones for the Promoter Region--
The
PCR primer pairs 951/952 and 910/911 amplify PS-1 specific sequences
(8) and were used with PCR to screen a human genomic library in a PAC
vector, kindly provided by Dr. Pannos A. Ioannu (50). Out of the three
PAC clones identified, PAC94 was digested with EcoRI,
and the restriction enzyme fragments were subcloned into
pBluescript-II-KS (+) phagemid vector (Stratagene) using a DNA ligation
kit (Stratagene). The oligonucleotide probe
(5'-TGGGACAGGCAGCTCCGGGGTCCGCG-3') from exon 1 of the human PS-1 gene,
labeled with Searching Transcription Factor Binding Sites--
Potential
binding sites of vertebrate transcription factors in the human PS-1
promoter were identified using the TFSEARCH (version 1.3) network
service available from Genome Net in Japan.
Isolating Nuclear Protein Extracts to Confirm Functional
Transcription Factor Binding Sites--
Nuclear extracts were prepared
from NMDA-stimulated SK-N-SH cells according to published methods
(51-52) with minor modifications. In brief, cells were stimulated with
100 µM NMDA for 30 min, harvested by scraping, and washed
with 0.5 volumes of ice-cold phosphate-buffered saline followed by a
gentle centrifugation. The cells were then washed in 0.1 volume of cold
buffer A (10 mM HEPES (pH 7.9), 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM
dithiothreitol). The washed cell pellets were then suspended in 50 µl
of buffer A plus 0.1% Nonidet P-40 supplemented with 1 µg/ml
leupeptin and 1 µg/ml aprotinin and incubated on ice for 10 min
before briefly vortexing to mix the pellets, followed by centrifugation
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 ice-cold buffer C (20 mM HEPES,
25% glycerol, 0.42 M NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) containing 1 µg/ml leupeptin and 1 µg/ml aprotinin and then
incubated on ice for 15 min with intermittent vortexing. The extracts
were then centrifuged at 10,000 rpm at 4 °C for 10 min, and the
supernatant was removed and divided into aliquots for freezing at
Electrophoretic Mobility Shift Assay--
Probes corresponding
to the CRE-like element of the human PS-1 promoter sequence (PS1-CRE;
5'-GAGCCGGAAATGACGACAACGGTGAGG-3') and to a mutated CRE-like site
containing three nucleotide substitutions (PS1-CREm;
5'-GAGCCGGAAATGATAGCAACGGTGAGG-3') were synthesized as complementary
double-stranded oligonucleotides. As a control probe, a consensus CRE
element contained in a double-strand oligonucleotide probe
(5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3') was purchased from Promega. The
probes were 5'-end labeled with [ Construction of PS-1 Promoter-Firefly Luciferase
Reporters--
The plasmid clone A4, which contains a human PS-1
genomic DNA fragment inserted into the EcoRI site of
pBluescript II KS (+) vector, was double-digested with NotI
and one of SalI, HindIII, or KpnI
restriction enzymes, respectively. The promoter fragments were purified
with Wizard PCRpreps DNA Purification System (Promega), blunted with a
DNA blunting kit (Takara Biochemicals), and inserted into the
SmaI site of promoterless pGL3-basic vector (Promega) to
make LUC5, LUC4, and LUC3. Similarly, clone A4 was digested with
HindIII alone or HindIII and KpnI,
blunted, and inserted into pGL3 to make LUC2 and LUC1, respectively.
Two synthetic oligonucleotides, PS1-CRE-S
(5'-TCGAGAGCCGGAAATGACGACAACGGT-3') and PS1-CRE-AS
(5'-AGCTACCGTTGTCGTCATTTCCGGCTC-3') were 5'-phosphorylated with ATP and
T4-polynucleotide kinase, mixed together, heated to 95 °C to
denature, gradually cooled down to room temperature to anneal, and then
inserted into the XhoI/HindIII site of pGL3-basic
vector to make LUC6. Similarly, two synthetic oligonucleotides,
PS1-CREm-S (5'-TCGAGAGCCGGAAATGATAGCAACGGT-3') and PS1-CREm-AS
(5'-AGCTACCGTTGCTATCATTTCCGGCTC-3'), which are identical to the
PS1-CRE-S and PS1-CRE-AS, respectively, except for the substitution of
three nucleotides at the CRE-like site, were inserted into the
XhoI/HindIII site of pGL3-basic vector to make LUC6m.
Transient Transfection and Luciferase Assays--
SK-N-SH cells
were plated in six-well tissue culture dishes at 9 × 104 cells/well and allowed to recover for 1 day. Cells
containing PS-1-promoter/reporter constructs were then cotransfected
with 0.3 pmol of one of the promoter-firefly luciferase plasmid
constructs, pGL3-basic vector or pGL3-promoter plasmid (which
contains an SV40 promoter upstream of the firefly luciferase gene;
Promega), and 0.3 pmol of pRL-TK plasmid (which contains a herpes
simplex virus thymidine kinase promoter upstream of the
Renilla luciferase gene; Promega), using the Lipofectin
procedure (Life Technologies, Inc.) as described in the manufacturer's
protocol. 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 the Dual-Luciferase Reporter Assay System (Promega)
and a model TD-20E luminometer (Turner Design). After measuring the
firefly luciferase signal (FL) and the Renilla (sea pansy)
luciferase signal (RL), the relative promoter activity (RPA) was
calculated as follows: RPA = FL/RL.
Northern Blot Hybridization Analysis--
Total RNA extracted
from SK-N-SH cells after incubation with or without NMDA for 24 h
was Northern blotted at 20 µg/lane. A 368-bp human PS-1 cDNA
probe sequence was prepared using PCR with sense primer
(5'-CGGGGAAGCGTATACCTAATC-3'), antisense primer (5'-TCAGCTCCTCATCTTCTTCCTCATC-3'), and human PS-1 cDNA as template. The probe was 32P-labeled using the Random Primed DNA
Labeling Kit (Roche Molecular Biochemicals) and hybridized to Northern blots.
Recombinant Adenovirus--
Constitutively active
mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase (MEK) adenovirus (Ad-caMEK) was constructed as follows. cDNA
fragments comprising the entire coding regions for human MEK1 cDNA
were isolated from HEK293 cell cDNAs by using PCR with sense
(sequence of sense) and antisense (sequence of antisense) primers.
Constitutively active MEK, which lacks its nuclear export signal (amino
acids 30-51; Ref. 53) and in which two phosphorylation sites, serine
218 and serine 222, are converted to glutamic acids, was prepared by
site-directed mutagenesis as described previously (54). Subsequently, a
c-Myc tag sequence was fused to its N-terminal by PCR. The fragment was
inserted into pAxCAwt cosmid vector (55) and was designated as
pAxCAca-MEK.
A dominant negative form of CREB with a mutation in the DNA binding
domain (K-CREB adenovirus or Ad-KCREB; Ref. 56) was constructed as
follows. A plasmid bearing wild type rat CREB cDNA was kindly
provided by Dr. Hagiwara (Tokyo Medical and Dental University, Japan).
The coding sequence was amplified, and a c-Myc tag sequence was fused
to its N terminus by PCR. Subsequently, leucine was substituted for
arginine 301 by site-directed mutagenesis using the QuickChange
Site-Directed Mutagenesis Kit (Stratagene). The fragment was subcloned
into pAxCALNLw, Cre-inducible expression cassette (16), and was
designated as pAxCALNL-KCREB.
The cosmids, pAxCAca-MEK and pAxCALNL-KCREB, were separately
transfected into 293 cells together with the EcoT221-digested adenoviral DNA-terminal protein complex (55) using the calcium phosphate precipitation method. The desired recombinant adenoviruses generated through homologous recombination were purified through a
CsCl2 gradient followed by dialysis.
The recombinant adenovirus, AxCANCre, which efficiently produces a
nuclear localization signal-tagged Cre recombinase under control of the CAG promoter (57), and AxCANLZ, which can express LacZ
under control of the CAG promoter (16), were generously provided by Dr.
Saito (University of Tokyo, Japan).
CREB-M1 adenovirus, a dominant negative form of CREB in which Ser-133
is converted to an alanine and that cannot be phosphorylated or
activated, was a generous gift from Dr. Anthony Zeleznik (University of
Pittsburgh School of Medicine; Ref. 58).
All of the viruses were grown in 293 cells and purified by
CsCl2 gradient centrifugation. Virus titers were determined
by plaque assay, and concentrated virus was stored at Metabolic Labeling and Electrophoresis--
Confluent SK-N-SH
cells and rat primary cultured hippocampal neurons were prepared in
six-well dishes. Medium was changed to the methionine-free Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) with 2% dialyzed
fetal bovine serum (Life Technologies, Inc.) and incubated for 7 h, followed by pulse labeling with 200 µCi/ml
[35S]methionine for 1 h. The cells were then lysed
immediately with ice-cold lysis buffer (50 mM Tris-HCl, pH
7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM
Na3VO4, 0.1% (v/v) 2-mercaptoethanol, 1%
Triton X-100, 50 mM sodium fluoride, 5 mM
sodium pyrophosphate, 10 mM sodium glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 1 µM
microcystin). For NMDA stimulation experiments, 10 or 100 µM NMDA was added to the medium 6 h prior to the
addition of radioisotope. For inhibition experiments, 100 µM MK801 or 20 µM nifedipine was added to
the medium when the medium was changed. For adenovirus transfection experiments, the cells were transfected with 10-50 multiplicity of
infection (m.o.i.) adenovirus 24 h prior to the change of medium. For time course experiments, radioisotope was added to the medium at
the indicated time after 100 µM NMDA was added, and then
the cells were lysed. Immunoprecipitation with polyclonal antibody against the N-terminal fragment of PS-1 was performed as described previously (59). Electrophoresis was performed with 10% Tris-glycine polyacrylamide gel. The gels were vacuum-dried and autoradiographed.
Quantification of PS-1 mRNA and Labeled PS-1
Protein--
The amount of PS-1 mRNA and labeled PS-1 protein was
quantified by scanning the density of radiolabeled or immunodetected bands on x-ray film using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).
Characterization of the Human PS-1 Promoter Sequence--
We
isolated and sequenced a 5571-bp human genomic DNA that contains the
human PS-1 promoter. This genomic DNA includes 4353 bp of the
5'-flanking sequence of exon 1 and exon 2 (Fig.
1 and GenBankTM Accession
number AF029701). The comparison of our sequence for the human PS-1
promoter with previously reported sequences (Ref. 60 and
GenBankTM accession number L76518) indicated general
agreement with several minor differences. This region contains
four Alu subfamily repeat sequences where the 3'-end point of the last
Alu sequences is position number Human PS-1 Proximal Promoter Binds to CREB--
To verify that the
CRE sequence appearing in the PS-1 proximal promoter functionally binds
to the CREB transcription factor, we synthesized the double-stranded
oligonucleotide probe with the PS-1 sequence (PS1-CRE) and the probe
identical to PS1-CRE except for the substitution of three nucleotides
at the CRE-like site (PS1-CREm) and used them for electrophoretic
mobility shift assays. A similar, but nonidentical double-stranded
oligonucleotide with a consensus sequence that binds CREB (CRE) was
used as a positive control. The PS1-CRE sequence contains a
TGACGACA element, the consensus CRE sequence contains a
TGACGTCA element, and the PS1-CREm sequence contains a
TGATAGCA element (Fig.
2A).
Each of these elements was radiolabeled and used to probe the binding
proteins present in nuclear extracts of cells. Radiolabeled CRE probe
incubated with a nuclear extract from NMDA-stimulated SK-N-SH cells
formed complexes that were observed on nondenaturing polyacrylamide
gels as one strong band and one weaker band (Fig. 2B,
bands a and b). To show that these
complexes contain the CREB protein, anti-CREB monoclonal antibody was
added to the reaction, and the observed band was supershifted to an
apparently higher molecular weight (Fig. 2C, band
ss). To show that the antibody reaction was specific for
CREB protein, a control antibody (anti-
Using this assay, we then tested whether the CRE-like element present
in the PS-1 promoter region could function to bind CREB proteins.
Incubation of radiolabeled PS1-CRE probe with nuclear extracts revealed
the same a and b bands that were observed when a
consensus CRE probe was employed (Fig. 2B). When anti-CREB
antibody was added to the PS1-CRE reaction, a supershifted band was
observed (Fig. 2D, band ss),
indicating that CREB is a component of this protein-PS1-CRE-probe
complex as well. As another control, the addition of a molar excess of
unlabeled PS1-CRE oligonucleotide probe to the nuclear extracts
inhibited detection of this protein-PS1-CRE-probe complex (Fig.
2D, lane 4). The specificity of the
complex formation between CREB proteins and PS1-CRE-probes was also
tested using a mutated PS1-CRE probe. Incubation of radiolabeled
PS1-CREm (mutated) probe with nuclear extracts did not reveal the
a or b band (Fig. 2B, lane
4), suggesting that this putative CRE element in the PS-1
promoter is crucial for forming CREB-DNA complexes.
CREB-responsive Element in the PS-1 Proximal Promoter Is
Functional--
To test whether the PS1-CRE element identified in the
proximal promoter could participate in transcriptional activity, we combined this element with a firefly luciferase reporter (Promega; pGL3-basic vector) to look for CREB-mediated activity in human SK-N-SH
cells of neuronal origin. As an internal standard, we used the pRL-TK
vector (Promega), which contains the Renilla luciferase gene
driven by a herpes simplex virus thymidine kinase promoter. Of all of
the DNA fragments tested for promoter activity, plasmid LUC-3
with the human PS-1 fragment
Since basal levels of activated CREB are rather low in these cells, we
asked whether NMDA treatment of these cells would activate CREB by
enhancing its phosphorylation at Ser-133, subsequently resulting in
increased PS-1 expression. 15 min after adding 100 µM
NMDA, CREB was phosphorylated at Ser-133 and remained significantly phosphorylated for another 360 min as shown on Western blots (Fig. 3B). We then tested whether phosphorylation of CREB led to
increased transcriptional activity when CRE element-containing
promoters were present. As a positive control, a CRE-SEAP reporter
plasmid (CLONTECH), which contains three copies of
a consensus CRE element fused to a TATA-like promoter from the herpes
simplex virus thymidine kinase promoter and a secreted alkaline
phosphatase (SEAP) gene, was transfected into SK-N-SH neuroblastoma
cells. NMDA treatment of these cells led to a dose- and
time-dependent increase of extracellular phosphatase
activity (Fig. 3C). These results suggest that NMDA treatment stimulates phosphorylation of CREB, which appears to function
and increase CRE-mediated levels of SEAP activity.
To test whether the PS1-CRE element would also respond to NMDA
treatments that activate CREB, we returned to our luciferase activity
system to measure functional responses. NMDA treatment of SK-N-SH cells
displayed 2.3-fold increase in LUC-3 (
As a control, cells were preincubated with the selective NMDA
receptor antagonist, MK-801, and the subsequent effect of NMDA to
induce PS-1 promoter-directed luciferase activity was significantly inhibited (Fig. 3E). These results suggest that the CRE
element contained in the 26-bp fragment of the PS-1 promoter spanning positions NMDA Treatment Induces PS-1 Gene Expression--
To extend our
finding that CREB activates PS-1 reporter gene expression, we also
evaluated the ability of activated CREB to increase expression of the
endogenous PS-1 gene products. Like the NMDA-dependent
phosphorylation of CREB, NMDA dose-dependently increases
the level of PS-1 mRNA in SK-N-SH neuroblastoma cells (Fig.
4). With respect to protein expression,
we found that treatment of SK-N-SH cells with NMDA for 6 h
followed by a 1-h pulse label with [35S]methionine
resulted in a dose-dependent increase of labeled PS-1
protein levels (Fig. 5A). In
these experiments, full-length PS-1 (PS-1FL) was observed as
a strong band at 45 kDa, and the amino-terminal fragment of PS-1
(PS-1NTF) was observed as faint bands at 30 kDa, suggesting
that full-length PS-1 was detected before its cleavage into
amino-terminal and C-terminal fragments (61, 62). PS-1 protein
synthesis appears to start at 4 h after NMDA treatment and lasts
until 18 h after the treatment (Fig. 5C). We could also
detect several bands that migrated more slowly than the full-length
monomeric PS-1 protein, suggesting that they were aggregated forms of
PS-1 and/or its fragments (PS-1ag).
In cells preincubated with MK801, a selective antagonist of the NMDA
receptor, or with nifedipine, a selective blocker of the L-type voltage
dependent Ca2+ channel (LVDCC), the effect of NMDA
treatment to increase PS-1 protein levels was inhibited. These
inhibitor treatments support a specific and selective role for NMDA and
calcium in stimulating PS-1 gene expression (Fig. 5A). Since
NMDA receptor activation can lead to signal transduction mediated
through the MEK kinase, then inhibition of MEK kinase activity may
modulate PS-1 expression. Preincubation with 10 µM U0126,
a selective MEK inhibitor (63), reduced PS-1 induction, suggesting that
this kinase activity was necessary for PS-1 induction at the protein
level (Fig. 5B).
Relationship of NMDA and CREB to PS-1 Induction--
To test
whether CREB directly regulates endogenous human PS-1 expression, we
used adenovirus vectors to express different dominant negative forms of
CREB before NMDA treatments of neuroblastoma cells and then measured
PS-1 expression levels. K-CREB has a mutation in the domain that binds
to CRE elements in DNA (56), and CREB-M1 contains a serine 133 to
alanine mutation (S133A) that cannot be phosphorylated (58), both of
which result in nonfunctional CREB proteins that fail to stimulate gene
transcription. Overexpression of K-CREB or CREB-M1 dominant negative
forms of CREB in SK-N-SH cells followed by NMDA treatment reduced the
induction of PS-1 gene products, indicating that CREB activation is
essential for NMDA induction of PS-1 gene expression (Fig.
5B). As a control, adenovirus expressing the
lacZ gene product, BDNF Induces PS-1 Expression--
BDNF was also reported to
stimulate phosphorylation at Ser-133, thereby activating CREB (64).
Unlike NMDA treatments, BDNF application does not stimulate influx of
Ca2+ from outside of the cell, but it does cause a slow
intracellular Ca2+ rise that is probably from an
internal Ca2+ store (64). Therefore, we used BDNF to
activate CREB to understand whether the influx of extracellular
Ca2+ from activated NMDA receptors or from L-type
voltage-dependent Ca2+ channel is essential for
the induction of PS-1. BDNF treatment of neuroblastoma cells for 6 h resulted in a dose-dependent induction of PS-1 expression
(Fig. 5D).
Human PS-1 and Rat PS-1 Are Regulated by Phosphorylation of
CREB--
Through the action of one or more kinases, phosphorylation
at Ser-133 is a key step in CREB activation leading to induction of
gene transcription. The mitogen-activated protein kinase/extracellular signal-regulated kinase kinase, MEK, apparently contributes to CREB
activation, because preincubation with 10 µM U0126, a
selective MEK inhibitor, reduced the induction of PS-1 by NMDA in
SK-N-SH cells. On the other side of the coin, adenovirus-mediated
overexpression of constitutively active MEK (54) in SK-N-SH cells
dose-dependently induced PS-1 gene expression, while
control adenovirus expressing Like the amyloid peptide precursor gene, PS-1 is associated with
Alzheimer's disease. Except for rare families with mutations, the
majority of Alzheimer's disease patients express both of these genes
and their normal protein products in a wide variety of cells throughout
the body (8, 27). This distribution raises the question as to how PS-1
appears to participate in a disease that primarily affects only the
brain. Our studies (30) and those of others (29, 34) show that PS-1 is
differentially distributed throughout the human brain. PS-1 is highly
expressed in hippocampal pyramidal neurons, dentate granule neurons,
and cerebellar Purkinje neurons, while other cortical regions display
lower immunoreactivity. To understand this distribution, we began by
examining the transcriptional regulation of the human PS-1 gene to look
for other factors that control PS-1 and other genes of potential
interest in Alzheimer's disease.
Human PS-1 has two independent transcription start sites that are
distinguished by position and activity (Ref. 60 and this report). Our
initial screening of a Marathon-ready human brain cDNA library
(CLONTECH) yielded several human PS-1 cDNA
clones whose 5' terminus was actually upstream of the major
transcription start site reported by Rogaev et al. at that
time (60).3 We performed
primer extension with an antisense primer (5'-AGCCGCTGTTTTGTTTCC-3', Fig. 1C) and total cellular RNA from SK-N-SH cells and found
the major transcription start site that is located 26 bp upstream of
the reported start site (60). Pastorcic et al. (65)
confirmed this new location of the major transcription start site of
PS-1. Similar to our work with the mouse PS-1 gene, we also identified a minor transcription start site some 343 bp downstream of the major
transcription start site. Thus, the majority of the transcripts include
exon 1, which is spliced to exon 3, and a minority of transcripts lack
exon 1 and include exon 2 spliced to exon 3. Although the 5'-ends of
the transcripts are different, the encoded PS-1 proteins are the same,
since the translation starts from the ATG codon in exon 3 (Fig.
1A).
In the human PS-1 promoter sequence (GenBankTM accession
number AF029701), several DNA elements of interest are found that probably control PS-1 gene expression at the transcriptional level. Four Alu repeats, the size of which varies from 178 to 267 bp, were
identified, suggesting to us that the majority of the active elements
in the PS-1 promoter were located between the Alu repeat ending at
The cAMP response element binding protein must be present and must be
activated to bind to CRE elements within the promoter and stimulate
PS-1 transcription. Using immunocytochemistry, we found that CREB and
PS-1 are similarly distributed in the rat hippocampus and dentate
gyrus, being particularly abundant in pyramidal neurons of the
hippocampus and granule neurons of dentate gyrus (data not shown). CREB
that actively binds its DNA element, CRE, is typically phosphorylated
on serine at position 133. We found that the distribution of
phosphorylated CREB was also similar to PS-1. As others have reported,
the presence of phosphorylated CREB is usually associated with the
products of genes that respond to CREB, such as c-Fos, Bcl-2,
and BDNF (45-48).
Activation of CREB by its phosphorylation at serine residue 133 is
modulated by a variety of effectors. NMDA receptors associated with
synapses are involved in neuronal functions like long term potentiation
(44). Stimulation of NMDA receptors has also been associated with
increased calcium influx into cells, potentiation of kinase activities,
and CREB activation. Like hippocampal neurons, the SK-N-SH neuronal
cell line expresses functional NMDA receptors (66). Using
electrophoretic mobility shift assay, we showed that CREB binds to the
human PS-1 proximal promoter. When SK-N-SH cells were stimulated with
10 or 100 µM NMDA, transcription from the human PS-1 gene
was dose-dependently induced as shown by
promoter-luciferase reporter assay (Fig. 3, D and
E), Northern blotting (Fig. 4), and metabolic pulse labeling
with [35S]methionine to detect PS-1 proteins (Fig.
5A). We detected full-length PS-1, the amino-terminal
fragment of PS-1, and aggregated PS-1 proteins, which is consistent
with previous reports showing that PS-1 was cleaved with an average
half-life of 50 min after pulse labeling (67). In these experiments,
most of the labeled PS-1 protein detected was full-length PS-1, so
little amino-terminal fragment of PS-1 was observed. This is because we
employed 1-h pulse labeling, which appeared to be shorter than the
half-life of PS-1 protein, and full-length PS-1 was detected before its cleavage into amino-terminal and C-terminal fragments. Another cause of
the lack of PS-1 amino-terminal fragment observed is that we
used several kinds of protease inhibitors after lysing the cells as
described under "Experimental Procedures," such as phenylmethylsulfonyl fluoride, aprotinin, pepstatin, and leupeptin. Preincubation with MK801, an antagonist of the NMDA receptor, and with
nifedipine, an inhibitor of L-type voltage-dependent calcium channels, inhibited the induction of PS-1, suggesting that the
Ca2+ influx contributes to PS-1 gene induction (Fig. 5,
A and B). Interestingly, BDNF is also known to
phosphorylate CREB at Ser-133 without the participation of NMDA
receptors (64). Even in the absence of NMDA, BDNF
dose-dependently induces the transcription from PS-1 gene
(Fig. 5D). As a whole, these data support our hypothesis that activation of CREB results in increased PS-1 gene expression.
To directly elucidate the effect of CREB on PS-1 gene expression, we
used two adenoviruses expressing dominant negative CREB cDNAs. As a
control, an adenovirus expressing LacZ cDNA was used. CREB-M1, in
which Ser-133 is converted to an alanine and cannot be phosphorylated
or activated, was expressed in neuroblastoma cells and reduced the
expression of PS-1. K-CREB with a mutation in the DNA binding domain
was expressed in neuroblastoma cells and reduced the expression of
PS-1, while control LacZ did not affect the expression (Fig.
5B). These data directly indicate that phosphorylation of
CREB and its binding to the CRE element in the PS-1 promoter are
required for induction of PS-1.
MEK has also been reported to activate extracellular signal-regulated
kinases that can phosphorylate proteins such as CREB, resulting in PS-1
induction. MEK phosphorylates extracellular signal-regulated kinases,
and these activated extracellular signal-regulated kinases
phosphorylate CREB (68). To verify whether MEK can stimulate PS-1
expression, we overexpressed a constitutively active MEK by adenoviral
transduction and observed the dose-dependent induction of
full-length PS-1 protein, while control LacZ adenovirus did not induce
PS-1 (Fig. 6A). The MEK inhibitor U0126 not only inhibited MEK activity, as reported previously (63), but also inhibited PS-1
expression induced by the constitutively active MEK (Fig. 6B). It was also decreased by preoverexpression of either of
the dominant negative CREBs, making it clearer that CREB
phosphorylation is critical for the induction of PS-1 (Fig.
6C). The discrepancy between the effects of the MEK
inhibitor and the dominant negative CREBs in blocking PS-1 induction in
SK-N-SH cells by the constitutively active MEK could be due to
participation of the transcription factor Elk-1, because MEK can
stimulate not only CREB but also Elk-1, and Elk-1 can up-regulate PS-1
transcription as previously described (65). While dominant negative
CREBs only partially inhibited the induction of PS-1 by constitutively
active MEK in SK-N-SH cells, they fully inhibited the induction of PS-1
in rat hippocampal neurons. (Fig. 6, D and E).
This could potentially be due to differences in the organization of the
PS-1 promoter between rat and human, although we do not know the exact
sequence of the rat PS-1 promoter.
CREB activation is associated with acquisition of learning and memory,
which can be demonstrated in the hippocampus (8, 22). Among other
types, glutaminergic neurons in the hippocampus display abundant
numbers of dendritic spines, and their synapses are rich in NMDA
receptors. The activation of NMDA receptors leads to CREB
phosphorylation and Ca2+ influx into these postsynaptic
spines, which is critical for maintenance of synaptic plasticity and
ultimately synaptic transmission (69). When glutamate is released from
the presynaptic membrane of glutaminergic neurons, NMDA receptors open
and cause Ca2+ influx into postsynaptic spines, resulting
in depolarization that triggers the opening of LVDCCS. The
activation of LVDCCs, which open during strongly depolarizing
conditions, permits the entry of a larger amount of Ca2+
along the dendrites and into the cell bodies. This calcium influx can
activate kinases such as extracellular signal-regulated kinase and
calmodulin kinase II that function to phosphorylate CREB (45), resulting in PS-1 induction. Our finding that preincubation with nifedipine almost completely inhibited the PS-1 induction stimulated by
NMDA treatment suggests that the relevant Ca2+ comes
through LVDCC, not the NMDA receptor channel. The importance of calcium
influx, however, is not clear, since overexpression of constitutively
active MEK does stimulate CREB activation and PS-1 expression,
and preoverexpression of dominant negative CREBs reduced the PS-1
induction by constitutively active MEK while failing to stimulate
calcium influx.
We have shown by molecular and cell biological methods that the
activation of the NMDA receptor-CREB pathway regulates PS-1 transcription. Considering that synaptic plasticity of hippocampal neurons is also associated with the activation of the CREB pathway, then the regulation of PS-1 itself may be associated with synaptic plasticity and long term memory.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-secretase activity that
facilitates amyloid-
peptide release (26), the precise function of
PS-1 proteins found in the synaptic membranes remains elusive and is
also a matter of active investigation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P and T4-polynucleotide kinase, was used
to identify plasmid subclones that contain the putative promoter region
by hybridization. One of the positively hybridizing plasmids, clone A4,
was sequenced using an Applied Biosystems model 373A automated DNA
sequencer with a dye terminator cycle sequencing kit and protocols (PE
Biosystems) recommended by the manufacturer.
70 °C and used as purified nuclear extract. Protein concentrations
were determined using the Bio-Rad protein assay kit.
-32P]ATP and
T4-polynucleotide kinase and purified with Nucleotrap Push Columns
(Stratagene). 5 fmol of labeled probes were incubated with 5 µg of
SK-N-SH nuclear extract in a volume of 25 µl containing 0.33 M urea, 0.1 M NaCl, 0.33% Nonidet P-40, 25 mM HEPES, 10 mM dithiothreitol, 10% glycerol,
5 µg of bovine serum albumin, and 2 µg of poly(dI-dC) (Amersham
Pharmacia Biotech) for 30 min at room temperature. For antibody
supershifts, nuclear extracts were incubated with anti-CREB monoclonal
antibody overnight at 4 °C prior to the addition of labeled probes.
For competition experiments, a 10-fold excess of cold competitor probe
was preincubated with nuclear extracts for 15 min at 4 °C prior to
the addition of labeled probes. Incubation mixtures were subjected to
6% polyacrylamide gel electrophoresis using high ionic strength buffer
(50 mM Tris-HCl, pH 8.5, 400 mM glycine, 2 mM EDTA) as running buffer. Gels were vacuum-dried and autoradiographed.
80 °C.
Infection was carried out by adding recombinant adenoviruses to
serum-containing medium. The cells were incubated 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 use unless otherwise stated.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
812. The region spanning from
position
811 to the end of exon 1 at position +140 contains several
specific sequence motifs that are known to serve as binding sites for
transcription factors. These include one Lyf-1 site, two CdxA sites,
one AML-1a site, one Nkx-2 site, one Pbx-1 site, one E47 site, one MZF1
site, two Sp1 sites, one Elk-1 site, and one CREB site for
transcription factors that may bind to these motifs and regulate
expression of the PS-1 gene (Fig. 1b).
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Fig. 1.
Structure of transcripts and promoter of PS-1
gene. A, structure of two different presenilin-1
transcripts from human brain. DNA sequencing of the cloned products of
5'-rapid amplification of cDNA ends on human brain cDNA
revealed the presence of two independent transcripts (transcripts A and
B), which appear to derive from two unique transcription start sites. A
single vertical arrow represents the
major transcription start site at position +1, while a
double vertical arrow represents the
minor transcription start site at position +344. The position of the
major start site was certified by the primer extension method (data not
shown). Exonic regions are labeled and are separated by unlabeled
introns. Splicing patterns are derived from comparison of cDNA
sequence with genomic DNA. The bent arrow
represents the translation initiation site. B, structure of
4.5-kb human presenilin-1 promoter region. The human 4.5-kb
presenilin-1 promoter region contains four Alu-subfamily sequences. The
4.5-kb full sequence appears in GenBankTM
(accession number AF029701). Hatched boxes
represent Alu subfamily sequences. The first Alu subfamily sequence
starts 812 bp upstream from the major transcription start site.
Sequence homologies to consensus motifs for transcription factor
binding sites are marked. C, putative transcription
factor binding sites on PS-1 proximal promoter around the major
transcription start sites. We performed primer extension with an
antisense primer (horizontal arrow) and total
cellular RNA from SK-N-SH cells and found the major transcription start
site. Human PS-1 transcription begins with "A" at position +1 of
exon 1 as indicated with a vertical arrow. Our
transcription start site (bent arrow) is located 26 bp
upstream of the reported start site (60). Pastorcic et
al. (65) confirmed this new location of the major transcription
start site of PS-1. Consensus binding sites for the transcription
factors Elk-1, CREB, and Sp1 are underlined and
labeled.
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Fig. 2.
Interaction of PS-1 proximal promoters and
CREB. Electrophoretic mobility shift assay revealed binding of
CREB to the human presenilin-1 proximal promoter containing putative
binding sites. A, the nucleotide sequences of the PS-1 CRE
probe (PS1-CRE), the control CRE probe (CRE), and the mutant PS-1 CRE
probe (PS1-CREm). The consensus CREB-binding site of CRE probe and the
putative CREB-binding site of PS1-CRE and PS1-CREm are
underlined. The PS1-CRE is identical to the CRE except for
the nucleotide substitution of A for T. The PS1-CREm is identical to
the PS1-CRE except for the substitution of three nucleotides at the
CRE-like site. B, binding of nuclear proteins from
NMDA-stimulated SK-N-SH cells to radiolabeled PS1-CRE probe, CRE probe,
and PS1-CREm probe in an electrophoretic mobility shift assay.
a and b, band of CREB-CRE complex. PS1-CREm probe
did not show either a or b, suggesting that the
putative CRE-binding sequence in PS1-CRE probe is crucial for binding
to CREB. C, binding of nuclear proteins from NMDA-stimulated
SK-N-SH cells to radiolabeled control CRE probe in an electrophoretic
mobility shift assay. a and b, bands of CREB-CRE
complex. ss, band that supershifted with anti-CREB
monoclonal antibody. Anti- -galactosidase polyclonal antibody was
used as a control antibody, and no supershift was observed. Excess cold
CRE probe inhibited the formation of CREB-CRE complex. D,
binding of nuclear proteins from NMDA-stimulated SK-N-SH cells to
radiolabeled PS1-CRE probe in an electrophoretic mobility shift assay.
a and b, band of CREB-PS1-CRE complex.
ss, band that supershifted with anti-CREB monoclonal
antibody. Excess cold PS1-CRE probe inhibited the formation of CREB-CRE
complex.
-gal) was added to the
reaction, and no supershift band was observed (Fig. 2C,
lane 4), indicating that the supershift effect of
the anti-CREB antibody is specific for CRE/CREB-containing complexes.
297 to +189 showed the greatest amount
of firefly luciferase activity relative to the Renilla luciferase internal control. This ratio of LUC-3 activity to
Renilla activity was defined as 100% of relative promoter
activity (100% RPA) (Fig.
3A). Larger DNA fragments such
as LUC-4 (
1191 to +189) and LUC-5 (
4311 to +189) displayed 84% and
25% of the LUC-3 activity (84% and 25% RPA). LUC-1 (
297 to +1258)
and LUC-2 (
1191 to +1258) each displayed less than 10% RPA,
suggesting that they contained negative elements that apparently reduce
overall promoter activity. Although LUC-6 (
18 to +8) was the shortest
fragment tested, it contains a putative CREB binding site and a
putative Elk-1 site and displayed 23% RPA. LUC6m, which is identical
to LUC-6 except for the substitution of three nucleotides at the
CRE-like site, displayed only 8.2% RPA, suggesting that the PS1-CRE
element accounts for about two-thirds of the functional activity of
this PS-1 promoter fragment.
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Fig. 3.
26-bp core element is critical for human
presenilin-1 promoter activation. A, human presenilin-1
promoter-reporter constructs and their relative luciferase activity
(%RLA) under unstimulated conditions. Of all the DNA
fragments tested for promoter activity, plasmid LUC-3 with the human
PS-1 fragment 297 to +189 showed the greatest ratio of firefly
activity to Renilla internal control, which was defined as
100% of relative luciferase activity (100% RLA). B,
phosphorylation of CREB at Ser-133 was induced with 100 µM NMDA in SK-N-SH cells. Western blots of cell lysates
from SK-N-SH cells stimulated with 100 µM NMDA for the
indicated time were probed with anti-CREB and anti-phospho-CREB
(p-CREB) antibodies. C, transcription activity was induced
with 100 µM NMDA in SK-N-SH cells. SK-N-SH cells were
transiently transfected with CRE-SEAP plasmid
(CLONTECH), which contains the SEAP reporter gene
downstream of three copies of the CRE-binding sequence fused to a
TATA-like promoter region from the herpes simplex virus thymidine
kinase promoter. Alkaline phosphatase activities in the media
at the indicated times were measured using Great EscApe SEAP
Chemiluminescence Detec tion Kit (CLONTECH). *, p < 0.01 compared with unstimulated control using Student's t
test. D, NMDA-transactivated PS-1 promoters. SK-N-SH cells
were transiently cotransfected with one of the human presenilin-1
promoter-reporter constructs and pRL-TK plasmid. Cells were cultured
for 24 h with (NMDA) or without 100 µM
NMDA (Unstimulated), FL and RL activities were measured
sequentially, and the RPA was calculated as follows: RPA = FL/RL.
-Fold inductions are shown. Each value represents the average of at
least four independent determinations, and the error
bars indicate the S.E. **, p < 0.01 using
Student's t test (n = 3). E,
MK801 inhibited the activation of PS-1 promoter by NMDA. The cells were
preincubated with 100 µM MK801, a selective NMDA
antagonist, prior to the addition of 100 µM NMDA. **,
p < 0.01 using Student's t test
(n = 3)
297 to +189) activity and
1.6-fold increase in LUC-4 (
1191 to +189) activity compared with
nonstimulated conditions (Fig. 3D). When LUC-6 (positions
18 to +8), the shortest PS-1 promoter fragment that contains a CRE
element, was tested, NMDA treatment displayed a 3.7-fold increase in
its activity compared with nonstimulated conditions. In contrast, the
mutated CRE sequence in LUC-6m displayed similar RPA levels with or
without NMDA treatment. On the other hand, NMDA treatments had no
significant effect in cells transfected with LUC-1 (positions
297 to
+1258), LUC-2 (positions
1191 to +1258), or LUC-5 (positions
4311
to +189) (Fig. 3D).
18 to +8 can bind phosphorylated CREB and activate transcription at the major transcription start site (position +1).
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Fig. 4.
Northern blot hybridization analysis.
The SK-N-SH cells were cultured with the indicated concentration of
NMDA for 6 h, RNA was extracted from the cells, and PS-1 mRNA
was quantified with Northern hybridization analysis. *,
p < 0.01 using Student's t test
(n = 3).
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Fig. 5.
Metabolic labeling with
[35S]Methionine Clarified the Induction of PS-1 by NMDA
and BDNF in SK-N-SH cells. The horizontal
arrows indicate aggregated PS-1 (PS-1ag),
full-length PS-1 (PS-1FL), and amino-terminal fragment of
PS-1 (PS-1NTF). A, NMDA induced PS-1, and both
MK801 and nifedipine inhibited the induction. Confluent SK-N-SH cells
were prepared in six-well dishes. Medium was changed to the
methionine-free Dulbecco's modified Eagle's medium with 2% dialyzed
fetal bovine serum and incubated for 1 h. Then NMDA was added to
the medium at the indicated concentration and incubated for 6 h,
followed by pulse labeling with 200 µCi/ml
[35S]methionine for 1 h. The cells were lysed
immediately, and immunoprecipitation with polyclonal antibody against
N-terminal fragment of PS-1 was performed. Electrophoresis was
performed with 10% Tris-glycine polyacrylamide gel. The gels were
vacuum-dried and autoradiographed. For inhibition experiments, 100 µM MK801 or 20 µM nifedipine was added to
the medium when the medium was changed. *, p < 0.01 compared with unstimulated control using Student's t test
(n = 3); **, p < 0.01 compared with
each other using Student's t test (n = 3).
B, U0126-inhibited and dominant negative CREBs reduced PS-1
induction by NMDA. 10 µM U0126 was added to the culture
medium when the medium was changed to the methionine-free one, or the
cells were transfected with 50 m.o.i. adenovirus bearing control
LacZ cDNA or one of the dominant negative CREB cDNAs (CREB-M1
and K-CREB) 24 h prior to change of the medium. C, time
course experiments. 200 µCi/ml [35S]methionine was
added to the medium at the indicated time after 100 µM
NMDA was added. D, induction of PS-1 by BDNF. SK-N-SH cells
were cultured with the indicated concentration of BDNF for 6 h,
followed by pulse labeling with 200 µCi/ml
[35S]methionine for 1 h.
-galactosidase, did not affect
NMDA-mediated increases in PS-1 gene expression.
-galactosidase did not change PS-1
gene expression levels (Fig. 6A). Like endogenous kinase,
the MEK-mediated induction of PS-1 protein expression was
reduced by treatment with the U0126 kinase inhibitor (Fig.
6B) and was reduced by adenovirus-mediated expression of
dominant negative CREBs (Fig. 6C). Adenovirus-mediated
expression of MEK leading to increased PS-1 expression was not
inhibited by MK801 treatment as might be expected, since NMDA receptor
activation is upstream of MEK in this signal transduction pathway (Fig.
6B). In a parallel experiment where rat primary hippocampal
neurons were transfected with an adenoviral vector expressing
constitutively active MEK, endogenous rat PS-1 expression was also
induced (Fig. 6D). The induction of PS-1 in these primary
rat neuronal cultures was fully inhibited by adenovirus-mediated
expression of dominant negative CREBs and the U0126 MEK inhibitor,
since it was also reduced in human SK-N-SH cells, suggesting the
conservation of CREB binding sites in the rat PS-1 promoter (Fig. 6,
D and E).
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Fig. 6.
Constitutively active MEK induced PS-1 in
SK-N-SH cells and rat hippocampal neurons. The
horizontal arrows indicate full-length PS-1
(PS-1FL) and amino-terminal fragment of PS-1
(PS-1NTF). A, SK-N-SH cells were transfected with
50 m.o.i. adenovirus bearing control LacZ cDNA or
constitutively active MEK cDNA 24 h prior to change of the
medium, followed by metabolic labeling with
[35S]methionine. *, p < 0.01 compared
with unstimulated control using Student's t test
(n = 3). B, U0126 inhibited PS-1 induction
by constitutively active MEK, but MK801 did not in SK-N-SH cells.
SK-N-SH cells were transfected with 50 m.o.i. adenovirus bearing
constitutively active MEK 24 h prior to change of the medium. 100 µM MK801 or 10 µM U0126 was added to the
culture medium when the medium was changed. The cells were then
incubated for 6 h, followed by pulse labeling with 200 µCi/ml
[35S]methionine. *, p < 0.01 compared
with unstimulated control using Student's t test
(n = 3); **, p < 0.01 compared with
each other using Student's t test (n = 3).
C, dominant negative CREBs reduced PS-1 induction by
constitutively active MEK in SK-N-SH cells. SK-N-SH cells were
transfected with 50 m.o.i. adenovirus bearing constitutively
active MEK, dominant negative CREB cDNAs (CREB-M1 and K-CREB),
and/or control LacZ cDNA. D, U0126 inhibited PS-1
induction by constitutively active MEK, but MK801 did not in rat
hippocampal neurons. Rat hippocampal neurons were transfected with
50 m.o.i. adenovirus bearing constitutively active MEK, 24 h
prior to change of the medium. 100 µM MK801 or 10 µM U0126 was added to the culture medium when the medium
was changed. The cells were then incubated for 6 h, followed by
the pulse-labeling with 200 µCi/ml [35S]methionine.
E, U0126 and dominant negative CREBs in rat hippocampal
neurons inhibited PS-1 induction by constitutively active MEK. Rat
hippocampal neurons were transfected with 50 m.o.i. adenovirus
bearing constitutively active MEK and/or dominant negative CREB
cDNAs (CREB-M1 and K-CREB), 24 h prior to change of the
medium. Alternatively, 100 µM MK801 or 10 µM U0126 was added to the culture medium when the medium
was changed. The cells were then incubated for 6 h, followed by
pulse labeling with 200 µCi/ml [35S]methionine.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
812 and the major transcription start site (Fig. 1). Several putative
transcription factor binding sites were identified in this region
including one Lyf-1 site, two CdxA sites, one AML-1a site, one Nkx-2
site, one Pbx-1 site, one E47 site, one MZF1 site, two Sp1 sites, one
Elk-1 site, and one CREB site. Among these transcription factor binding
sites is a CREB binding site, which overlaps with the putative
Elk-1 binding site. To prove our hypothesis that CREB activation
controls PS-1 gene expression, which may participate in synaptic
function, we closely examined the relationship between CREB and
PS-1.
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ACKNOWLEDGEMENTS |
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We thank Dr. Anthony Zeleznik for the generous gift of CREB-M1 adenovirus; Izumu Saito for AxCANCre and AxCANLZ recombinant adenovirus; Dr. Masatoshi Hagiwara for wild-type CREB cDNA; and Drs. Takashi Kudo, Hana Dawson, Kazunori Imaizumi, and Taisuke Katayama for helpful and informative discussions.
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FOOTNOTES |
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* This work was supported by grants from CREST (Japan Science and Technology); the Ministry of Education, Culture, Sports, Science and Technology, Japan (Priority Area (c)-Advanced Brain Science Project); and Norvatis Foundation for Gerontological Research.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.
¶ To whom correspondence should be addressed: Dept. of Physiology, Ehime University School of Medicine, Shitsukawa, Shigenobu-cho, Ehime 791-0295, Japan. Tel.: 81-89-960-5245; Fax: 81-89-960-5246; E-mail: mitsuda@m.ehime-u.ac.jp.
Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M006153200
2 N. Mitsuda, N. Ohkubo, M. Tamatani, Y.-D. Lee, M. Taniguchi, K. Namikawa, H. Kiyama, A. Yamaguchi, N. Sato, K. Sakata, T. Ogihara, M. P. Vitek, and M. Tohyama, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: PS-1 and -2, presenilin-1 and -2, respectively; CREB, cAMP-response element-binding protein; BDNF, brain-derived neurotrophic factor; NMDA, N-methyl-D-aspartate; PCR, polymerase chain reaction; FL, firefly luciferase; RL, Renilla luciferase; RPA, relative promoter activity; bp, base pair(s); kb, kilobase pair(s); m.o.i., multiplicity of infection; CRE, cAMP-response element; SEAP, secreted alkaline phosphatase; LVDCC, L-type voltage dependent Ca2+ channel.
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