Disrupted intracellular calcium regulates BACE1 gene expression via nuclear factor of activated T...

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: inhee@snu.ac.kr

Accepted for publication

20 November 2007

NFAT1 activation regulates BACE1 expression, H. J. Cho

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© 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

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© 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

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© 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

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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.

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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.

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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.

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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).

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