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Aging Cell
(2008)
7,
pp137–147 Doi: 10.1111/j.1474-9726.2007.00360.x
© 2008 The Authors
137
Journal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2008
Blackwell Publishing Ltd
Disrupted intracellular calcium regulates BACE1 gene expression via nuclear factor of activated T cells 1 (NFAT 1) signaling
Hyun Jin Cho,
1,2
Seok Min Jin,
1
Hong Deuk Youn,
1
Kyoon Huh
2
and Inhee Mook-Jung
1
1
Department of Biochemistry and Cancer Research Institute, Seoul National University College of Medicine, Seoul, South Korea
2
Neuroscience Graduate Program, Ajou University School of Medicine, Suwon, South Korea
Summary
Beta-site APP-cleaving enzyme 1 (BACE1) expression iselevated in the brains of Alzheimer’s disease (AD) patientsand in aged-animal models. Because both AD and agingare associated with disrupted calcium homeostasis, weinvestigated the role of nuclear factor of activated T cells(NFAT) – a transcription factor regulated by the calcium-and calmodulin-dependent phosphatase calcineurin – inBACE1 expression. BACE1 expression was stimulatedby a calcium ionophore in primary cortical cultures, andby SH-SY5Y neuroblastoma cells, which was both blockedby pretreatment with either cyclosporin A, an inhibitor ofcalcineurin, or ethyleneglycotetraacetic acid, a calciumchelator. Gel shift assays revealed direct binding of NFAT1to specific DNA sequences within the BACE1 gene promoterregion. Treatment with amyloid beta (Aββββ
), one of themajor factors in AD pathogenesis, stimulated activationand nuclear translocation of NFAT1 following up-regulationof BACE1 expression. In addition, primary cortical culturesfrom Tg2576 mouse brains generated more Aββββ
byionophore stimulation, which was reversed by cyclosporinA treatment. Furthermore, NFAT1 activation was observedin Tg2576 mouse brains. These results suggest that calciumionophore- or Aββββ
-induced increases in intracellular calciumconcentration stimulate BACE1 expression, resulting inaccelerated Aββββ
generation, and that this process ismediated through the calcineurin-NFAT1 signalingpathway. This process may play a significant role in thepathogenesis of AD and aging.Key words: Alzheimer’s disease; amyloid beta; beta-secretase; calcium ionophore; NFAT.
Introduction
One important feature of Alzheimer’s disease (AD) is extracellulary
deposited senile plaques in the brain (Mattson, 2004), which
are composed of amyloid beta (A
β
) peptide, a proteolytic product
of amyloid precursor protein (APP) processing (Haass
et al
.,
1992; Sisodia & Price, 1995). As a critical protease in generating
A
β
,
β
-site APP-cleaving enzyme 1 (BACE1) is considered as a
key player in the pathogenesis of AD (Vassar
et al
., 1999; Yan
et al
., 1999).
BACE1 expression and enzymatic activity are elevated in the
brains of AD patients and in animal models of AD (Fukumoto
et al
., 2002). The Tg2576 mouse, which is an AD animal model
that overexpresses the Swedish form of APP in neurons, shows
an age-dependent increase in BACE1 expression (Apelt
et al
.,
2004). Several lines of evidence indicate that stress-related
events, such as inflammatory responses and reactive oxygen
species generation, are associated with BACE1 expression.
Oxidative molecules and nonsteroidal anti-inflammatory drugs
are known to regulate BACE1 expression in
in vitro
model systems
(Tamagno
et al
., 2002; Sastre
et al
., 2006). Interferon-gamma,
a proinflammatory cytokine, induces BACE1 expression (Hong
et al
., 2003; Cho
et al
., 2007), and changes in BACE1 protein
levels are associated with insulin-like growth factor 1 signaling,
which is one of the major regulators of age-dependent events
(Costantini
et al
., 2006).
Disrupted calcium homeostasis has been reported in the brains
of AD patients and normal-aged subjects. Overall levels of free
and protein-bound calcium (Palotas
et al
., 2002) and calcium-
activated transglutaminase activity (Johnson
et al
., 1997) were
elevated in tissues obtained from AD patients. Altered calcium
homeostasis and disrupted calcium signaling also mediate the
expression of biological markers of the aging brain (Foster &
Kumar, 2002). Therefore, it is possible that disruption of calcium
homeostasis and elevated BACE1 expression have a causal
relationship in AD pathogenesis and aging. Because A
β
exerts
its neurotoxic effects by disrupting calcium homeostasis
(Mattson
et al
., 1992), and because disrupted calcium levels
cause more production of A
β
peptide (Mattson
et al
., 1993a;
Querfurth & Selkoe, 1994), we hypothesized that disrupted
calcium homeostasis might enhance BACE1 expression through
a calcium-dependent signaling cascade, leading to increased
production of A
β
.
We are particularly interested in the role of nuclear factor of
activated T cells (NFAT) in this process. NFAT is a transcription
factor activated by calcineurin, a calcium- and calmodulin-
dependent phosphatase (Macian, 2005). NFAT has a pivotal role
Correspondence
Inhee Mook-Jung, PhD, Department of Biochemistry and Cancer Research
Institute, Seoul National University College of Medicine, 28 Yungun-dong,
Jongro-gu, Seoul 110–799, South Korea. Tel.: +82-2-740-8245;
fax: +82-2-744-4534; e-mail: [email protected]
Accepted for publication
20 November 2007
NFAT1 activation regulates BACE1 expression, H. J. Cho
et al.
© 2008 The AuthorsJournal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2008
138
in the inducible gene transcription of cytokines during immune
responses (Rao
et al
., 1997; Shaw
et al
., 1998). In resting cells,
NFAT is phosphorylated and resides in the cytoplasm. Upon an
increase in intracellular calcium, calcineurin is activated and
dephosphorylates NFAT, promoting its translocation to the
nucleus (Ruff & Leach, 1995; Loh
et al
., 1996).
The NFAT family includes multiple isoforms, derived primarily
from alternative splice variants: NFAT1 (NFATp or NFATc2),
NFAT2 (NFATc or NFATc1), NFAT3 (NFATc4), NFAT4 (NFATc3),
and NFAT5 (Rao
et al
., 1997). Although NFAT was first identified
in T cells, recent studies have demonstrated NFAT protein
expression in other immune-related cells, as well as in many
other tissues, including the spleen, thymus, lymph node, and
brain (Ochi
et al
., 1994; Masuda
et al
., 1995; Plyte
et al
., 2001;
Eberl & Littman, 2003). In the brain, NFAT1 is normally detected
in neuronal cells (Plyte
et al
., 2001), and NFAT4 is also expressed
and activated in astrocytes in the presence of elevated intracellular
calcium (Jones
et al
., 2003), inducing target gene expression.
Here, we demonstrate that BACE1 gene expression is modulated
by intracellular calcium via the calcineurin-NFAT signal transduction
pathway in mouse primary cortical cells, as well as in human
neuroblastoma cells. In addition, we identified a direct NFAT1-
binding region in the BACE1 promoter. Furthermore, treatment
with A
β
, which is known to disrupt intracellular calcium
homeostasis, regulated BACE1 gene expression through NFAT1
activation. These results suggest that elevated intracellular
calcium, which is a well-known physiological correlate of
age-related neurodegenerative disorders, activates BACE1 gene
expression and that this process is mediated by calcineurin-
NFAT1 activation.
Results
Treatment with ionomycin facilitates BACE1 promoter activity and protein expression
To examine the effect of intracellular calcium levels on BACE1 gene
expression, we stimulated cells with ionomycin and performed
promoter activity assays using a luciferase reporter gene system.
Ionomycin treatment enhanced BACE1 promoter activity in a
dose-dependent manner (0.25 and 0.5
µ
M
,
P
< 0.01 and 0.001,
respectively; Fig. 1A). Western blotting showed increased
BACE1 protein expression following ionomycin stimulation in
SH-SY5Y cells (Fig. 1B). These results indicate that increased
Fig. 1 Ionomycin-induced BACE1 expression and NFAT1 activation. (A) BACE1 promoter activity in uBACE-2K-transfected SH-SY5Y cells and (B) BACE1 protein levels were elevated by ionomycin. (C) Ionomycin-induced BACE1 promoter activity was blocked by EGTA pretreatment. (D) Western blotting of cells pretreated with EGTA showed the recovery of inactive NFAT1, while NFAT4 was unchanged. (E) CsA inhibited ionomycin-induced BACE1 promoter activity. (F) CsA pretreatment increased inactive NFAT1, compared with ionomycin-treated cells. NFAT4 was unchanged under the same conditions. Data are mean ± SEM of triplicate experiments. **P < 0.01, ***P < 0.001.
NFAT1 activation regulates BACE1 expression, H. J. Cho
et al.
© 2008 The AuthorsJournal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2008
139
intracellular calcium facilitates BACE1 promoter activity and
protein expression.
To confirm that the ionomycin-induced BACE1 expression
was caused by increased intracellular calcium, we measured the
effect of ionomycin on BACE1 promoter activity with ethylene-
glycotetraacetic acid (EGTA) added to the culture to chelate
extracellular calcium ions. EGTA significantly inhibited ionomycin-
induced BACE1 promoter activity (Fig. 1C).
To investigate the NFAT response by disrupted calcium
homeostasis in the cells, inactive forms of NFAT1 and NFAT4
were examined when ionomycin was administered. The inactive
form of NFAT1 was markedly decreased when ionomycin was
added, while EGTA pretreatment with ionomycin restored the
level of inactive NFAT1 (Fig. 1D). However, inactive NFAT4
showed no change with either ionomycin or EGTA treatment
(Fig. 1D).
In addition, cyclosporin A (CsA), a calcineurin inhibitor, was
given to the cells to examine the possible role of calcineurin-
NFAT signaling in BACE1 expression. CsA significantly blocked
ionomycin-induced BACE1 promoter activity (
P <
0.001, Fig. 1E)
and markedly elevated the level of inactive NFAT1, compared
with ionomycin-treated cells, while having no effect on NFAT4
(Fig. 1F), indicating that BACE1 gene expression is regulated by
calcineurin-NFAT1-mediated signal transduction.
NFAT1 protein interacts directly with a specific site in the BACE1 promoter
Because putative NFAT1-binding sequences exist in the BACE1
promoter region (–500 to –508 bp; Fig. 2A), we examined
whether NFAT1 interacts directly with these specific sequences
by testing three constructs, using a luciferase reporter gene
assay. Enhanced promoter activity was observed in SH-SY5Y
cells containing uBACE-1Ka and uBACE-2K following ionomycin
treatment, whereas no enhancement was observed in those
cells containing uBACE-1Kb (Cho
et al
., 2007), indicating a
putative NFAT1-binding site within the uBACE-1Ka region of
the BACE1 promoter (Fig. 2B).
To test the direct interaction between NFAT1 and the BACE1
promoter, gel shift assays were performed using biotin-labeled
oligonucleotides corresponding to the putative NFAT1-binding
consensus sequences (TGGAAAAAC, at –500 to –508 bp;
Fig. 2A) within the BACE1 gene promoter region. The shifted
band, denoting a complex of NFAT1-DNA, was present in the
ionomycin-treated nuclear extracts incubated with biotin-labeled
NFAT1 probe (arrowhead in lane 2, Fig. 2C).
We confirmed the specificity of the shifted band by preincuba-
tion with a 100-fold excess of an unlabeled NFAT1 competitor
probe (comp), which abolished the shifted band (lane 3,
Fig. 2 Direct binding of NFAT1 to BACE1 promoter region. (A) Schematic diagram of the predicted NFAT1-binding site within the BACE1 gene promoter region. Putative NFAT1-binding sequences (black box), putative MEF2-binding sequences (gray boxes), STAT1-binding sequences (white box). GenBank accession number AY542689. (B) Expression of uBACE-1Ka (1Ka) and uBACE-2K (2K) increased ionomycin-induced BACE1 promoter activity, but uBACE-1Kb (1Kb) had no effects. Open bars, vehicle-treated cells; solid bars, 0.5 µM ionomycin-treated cells. (C) Gel shift assays with biotin-labeled NFAT1 probes. The probes were incubated with nuclear extracts (NE) of cells stimulated with ionomycin. The shifted band of NFAT1-DNA complex (lane 2, arrowhead) was abolished by competition assay (lane 3). Arrows, nonspecific bands. (D) NFAT1-DNA complex (arrowhead) was induced by ionomycin (I) treatment (lane 3) and was blocked by CsA (lane 4). In the presence of cold probes as a competitor (Comp), this specific band was abolished (lane 1). Data are mean ± SEM of triplicate experiments. ***P < 0.001.
NFAT1 activation regulates BACE1 expression, H. J. Cho
et al.
© 2008 The AuthorsJournal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2008
140
Fig. 2C). In addition, binding assays using nuclear extracts from
ionomycin-treated cells showed that the ionomycin-induced
shifted band was ablated by CsA pretreatment (lane 4, Fig. 2D),
suggesting that calcineurin function is necessary for the
interaction between NFAT1 and the BACE1 promoter.
NFAT1, but not NFAT4, regulates BACE1 promoter activity by binding directly to the BACE1 promoter
To examine the specificity of binding between NFAT1 and the
BACE1 promoter, a super-shift analysis using specific antisera
against NFAT1 or NFAT4 was performed. Preincubation with the
NFAT1-specific antibody abolished the protein-DNA complex
(arrowhead in lane 2, Fig. 3A), while preincubation with NFAT4-
specific antibody had no effect on the shifted complex band
(lane 3, Fig. 3A), indicating that NFAT1, but not NFAT4, binds
to the BACE1 gene promoter in SH-SY5Y cells.
To confirm that ionophore-induced BACE1 promoter activity
is mediated by NFAT1, we transfected NFAT1 cDNA into the
cells to overexpress NFAT1 protein and then measured BACE1
promoter activity. Cotransfection of various concentrations of
NFAT1 cDNA with the uBACE-2K construct enhanced BACE1
promoter activity in a dose-dependent manner following
ionomycin treatment (black bars, Fig. 3B), whereas coexpression
of a vector encoding NFAT4 with the uBACE-2K construct had
no additional effect on BACE1 promoter activity even in the
presence of ionomycin (black bars, Fig. 3C). Similarly, protein
levels of NFAT1 or NFAT4 increased dose-dependently (data not
shown). These results indicate that ionophore-induced activation
of NFAT1, but not NFAT4, enhances BACE1 gene expression
by binding directly to the BACE1 promoter.
Treatment with Aββββ
1–42
activates NFAT1 and induces its translocation to the nucleus
Several lines of evidence suggest that A
β
treatment increases
intracellular calcium concentration and affects calcium-mediated
signaling in neuronal cells (Mattson
et al
., 1992; Abramov
et al
.,
2004). To investigate whether NFAT1 activation is affected by
A
β
treatment, we used Western blotting to measure NFAT1
dephosphorylation following treatment with A
β
1–42
for 48 h
(Fig. 4A). The levels of the phosphorylated form of NFAT1 (pNFAT1,
inactive form) decreased in cells with increasing doses of A
β
1–42
(4 and 8
µ
g mL
–1
,
P
< 0.01 and 0.001, respectively; Fig. 4B).
However, A
β
1–42
had no effects on NFAT4 dephosphorylation
under the same conditions (data not shown).
A fractionation experiment showed that dephosphorylated
NFAT1 (active form of NFAT1) levels were increased in the nuclear
fraction when SH-SY5Y cells were stimulated with 8
µ
g mL
–1
A
β
1–42
for 48 h (right panel, Fig. 4C), while pNFAT1 was decreased
in the cytosolic fraction (left panel, Fig. 4C). Treatment of the cells
with reverse peptide A
β
42–1
had no effect on NFAT1 activation
(Fig. 4D), indicating the specificity of the effect of A
β
1–42
.
To examine whether A
β
-induced NFAT1 activation is mediated
by calcineurin, CsA was added to SH-SY5Y cells in the presence
or absence of A
β
1–42
. CsA treatment resulted in the recovery
of pNFAT1, compared with that of A
β
-treated cells (Fig. 4E).
Because A
β
1–42
-activated NFAT1 is expected to translocate
into the nucleus, localization of NFAT1 was examined in the
presence of A
β
with or without CsA treatment by immunostain-
ing with an NFAT1-specific antibody (Fig. 4F). As expected,
A
β
1–42
-treated cells treated cells showed intense NFAT1 signals
in the nucleus, and NFAT1 proteins localized in the cytosol with
Fig. 3 Ionomycin-induced BACE1 expression is associated with NFAT1, but not NFAT4. (A) Nuclear extracts from the cells treated with ionomycin were preincubated with antisera specifically recognizing the NFAT1 or NFAT4 isoform (α-NFAT). The protein-DNA band (arrowhead) was abolished by the anti-NFAT1 antibody (2 µg) (lane 2), while the anti-NFAT4 antibody had no effect (lane 3). This specific band was inhibited by cold NFAT1 probes as a competitor (lane 4). Arrows, nonspecific bands. (B) Overexpression of NFAT1 enhanced ionomycin-induced BACE1 promoter activity in a dose-dependent manner. SH-SY5Y cells were cotransfected transiently with uBACE-2K and NFAT1 expression vectors (0.5, 1, 2, 4 ratio vs. uBACE-2K) in the presence of ionomycin. (G) Transfection of NFAT4 expression vector (0.5, 1, 2, 4 ratio vs. uBACE-2K) had no effect on ionomycin-induced BACE1 promoter activity. Data are mean ± SEM of triplicate experiments. *P < 0.5, **P < 0.01, ***P < 0.001.
NFAT1 activation regulates BACE1 expression, H. J. Cho
et al.
© 2008 The AuthorsJournal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2008
141
CsA pretreatment. Anti-NFAT1 signals were observed in the
cytosol in both vehicle- and CsA-treated cells. These results
indicate that NFAT1 proteins were translocated to the nucleus
by A
β
treatment and that this process was inhibited by CsA.
Aββββ
1–42
up-regulates BACE1 expression and activity through activation of the calcineurin-NFAT1 signaling pathway
Because BACE1 expression was induced by ionomycin through
NFAT1 activation (Figs 1 and 2), we examined changes in BACE1
expression and activity by A
β
1–42
. Treatment with A
β
1–42
(8
µ
g mL
–1
for 48 h) increased the level of BACE1 protein
(Fig. 5A) and BACE1 promoter activity in SH-SY5Y cells (
P <
0.01,
Fig. 5B), while treatment of the reverse peptide A
β
42–1
had no
effect. Furthermore, A
β
-induced BACE1 protein expression was
blocked by CsA pretreatment (Fig. 5C). CsA also reduced A
β
-
induced BACE1 promoter activity significantly (
P
< 0.05, Fig. 5D).
To confirm whether BACE1 enzymatic activity was affected
by calcineurin-NFAT1 signaling,
β
-secretase activity was measured,
using membrane fractions prepared from A
β
-treated SH-SY5Y
cells. BACE1 enzymatic activity was increased by A
β
treatment
(
P
< 0.001, Fig. 5E) and reduced by CsA pretreatment significantly
(
P
< 0.05, Fig. 5E). In addition, a gel shift assay showed that A
β
-
activated NFAT1 bound directly to the BACE1 promoter region
(arrowhead, Fig. 4F), suggesting that A
β
induces BACE1 expression
and activity by activated NFAT1 protein, which in turn interacts
directly with the BACE1 promoter.
Up-regulation of BACE1 expression by ionomycin enhances Aββββ generation in primary cortical cells from Tg2576 mouse brains
Because BACE1 plays a major role in the generation of Aβ from
APP, it was important to examine whether up-regulation of
BACE1 expression by ionomycin affects Aβ generation. We
Fig. 4 Aβ1–42 induced NFAT1 activation through calcineurin activation. (A) Aβ1–42 treatment for 48 h showed a dose-dependent decrease in pNFAT1. (B) The ratio of pNFAT1/actin was reduced at 4 and 8 µg mL–1 of Aβ1–42 treatment. **P < 0.01; ***P < 0.001. (C) Crude nuclear (Nucleus) and cytosolic extracts (Cytosol) were isolated from cells stimulated with Aβ1–42 (8 µg mL–1), which induced an increase of NFAT1 in the nucleus and a decrease of pNFAT1 in the cytosol. (D) pNFAT1 was unchanged by treatment with reverse peptide Aβ42–1 (8 µg mL–1). (E) Aβ1–42-induced NFAT1 activation (lane 2) was blocked by 1 µM CsA pretreatment (lane 3). (F) While NFAT1 proteins (anti-NFAT1-FITC, green) were mostly detected in cytosol (Ctrl), Aβ-treated cells showed intense NFAT1 signals in the nucleus. Pretreatment with CsA blocked Aβ-induced translocation of NFAT1 proteins (Aβ42 + CsA). 4′-6-Diamidino-2-phenylindok (blue) stained the nuclei of the cells.
NFAT1 activation regulates BACE1 expression, H. J. Cho et al.
© 2008 The AuthorsJournal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2008
142
analyzed the level of Aβ in primary cortical cultures from the
brains of Tg2576 mice, which overexpress the Swedish form of
APP in neurons. The levels of Aβ40 and Aβ42 from cell extracts
prepared using RIPA buffer were significantly increased in
ionomycin-treated cells, compared with control cells (P < 0.05,
Fig. 6A). However, pretreatment of CsA completely blocked
ionomycin-induced Aβ generation (Fig. 6A). Furthermore,
extracellular Aβ40 and Aβ42 levels from conditioned media were
significantly changed by ionomycin or CsA treatment (P < 0.01
and P < 0.05, Aβ40 and Aβ42, respectively; Fig. 6B).
Aβ42 treatment, as well as ionomycin, induced BACE1 expression
and dephosphorylation of pNFAT1; both processes were inhibited
by CsA in primary cortical cultures (Fig. 6 C,D). These results
indicate that up-regulation of BACE1 expression by ionomycin
or Aβ42 elevates Aβ generation through the calcineurin-NFAT1
signaling pathway in primary cortical cultures from Tg2576
mouse brains.
NFAT1 is activated in Tg2576 mouse brains
Since NFAT1 is activated by Aβ (Fig. 4), we examined the level
of NFAT1 activation in Tg2576 mouse brains. The ratio of NFAT1
(active) to pNFAT1 (inactive) increased significantly in Tg2576
mouse brains (P < 0.01, Fig. 7A). With regard to confirmation
of NFAT1 activation in Tg2576 mouse brains, when nuclear
extracts from mouse brains were analyzed using immunoblotting,
active NFAT1 levels were higher in Tg2576 brains compared
with wild-type mouse brains, indicating endogenous activation
of NFAT1 in Tg2576 mouse brains (Fig. 7B).
Discussion
Previous reports have shown that BACE1 expression and enzymatic
activity are enhanced in the brains of AD model animals, and
normal-aged and AD subjects, although the molecular mechanisms
underlying these phenomena are not well understood (Fukumoto
et al., 2002; Yang et al., 2003; Li et al., 2004). Additionally,
dysregulation of intracellular calcium has been shown to be
associated with pathogenic mechanisms of AD (Mattson et al.,1993b; LaFerla, 2002; Kawahara, 2004). Calcium-dependent
enzymes – such as calpain, which is a family of calcium-activated
intracellular cysteine proteases, and calcineurin, which is a calcium-
and calmodulin-dependent protein phosphatase – are also
activated in the brains of AD patients (Liu et al., 2005). Cytosolic
NFAT proteins are dephosphorylated by calcineurin and
translocate to the nucleus to alter gene transcription (Ruff &
Leach, 1995; Rao et al., 1997; Macian, 2005).
In this study, NFAT1, but not NFAT4, was activated by either
ionomycin or Aβ42 peptide, which was followed by the induction
of BACE1 gene expression at the transcription level. These
events were blocked by CsA and EGTA, and overexpression of
NFAT1 enhanced ionomycin-stimulated BACE1 promoter activity.
NFAT1 has been found in the brain and in neuronal cells (Plyte
et al., 2001), and NFAT4 is expressed and activated in astrocytes
Fig. 5 Aβ-induced BACE1 expression through calcineurin-NFAT1 activation. (A) 8 µg mL–1 Aβ42 treatment for 48 h elevated the level of BACE1 protein and (B) luciferase promoter activity. Reverse peptide Aβ42–1 (8 µg mL–1) showed no effects on BACE1 expression. (C) BACE1 protein expression and (D) Aβ-induced BACE1 promoter activity were blocked by CsA pretreatment. (E) The in vitro cleavage assay for β-secretase activity measurement showed an increase in BACE1 enzymatic activity when Aβ42 was administered to SH-SY5Y cells, but was blocked by CsA. (F) The shifted band (arrow head) of the NFAT1-DNA complex was enhanced by incubation with a biotin-labeled NFAT1 probe and nuclear extracts from Aβ42 (8 µg mL–1)-treated cells. Data are mean ± SEM of triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
NFAT1 activation regulates BACE1 expression, H. J. Cho et al.
© 2008 The AuthorsJournal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2008
143
in the presence of elevated intracellular calcium (Jones et al.,2003). Interestingly, although we found both NFAT1 and NFAT4
in SH-SY5Y cells and primary cortical cultures, only NFAT1 was
activated by disruption of intracecullar calcium levels.
Several lines of evidence indicate that NFAT isoforms are differ-
entially regulated. Under hypertonic stress conditions, NFAT5 binds
directly to the tumor necrosis factor promoter, and it is distinctly
regulated by NFATp, c, 3, and 4 (Esensten et al., 2005). Barlic
et al. (2004) reported that NFAT1, but not NFAT2, is recruited
to the CX3CR1 promoter to regulate gene expression in leukocytes
(Barlic et al., 2004). In addition, NFAT1, but not NFAT4, is involved
in depolarization-induced activation in growth hormone-releasing
hormone (GHRH) gene transcription in neuronal cells, despite
the presence of both NFAT1 and NFAT4 (Asai et al., 2004).
Our data provide another example of differential regulation
of NFAT isoforms. Although the exact underlying mechanism
has yet to be elucidated, it is possible that an NFAT4-specific
phosphatase, if any exists, is absent or not activated in our
experimental system. Gel shift assays using probes for a putative
NFAT-binding site within the BACE1 gene promoter region
confirmed direct binding of NFAT1 to the BACE1 gene promoter.
These results show a molecular link between intracellular calcium
changes and BACE1 expression and explain why BACE1 expression
is elevated in the brains of aged individuals and AD patients.
Myocyte enhancer factor 2 (MEF2) is another calcium signaling
transducer that functions as a transcription factor (McKinsey
Fig. 6 Aβ generation via ionomycin-induced BACE1 expression in primary cortical cultures from Tg2576 mouse brains. (A) The levels of Aβ40 and Aβ42 were measured by sandwich ELISA from cell lysates and (B) conditioned media of primary cortical cultures from Tg2576 mouse brains. 0.5 µM ionomycin treatment for 48 h increased both Aβ40 and Aβ42 levels, which were inhibited by CsA effectively. (C) Both 8 µg mL−1 Aβ42 and (D) Ionomycin treatments increased the levels of BACE1 protein expression and decreased levels of inactive NFAT1 in primary cortical cultures, both processes of which were inhibited by CsA. Actin was used as a loading control. Data are mean ± SEM of triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 7 NFAT1 activation in Tg2576 mouse brains. (A) Brain extracts from Tg2576 mice (n = 4) and littermates (n = 4) were examined to observe the status of NFAT1 activation using Western blotting, followed by densitometric quantification. The ratio of NFAT1 to pNFAT1 was presented as mean ± SEM. **P < 0.01. (B) Nuclear extracts from mouse brain cortices were analyzed by immunoblotting. The bands of active NFAT1 were enhanced in Tg2576 brains. Lamin B is a nuclear marker used as a loading control.
NFAT1 activation regulates BACE1 expression, H. J. Cho et al.
© 2008 The AuthorsJournal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2008
144
et al., 2002). Both NFAT and MEF2 are known to be involved
in the regulation of interleukin-2 gene transcription during T-
cell activation (Pan et al., 2004). Putative MEF2-binding sites
(ctaaaaata) in the BACE1 promoter are also predicted by a
GenBank search based on a previous report (–1085 to –1093 bp,
and –1494 to –1502 bp; Fig. 2A) (Andres et al., 1995). It is possible
that MEF2 acts as a synergistic regulator of NFAT1 during
calcium-induced BACE1 gene expression. This needs to be
clarified in future work.
A STAT1-binding site in the BACE1 promoter region has been
previously reported (Cho et al., 2007). This site is responsive to
the interferon-gamma-induced Janus kinase-signal transducers
and activators of transcription (STAT) signal transduction
pathway, and is involved in BACE1 expression in astrocytes.
Considering that the brains of aged individuals and AD patients
have severely disrupted intracellular calcium homeostasis, as
well as brain inflammation, binding of NFAT1 and STAT1 to the
BACE1 promoter is likely to play a role in elevating BACE1
expression and activity in the brains of these subjects.
Aβ-induced cell toxicity has been reported to be associated
with the disruption of intracellular calcium signal transduction
(Mattson et al., 1993b), and Aβ treatment elevates cytosolic
calcium levels (Mattson et al., 1992; Brorson et al., 1995). This
study demonstrates that Aβ regulates BACE1 gene expression
by activating NFAT1. Aβ treatment induced nuclear translocation
of NFAT1, and translocated NFAT1 interacted directly with the
BACE1 promoter. These results suggest the existence of a positive
feedback loop between Aβ production and NFAT1-mediated
BACE1 expression, which might accelerate Aβ generation in AD
pathogenesis. In addition to ionomycin, Aβ42 had a similar effect
on NFAT1 activation followed by BACE1 expression and activation,
resulting in further generation of Aβ. Although calcium stimulates
the intracellular secretory pathway (Barclay et al., 2005), further
Aβ generation in our results was not due to stimulation of
the calcium-induced intracellular secretory pathway, because
the amount of Aβ in cell lysates was markedly increased. If the
calcium-induced secretory pathway had been the primary
mechanism for generating more Aβ, only extracellular Aβ levels
– not intracellular Aβ – should have increased. Our results show
that both intracellular and extracellular Aβ levels were enhanced,
suggesting that elevated Aβ was due to increased BACE1 activity
by NFAT1 binding. It has been reported that Aβ peptides increase
the level of intracellular calcium via several mechanisms, including
activation of intracellular calcium stores (Cowburn et al., 1995;
Singh et al., 1995), voltage-dependent calcium channels (VDCC)
(Ueda et al., 1997), and N-methyl-D-aspartic acid (NMDA)
glutamate receptors (Le et al., 1995). To investigate the route
of calcium into the cells on Aβ42 treatment, several inhibitors that
block calcium entry were examined. MK801, an NMDA receptor
blocker, and nifedipine, a VDCC blocker, failed to inhibit Aβ-
induced NFAT1 activation (data not shown), while the calcium
chelator EGTA inhibited Aβ-induced NFAT1 activation completely,
suggesting that Aβ-induced NFAT1 activation does not occur
through the NMDA receptor or VDCC, but from another
extracellular source whose mechanism is unknown at this time.
Because several reports suggest that aggregated Aβ acts as
a calcium channel in cell membranes, permeable to calcium ion
(Oyama et al., 1995), it is possible that Aβ peptide treatment
creates calcium-permeable pores in cells, a concept supported
by the long delay of Aβ-induced NFAT1 activation. Considering
that NFAT1-mediated BACE1 expression was similarly observed
following treatment with either calcium ionophore or Aβ, it is
likely that NFAT1-mediated BACE1 expression was caused by
disruption of intracellular calcium homeostasis. Because
disruption of calcium homeostasis plays a crucial role in aging
and AD pathogenesis, our study suggests that the calcineurin-
NFAT1 signaling pathway, which regulates BACE1 gene
expression, is a potential therapeutic target against Aβ-induced
pathogenesis of AD and aging.
Experimental procedures
Cell culture and drug treatments
Mouse primary cortical cultures were prepared from brains of
day 1 postnatal pups of Tg2576 (gifted by Dr Karen Hsiao-Ashe,
University of Minnesota, Minneapolis, MN, USA) (Hsiao et al.,1996) or wild-type mice. Cells were maintained in a 37 °C CO2
incubator for 7 days for the assay. Human SH-SY5Y neuroblastoma
cells were maintained in Dulbecco’s modified Eagle’s medium
(DMEM; HyClone, Salt Lake City, UT, USA) supplemented with
10% fetal bovine serum (FBS; HyClone, Irvine, CA, USA) and a
1% penicillin/streptomycin antibiotic mixture at 37 °C in a
humid atmosphere of 5% CO2. Cells were treated with ionomycin
(Sigma, St. Louis, MO, USA) in DMEM supplemented with 1%
FBS. CsA (1 µM, Sigma) or EGTA (300 nM, Sigma) was added for
30 min before the ionomycin treatment. Cells were treated with
aggregated Aβ1–42 (Bachem, Bubendorf, Switzerland). Aggregated
Aβ1–42 was obtained by incubation at 37 °C for 16 h.
DNA constructs and luciferase assay
Genomic DNA purified from HEK293 cells was used as a
template to clone the BACE1 promoter region as described in
Cho et al. (2007). Three constructs were used: uBACE-1Ka, –1 bp
to –994 bp; uBACE-1Kb, –930 bp to –1876 bp; and uBACE-2K,
+50 bp to –2100 bp. Luciferase assay was conducted according
to instructions provided by the manufacturer (Dual Luciferase
Kit, Promega, Madison, WI, USA) using a luminometer (LUMAT
LB9507; EG & G Berthold, Bad Wildbad, Germany). Firefly
luciferase activity was normalized to renilla luciferase activity.
For overexpression of NFAT, expression constructs encoding
NFAT1 or NFAT4 were transiently transfected, and the plasmid
amounts were normalized with mock vector.
Preparation of nuclear extracts
Harvested cells or brain tissues were resuspended in hypotonic
buffer [10 mM Tris (pH 7.4), 1 mM ethylenediaminetetraacetic
acid, and 1 mM EGTA] including protease inhibitor cocktail
NFAT1 activation regulates BACE1 expression, H. J. Cho et al.
© 2008 The AuthorsJournal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2008
145
[100 mg mL–1 phenylmethylsulfonyl fluoride (PMSF), 2 mg mL–1
leupeptin, and 2 mg mL–1 aprotinin, all from Sigma]. After
incubation on ice for 30 min, swelled cells were disrupted with
25 strokes of a tight-fitting pestle in a Dounce homogenizer,
followed by centrifugation at 500 g for 15 min. Pellets were
then incubated with RIPA buffer (150 mM NaCl, 1% NP-40, 0.5%
deoxycholic acid, 0.1% SDS, 50 mM Tris, pH 7.4) on ice for
15 min and centrifuged (nuclear extracts). Supernatants were
centrifuged at 17 000 g for 30 min and upper layers (cytosolic
extracts) were isolated. Membrane extracts were extracted from
pellets with RIPA buffer. All solutions included protease inhibitor
cocktail, PMSF, and dithiothreitol. The amount of protein was
measured by a bicinchoninic acid protein assay kit (Amersham
Pharmacia, Arlington Heights, IL, USA).
Electrophoretic mobility shift assay
Double-stranded probe was generated by annealing two
biotin-labeled oligonucleotides against the putative NFAT-binding
site (TGGAAAAAC) within the human BACE1 gene promoter
region; BACE1-NFAT forward probe (5′-biotin-TGCAGCCT-
GGAAAA ACTCTTC-3′) and BACE1-NFAT reverse probe (5′-biotin-GAAGAGTTTTTCCAGGC TGCA-3′). For binding reactions,
5 µg of nuclear extracts were preincubated with 1 µg of poly
(dI-dC) (Sigma) for 5 min and then incubated with biotin-labeled
BACE1-NFAT probes at room temperature for 30 min. For
competition experiments, a 100-fold molar excess of unlabeled
BACE1-NFAT1 cold probes was preincubated with nuclear
extracts for 10 min. For supershift assays, 2 µg of anti-NFAT1
(Affinity BioReagents, Golden, CO, USA) or anti-NFAT4 (Santa
Cruz Biotechnologies, Santa Cruz, CA, USA) monoclonal antibody
(Upstate Biotechnology, Lake Placid, NY, USA) were added to
the extracts 30 min before the addition of biotin-labeled probes.
Protein-DNA complexes were analyzed by 5% nondenaturing
polyacrylamide gel electrophoresis (PAGE) in 0.5× TBE buffer
and transferred to Biodyne B Nylon Membranes (Pierce, Rockford,
IL, USA). Signals were detected using a Light Super Shifted Module
Kit (Pierce) according to the manufacturer’s instructions.
Antibodies
A monoclonal antibody against the C-terminus of BACE1
(Chemicon, Temecula, CA, USA); BACE AB-2 (Oncogene,
Darmstadt, Germany), a polyclonal antibody against amino
acids 485–501; an antiactin monoclonal antibody (Sigma); and
an antilamin B antibody (Santa Cruz Biotechnologies) were used
for immunoblotting. A monoclonal antibody against NFAT1
(Affinity BioReagents) and the anti-NFAT4 monoclonal antibody
(Santa Cruz Biotechnologies) were used at 1 : 1000 and 1 : 500,
respectively, for Western blotting.
Immunocytochemistry
Cells were fixed and then incubated with anti-NFAT1 monoclonal
antibody, followed by tetramethylrhodamine isothiocyanate-
conjugated secondary antibody (Jackson Laboratories, Westchester,
PA, USA). The labeled cells were analyzed with a fluorescence
microscope (Olympus DP50, Tokyo, Japan).
ELISA for Aββββ
Conditioned media or total protein extract prepared from cells
with RIPA buffer (Jin et al., 2007) was subjected to sandwich
ELISA using an N-terminal-specific anti-Aβ antibody and a
C-terminal-specific anti-Aβ40 or -Aβ42 antibody according to the
manufacturer’s instruction (Human β-amyloid Immunoassay Kit,
BioSource, Carlsbad, CA, USA).
In vitro peptide cleavage assay for measurement of BACE1 enzymatic activity
BACE1 enzymatic activity assays were performed using 10 µg mL–1
synthetic peptide substrates, MCA-S-E-V-N-L-D-A-E-F-R-K(DNP)-
R-R-NH2 (Bachem). Protein extracts of membrane fractions and
fluorescent-labeled peptides were incubated in 0.15 M Na-
Acetate (pH 5.2) at 37 °C. The mixtures were quenched and the
signals were measured (excitation 325 nm, emission 393 nm)
by a fluorescence luminometer (LS-55, PerkinElmer, Norwalk,
CT, USA).
Statistical analysis
All data were expressed as mean ± SEM. Differences between
groups were examined for statistical significance using the Tukey–
Kramer multiple comparisons test. A P-value less than 0.05
denoted the presence of a statistically significant difference.
Acknowledgments
This work was supported by grants from 21C Frontier Functional
Proteomics Project (FPR05C2-010), Molecular & Cellular
Biodiscovery, and KOSEF (RO1-2004-000-10271-0 and R11-
2002-097-05001-2).
References
Abramov AY, Canevari L, Duchen MR (2004) Calcium signals inducedby amyloid β peptide and their consequences in neurons and astrocytesin culture. Biochim. Biophys. Acta 1742, 81–87.
Andres V, Cervera M, Mahdavi V (1995) Determination of the consensusbinding site for MEF2 expressed in muscle and brain reveals tissue-specific sequence constraints. J. Biol. Chem. 270, 23246–23249.
Apelt J, Bigl M, Wunderlich P, Schliebs R (2004) Aging-related increasein oxidative stress correlates with developmental pattern of beta-secretase activity and beta-amyloid plaque formation in transgenicTg2576 mice with Alzheimer-like pathology. Int. J. Dev. Neurosci. 22,475–484.
Asai M, Iwasaki Y, Yoshida M, Mutsuga-Nakayama N, Arima H, Ito M,Takano K, Oiso Y (2004) Nuclear factor of activated T cells (NFAT) isinvolved in the depolarization-induced activation of growth hormone-releasing hormone gene transcription in vitro. Mol. Endocrinol. 18,3011–3019.
NFAT1 activation regulates BACE1 expression, H. J. Cho et al.
© 2008 The AuthorsJournal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2008
146
Barclay JW, Morgan A, Burgovne RD (2005) Calcium-dependent regulationof exocytosis. Cell Calcium 38, 343–353.
Barlic J, McDermott DH, Merrell MN, Gonzales J, Via LE, Murphy PM(2004) Interleukin (IL)-15 and IL-2 reciprocally regulate expressionof the chemokine receptor CX3CR1 through selective NFAT1- andNFAT2-dependent mechanisms. J. Biol. Chem. 279, 48520–48534.
Brorson JR, Bindokas VP, Iwama T, Marcuccilli CJ, Chisholm JC, Miller RJ(1995) The Ca2+ influx induced by beta-amyloid peptide 25–35in cultured hippocampal neurons results from network excitation.J. Neurobiol. 26, 325–338.
Cho HJ, Kim SK, Jin SM, Hwang EM, Kim YS, Huh K, Mook-Jung I (2007)IFN-γ-induced BACE1 expression is mediated by activation of JAK2and ERK1/2 signaling pathways and direct binding of STAT1 to BACE1promoter in astrocytes. Glia 55, 253–262.
Costantini C, Scrable H, Puglielli L (2006) An aging pathway controlsthe TrkA to p75NTR receptor switch and amyloid β-peptide generation.EMBO J. 25, 1997–2006.
Cowburn RF, Wiehager B, Sundstro ME (1995) β-Amyloid peptidesenhance binding of the calcium mobilising second messengers inositol(1,4,5) trisphosphate and inositol (1,3,4,5)–tetrakisphosphate to theirreceptor sites in rat cortical membranes. Neurosci. Lett. 191, 31–34.
Eberl G, Littman DR (2003) The role of the nuclear hormone receptorRORγt in the development of lymph nodes and Peyer’s patches. Immunol.Rev. 195, 81–90.
Esensten JH, Tsytsykova AV, Lopez-Rodriguez C, Ligeiro FA, Rao A,Goldfeld AE (2005) NFAT5 binds to the TNF promoter distinctly fromNFATp, c, 3 and 4, and activates TNF transcription during hypertonicstress alone. Nucleic Acids Res. 33, 3845–3854.
Foster TC, Kumar A (2002) Calcium dysregulation in the aging brain.Neuroscientist 8, 297–301.
Fukumoto H, Cheung BS, Hyman BT, Irizarry MC (2002) β-Secretaseprotein and activity are increased in the neocortex in Alzheimerdisease. Arch. Neurol. 59, 1381–1389.
Haass C, Schlossmacher MG, Hung AY, Vigo-Pelfrey C, Mellon A,Ostaszewski BL, Lieberburg I, Koo EH, Schenk D, Teplow DB (1992)Amyloid β-peptide is produced by cultured cells during normalmetabolism. Nature 359, 322–325.
Hong HS, Hwang EM, Sim HJ, Cho HJ, Boo JH, Oh SS, Kim SU, Mook-Jung I (2003) Interferon γ stimulates β-secretase expression and sAPPβproduction in astrocytes. Biochem. Biophys. Res. Commun. 307, 922–927.
Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, Yang F,Cole G (1996) Correlative memory deficits, Aβ elevation, and amyloidplaques in transgenic mice. Science 274, 99–102.
Jin SM, Cho HJ, Jung MW, Mook-Jung I (2007) DNA damage-inducingagent-elicited γ-secretase activity is dependent on Bax/Bcl-2 pathwaybut not on caspase cascades. Cell Death Differ. 14, 189–192.
Johnson GV, Cox TM, Lockhart JP, Zinnerman MD, Miller ML, PowersRE (1997) Transglutaminase activity is increased in Alzheimer’s diseasebrain. Brain Res. 751, 323–329.
Jones EA, Sun D, Kobierski L, Symes AJ (2003) NFAT4 is expressed inprimary astrocytes and activated by glutamate. J. Neurosci. Res. 72,191–197.
Kawahara M (2004) Disruption of calcium homeostasis in the patho-genesis of Alzheimer’s disease and other conformational diseases.Curr. Alzheimer Res. 1, 87–95.
LaFerla FM (2002) Calcium dyshomeostasis and intracellular signallingin Alzheimer’s disease. Nat. Rev. Neurosci. 3, 862–872.
Le WD, Colom LV, Xie W, Smith RG, Alexianu M, Appel SH (1995) Celldeath induced by β-amyloid 1–40 in MES 23.5 hybrid clone: the roleof nitric oxide and NMDA-gated channel activation leading to apoptosis.Brain Res. 686, 49–60.
Li R, Lindholm K, Yang LB, Yue X, Citron M, Yan R, Beach T, Sue L,Sabbagh M, Cai H et al. (2004) Amyloid β peptide load is correlatedwith increased β-secretase activity in sporadic Alzheimer’s diseasepatients. Proc. Natl Acad. Sci. USA 101, 3632–3637.
Liu F, Grundke-Iqbal I, Iqbal K, Oda Y, Tomizawa K, Gong CX (2005)Truncation and activation of calcineurin A by calpain I in Alzheimerdisease brain. J. Biol. Chem. 280, 37755–37762.
Loh C, Shaw KT, Carew J, Viola JP, Luo C, Perrino BA, Rao A (1996)Calcineurin binds the transcription factor NFAT1 and reversibly regulatesits activity. J. Biol. Chem. 271, 10884–10891.
Macian F (2005) NFAT proteins: key regulators of T-cell developmentand function. Nat. Rev. Immunol. 5, 472–484.
Masuda ES, Naito Y, Tokumitsu H, Campbell D, Saito F, Hannum C,Arai K, Arai N (1995) NFATx, a novel member of the nuclear factorof activated T cells family that is expressed predominantly in thethymus. Mol. Cell Biol. 15, 2697–2706.
Mattson MP (2004) Pathways towards and away from Alzheimer’sdisease. Nature 430, 631–639.
Mattson MP, Barger SW, Cheng B, Lieberburg I, Smith-Swintosky VL,Rydel RE (1993a) β-Amyloid precursor protein metabolites and lossof neuronal Ca2+ homeostasis in Alzheimer’s disease. Trends Neurosci.16, 409–414.
Mattson MP, Cheng B, Davis D, Bryant K, Lieberburg I, Rydel RE (1992)β-Amyloid peptides destabilize calcium homeostasis and renderhuman cortical neurons vulnerable to excitotoxicity. J. Neurosci. 12,376–389.
Mattson MP, Rydel RE, Lieberburg I, Smith-Swintosky VL (1993b) Alteredcalcium signaling and neuronal injury: stroke and Alzheimer’s diseaseas examples. Ann. N. Y. Acad. Sci. 679, 1–21.
McKinsey TA, Zhang CL, Olson EN (2002) MEF2: a calcium-dependentregulator of cell division, differentiation and death. Trends Biochem.Sci. 27, 40–47.
Ochi Y, Koizumi T, Kobayashi S, Phuchareon J, Hatano M, Takada M,Tomita Y, Tokuhisa T (1994) Analysis of IL-2 gene regulation in c-fostransgenic mice. Evidence for an enhancement of IL-2 expression insplenic T cells stimulated via TCR/CD3 complex. J. Immunol. 153,3485–3490.
Oyama Y, Chikahisa L, Ueha T, Hatakeyama Y, Kokubun T (1995) Changein membrane permeability induced by amyloid β-protein fragment25–35 in brain neurons dissociated from rats. Jpn. J. Pharmacol. 68,77–83.
Palotas A, Kalman J, Palotas M, Juhasz A, Janka Z, Penke B (2002)Fibroblasts and lymphocytes from Alzheimer patients are resistant toβ-amyloid-induced increase in the intracellular calcium concentration.Prog. Neuropsychopharmacol. Biol. Psychiatry 26, 971–974.
Pan F, Ye Z, Cheng L, Liu JO (2004) Myocyte enhancer factor 2 mediatescalcium-dependent transcription of the interleukin-2 gene in Tlymphocytes: a calcium signaling module that is distinct from butcollaborates with the nuclear factor of activated T cells (NFAT). J. Biol.Chem. 279, 14477–14480.
Plyte S, Boncristiano M, Fattori E, Galvagni F, Paccani SR, Majolini MB,Oliviero S, Ciliberto G, Telford JL, Baldari CT (2001) Identification andcharacterization of a novel nuclear factor of activated T-cells-1 isoformexpressed in mouse brain. J. Biol. Chem. 276, 14350–14358.
Querfurth HW, Selkoe DJ (1994) Calcium ionophore increases amyloidbeta peptide production by cultured cells. Biochem. 33, 4550–4561.
Rao A, Luo C, Hogan PG (1997) Transcription factors of the NFAT family:regulation and function. Annu. Rev. Immunol. 15, 707–747.
Ruff VA, Leach KL (1995) Direct demonstration of NFATp dephospho-rylation and nuclear localization in activated HT-2 cells using aspecific NFATp polyclonal antibody. J. Biol. Chem. 270, 22602–22607.
NFAT1 activation regulates BACE1 expression, H. J. Cho et al.
© 2008 The AuthorsJournal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2008
147
Sastre M, Dewachter I, Rossner S, Bogdanovic N, Rosen E, BorghgraefP, Evert BO, Dumitrescu-Ozimek L, Thal DR, Landreth G et al. (2006)Nonsteroidal anti-inflammatory drugs repress β-secretase genepromoter activity by the activation of PPARγ. Proc. Natl Acad. Sci. USA103, 443–448.
Shaw JP, Utz PJ, Durand DB, Toole JJ, Emmel EA, Crabtree GR (1998)Identification of a putative regulator of early T cell activation genes.Science 241, 202–205.
Singh IN, McCartney DG, Kanfer JN (1995) Amyloid beta protein (25–35) stimulation of phospholipases A, C and D activities of LA-N-2 cells.FEBS Lett. 365, 125–128.
Sisodia SS, Price DL (1995) Role of the β-amyloid protein in Alzheimer’sdisease. FASEB J. 9, 366–370.
Tamagno E, Bardini P, Obbili A, Vitali A, Borghi R, Zaccheo D, Pronzato MA,Danni O, Smith MA, Perry G et al. (2002) Oxidative stress increasesexpression and activity of BACE in NT2 neurons. Neurobiol. Dis. 10,279–288.
Ueda K, Shinohara S, Yagami T, Asakura K, Kawasaki K (1997) Amyloidβ protein potentiates Ca21 influx through L-type voltage-sensitiveCa2+ channels: a possible involvement of free radicals. J. Neurochem.68, 265–271.
Vassar R, Bennett BD, Babu-khan S, Kahn S, Mendiaz EA, Denis P,Teplow DB, Ross S, Amarante P, Loeloff R et al. (1999) β-Secretasecleavage of Alzheimer’s amyloid precursor protein by the transmembraneaspartic protease BACE. Science 286, 735–741.
Yan R, Bienkowski MJ, Shuck ME, Miao H, Tory MC, Pauley AM,Brashier JR, Stratman NC, Mathews WR, Buhl AE et al. (1999)Membrane-anchored aspartyl protease with Alzheimer’s disease β-secretase activity. Nature 402, 533–537.
Yang LB, Lindholm K, Yan R, Citron M, Xia W, Yang XL, Beach T, Sue L,Wong P, Price D et al. (2003) Elevated β-secretase expression andenzymatic activity detected in sporadic Alzheimer disease. Nat. Med.9, 3–4.