Calcium/Calcineurin Signaling in Primary Cortical
Astrocyte Cultures: Rcan1-4 and Cyclooxygenase-2
as NFAT target genes
ANDREA CANELLADA,1BEL?EN G. RAMIREZ,1TAKASHI MINAMI,2JUAN MIGUEL REDONDO,1* AND EVA CANO1*
1Department of Vascular Biology and Inflammation. Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
2Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan
glia; calcium; calcineurin; NFAT; gene transcription; throm-
bin signaling; L-VGCC; ATP; glutamate; Rcan; Cox-2
The calcineurin/nuclear factor of activated T cells (NFAT)
signaling pathway mediates important cell responses to cal-
cium, but its activity and function in astrocytes have
remained unclear. We show that primary cortical astrocyte
cultures express the regulatory and catalytic subunits of
the phosphatase calcineurin as well as the calcium-regu-
lated NFAT family members (NFATc1, c2, c3, and c4).
NFATs are activated by calcium-mobilizing agents in astro-
cytes, and this activation is blocked by the calcineurin in-
hibitor cyclosporine A. Microarray screening identified cy-
clooxygenase-2 (Cox-2), which is implicated in brain injury,
and Rcan 1-4, an endogenous calcineurin inhibitor, as genes
up-regulated by calcineurin-dependent calcium signals in
astrocytes. Mobilization of intracellular calcium with iono-
phore potently augments the promoter activity and mRNA
and protein expression of Rcan 1-4 and Cox-2 induced by
combined treatment with phorbol esters. Moreover, Rcan
1-4 expression is efficiently induced by calcium mobilization
alone. For both the genes, the calcium signal component is
dependent on calcineurin and is replicated by exogenous
expression of a constitutively active NFAT, strongly sug-
gesting that the calcium-induced gene activation is medi-
ated by NFATs. Finally, we report that calcineurin-depend-
ent expression of Cox-2 and Rcan 1-4 is induced by physio-
agonists of purinergic and glutamate receptors, and L-type
voltage-gated calcium channels. These findings provide
insights into calcium-initiated gene transcription in astro-
cytes, and have implications for the regulation of calcium
responses in astrocytes.
C2008 Wiley-Liss, Inc.
Astrocytes are the most abundant cell type in the
brain, where they provide neurons with metabolic and
trophic support and regulate neural activity (Takuma et
al., 2004). Astrocytes express receptors for numerous
neurotransmitters, such as glutamate, gamma aminobu-
tyric acid (GABA), acetylcholine, ATP, and bradykinin
(Kettenmann, 1996; Porter and McCarthy, 1995; Verkh-
ratsky et al., 1998). Astrocytes and microglia are acti-
vated by many forms of injury and disease in the central
nervous system (CNS) (Griffin, 2006; Wisniewski et al.,
1997), and activated glia also increase as a normal conse-
quence of aging (Vaughan and Peters, 1974; Wisniewski
and Terry, 1973). Astrocytes are moreover implicated in
the response to cerebral hypoxia (Smith et al., 2003), and
astrocyte activation has been shown to modulate synaptic
plasticity and neuronal apoptosis (Takuma et al., 2004).
The phenotype of activated astrocytes is known as reac-
tive gliosis, and is characterized by hypertrophy of the
soma and cellular processes and by up-regulated expres-
sion of a characteristic set of markers, which includes the
(GFAP) and vimentin (Vim), as well as extracellular ma-
trix (ECM) molecules, growth factors, inflammatory cyto-
kines, and oxidative stress markers (Eddleston et al.,
1993; Eng et al., 2000; Ridet et al., 1997).
Communication among astrocytes and the physiologi-
cal activity of individual cells are highly dependent on
calcium signaling (Verkhratsky and Kettenmann, 1996).
Indeed, astrocyte activation is usually triggered by a
rise in intracellular calcium concentration ([Ca21]i) be-
cause of release from intracellular stores and uptake
from the extracellular space (Araque et al., 2001; Bezzi
et al., 2001; Deitmer et al., 1998; Grosche et al., 1999).
Dysregulated intracellular calcium signaling is a feature
of many neurodegenerative diseases in which astrocytes
become activated, such as neurotrauma, ischemia, tumor
growth, and neurodegeneration [reviewed in (LaFerla,
2002)]. However, despite the increasing evidence sup-
porting the functional importance of calcium-mediated
astrocyte activation [reviewed in (Takuma et al., 2004;
Zawadzka and Kaminska, 2005)], the precise role of ac-
tivated astrocytes and the particular gene expression
This article contains supplementary material available via the Internet at http://
Grant sponsor: Ministerio de Sanidad y Consumo, Spain; Grant number:
PI060491; Grant sponsor: Ministerio de Educaci? on y Ciencia, Spain; Grant numbers:
SAF2006-08348 and RD06/0014/005; Grant sponsor: European Union; Grant num-
ber: LSHM-CT-2004-0050033; Grant sponsor: Pro-CNIC Foundation, Mapfre Foun-
dation, Fundaci? on Carolina.
Andrea Canellada is currently at Instituto de Estudios en Inmunidad Humoral
(IDEHU), CONICET-UBA, Jun? ın 956, 4P-1113 Buenos Aires, Argentina.
*Correspondence to: Eva Cano (or) Juan Miguel Redondo, Centro Nacional de
Investigaciones Cardiovasculares (CNIC), Melchor Fern? andez Almagro 3, 28029
Madrid, Spain. E-mail: firstname.lastname@example.org (or) email@example.com
Received 3 September 2007; Accepted 21 January 2008
Published online 21 February 2008 in Wiley InterScience (www.interscience.
GLIA 56:709–722 (2008)
C2008 Wiley-Liss, Inc.
program initiated by calcium signaling in these cells
remain to be elucidated.
A key element of the cellular response to Ca21signals
is the action of the phosphatase calcineurin (CN). CN is a
Ca21- and calmodulin-dependent phosphatase that was
first discovered in brain tissue, where it has its greatest
abundance (Klee et al., 1979). CN has been found in
many other tissues [reviewed in (Mansuy, 2003; Marti-
nez-Martinez and Redondo, 2004)], notably in lympho-
cytes, where most studies of CN-regulated signaling path-
ways have been conducted. The CN holoenzyme is a het-
erodimer, consisting of a 61-kDa catalytic subunit (CN A)
and a 19-kDa regulatory subunit (CN B). The main mode
of action characterized so far for this phosphatase is the
regulation of nuclear factor of activated T cells (NFAT)
family of transcription factors. CN-mediated dephospho-
rylation promotes translocation of NFAT proteins to the
nucleus, where they bind specific elements within target
gene promoters, in many cases through association with
other transcription factors [reviewed in (Crabtree and
Olson, 2002; Hogan et al., 2003)]. The pharmacological
action of immunosuppressive drugs such as cyclosporin A
(CsA) and FK506 is based on their inhibition of CN in
immune effector cells (Liu et al., 1991).
Activated astrocytes express several proinflammatory
et al., 1995), IL6 (Pousset, 1994), tumor necrosis factor
alpha (TNFa) (Pousset et al., 1996), and interferon-
gamma (IFN-g) (Dong and Benveniste, 2001), which are
inducedby the calcium/CN/NFAT
immune system (Crabtree and Olson, 2002; Hogan et al.,
2003). Astrocytes are also one of the most abundant sour-
ces in the brain of prostaglandin E2 (PGE2), the key
metabolite of cyclooxygenase-2 (Cox-2), another target of
NFAT signals, both in lymphocytes and endothelial cells
(Iniguez et al., 2000). Cox-2 plays an important role in
the response to injury in the brain, where its expression
is induced by excitotoxins, ischemia, and seizure-like
nerve activity (Chen et al., 1995; Collaco-Moraes et al.,
1996; Yamagata et al., 1993). However, although cul-
tured astrocytes express Cox-2 in response to inflamma-
tory mediators (Hirst et al., 1999; Koyama et al., 1999;
O’Banion, 1999), direct induction of Cox-2 by calcium
signals in astrocytes has not been reported previously.
More recent evidence highlights the importance of
autoregulation of NFAT signaling. The recently identi-
fied calcineurin-NFAT-regulated target genes, regulators
of calcineurin (Rcan) belong to a family of calcineurin
inhibitors, previously named DSCR, MCIP, and Adapt78
in human (Davies et al., 2007; Rothermel et al., 2003),
which are regulated by increases in [Ca21]i in several
cell types (Cano et al., 2005; Hesser et al., 2004; Minami
et al., 2004; Yang et al., 2000). In the brain, Rcan 1
mRNA and protein expression has been detected mainly
in neurons (Ermak et al., 2001; Hoeffer et al., 2007;
Porta et al., 2007a); and Rcan 1 mRNA accumulates in
the brains of patients with Alzheimer’s disease (Ermak
et al., 2001). Very recently, the expression of mRNA and
protein for another family member, Rcan 2, has been
detected in oligodendrocytes (Porta et al., 2007a).
2 (IL2) (Eizenberg
Gene targets of the calcium/CN/NFAT pathway identi-
fied in the CNS include neuronal expression of the inosi-
tol-3-phosphate receptor (IP3R), brain-derived neuronal
factor (BDNF), and TNF-a (Canellada et al., 2006; Gen-
azzani et al., 1999; Graef et al., 1999; Groth et al.,
2003). Moreover, neuroprotective actions have been
reported for FK506 and CsA [see (Guo et al., 2001) and
references therein]; but although there is some sugges-
tion that CN inhibition might be neuroprotective in the
ischemic brain (Butcher et al., 1997), the exact mecha-
nism through which this is achieved is currently
In spite of the abundance of CN and NFATs in the
CNS and the importance of calcium-mediated astrocyte
activation, little is known about the activity of the Ca/
CN/NFAT pathway and its potential targets in astro-
cytes. CN is expressed primarily in neurons and only
weakly or not at all in astrocytes in healthy brain
(Dawson et al., 1994a; Goto et al., 1986); however,
increased expression of a specific CN A isoform has been
reported in activated astrocytes after transient ischemia
(Hashimoto et al., 1998). Although the effect of inhibi-
ting CN in glial cells differs (Matsuda et al., 1998; Pyr-
zynska et al., 2001), a recent report proposes a role for
CN in the appearance of the astrocyte activation pheno-
type (Norris et al., 2005). Primary cultured astrocytes
expressing elements of the reactive phenotype have been
reported to express both CN A and CN B subunits
(McMillian et al., 1994). Furthermore, very recently it
expressed in glial cells has a dual action, being able to
direct the neuroinflammatory process toward its resolu-
tion or its progression (Fernandez et al., 2007). NFATc3
is present in the U373 astrocytoma cell line and C6 gli-
oma cells, and there is evidence for its expression in pri-
mary astrocyte cultures (Jones et al., 2003; Mosieniak
et al., 1998).
Here,we show thatprimary
express all components of the Ca21/CN/NFAT pathway,
and that this pathway is activated by pharmacological
and physiological Ca21-mobilizing agents. Based on the
results of a targeted microarray screen, we also identify
the proinflammatory response gene Cox-2 and the calci-
neurin regulator Rcan 1-4 as neurologically relevant tar-
get genes that are up-regulated by this pathway in
astrocytes. These findings support an important role for
responses to increases in intracellular calcium.
MATERIALS AND METHODS
Cell Culture and Reagents
Raw 264.7 cells were obtained from the ATCC and cul-
tured as indicated. Astrocytes cultures were obtained
from neonatal Wistar rat or C57BL/6 mouse brain corti-
ces by a modification of the method of McCarthy and de
removed by shaking cultures at 200 rpm overnight and
washing twice with phosphate-buffered saline (PBS),
CANELLADA ET AL.
and the astrocyte layer was covered with fresh DMEM
medium containing 10% fetal calf serum. Cortical astro-
cyte cultures were plated at 4 3 105cells per well in 6-
well plates or 1 3 106cells per 10-cm plate and grown
to confluence, with medium changes every 3 days. At
17–20 days in culture, immunocytochemical analysis
indicated that (98% of the cells in the culture were
GFAP-positive (Raff et al., 1983).
The Ca21ionophore A23187 was from Calbiochem.
Phorbol 12-myristate 13-acetate (PMA), (1/2) Bay K
8644, thrombin, ATP, glutamate, nifedipine, verapamil,
anisomycin, and actinomycin D (ActD) were all from
Sigma. CsA was from Sandoz (East Hannover, NJ) and
LC Laboratories (Woburn, MA). All inhibitor pretreat-
ments were for 1 h.
Murine astrocytes on poly-L-lysine-coated glass cover-
slips were immunostained as in Cano et al. (2005), using
a 1:500 dilution of rabbit polyclonal anti-GFAP antibody
(Dako). Cells were incubated for 30 min at room temper-
ature with secondary antibody (Alexa Fluor 488-labeled
goat anti-rabbit IgG, Molecular Probes, Eugene, OR) in
the presence of the nucleic acid dye DAPI (Molecular
Probes). Cells were mounted and analyzed on a LSM510
metaconfocal laser microscope (Zeiss, Germany).
Raw 264.7 and murine primary astrocytes were trip-
synized and washed twice with PBS. Cell suspensions
were prepared in cold staining medium (PBS, 2% fetal
calf serum) and incubated with anti-CD16/CD32 mAb to
washing, cells were incubated with a 1:100 dilution of
Alexa Fluor 647-conjugated rat anti-mouse CD11b/Mac-1
mAb (BD Biosciences, San Jos? e CA). Dead cells and de-
bris were excluded by light-scattering parameters and
propidium iodide gating. Positively stained cells were
detected by comparison with the background fluores-
cence of samples stained with an isotype-specific control
mAb. At least 104gated cells were analyzed per sample.
Flow cytometry was performed on a CyAn ADP (Dako
Cytomation) equipped with three lasers: 405, 488, and
635 nm. Data was analyzed with Summit 4.3 (Dako).
Cell Lysis and Immunoblot Analysis
For whole-cell extracts, cells grown and stimulated in
6-well plates, or as indicated, were washed twice with
cold PBS and lysed for 30 min on ice in 100 lL hyper-
tonic buffer with occasional mild agitation, as described
(Cano et al., 2005). Total extracts were boiled in
Laemmli buffer and resolved by 10% SDS-PAGE. Pro-
teins were transferred to nitrocellulose membranes that
were then immunoblotted as described (Cano et al.,
2005). The following antibodies were used: rabbit poly-
clonal anti-calcineurin A (Chemicon) and anti-calci-
neurin B clone VA1 (Upstate); mouse monoclonal anti-
human Rcan (Minami et al., 2004); goat polyclonal anti-
rat Cox-2 C20 (Santa Cruz Biotechnologies); anti-NFAT
clone 7A6 (Alexis Biochemicals); anti-NFATc2 antiserum
672 (Cano et al., 2005); and anti NFATc3 polyclonal M75
and anti-NFATc4 polyclonal H74 (both from Santa
Cruz). All the antibodies were used at 1:1,000 except for
anti-Cox-2, which was diluted 1:2,500.
Calcineurin-Rcan Binding Assay
A GST-Rcan1 fusion protein was expressed in the pro-
tease-deficient BL21(DE3) Escherichia coli strain and
purified with glutathione (GSH)-sepharose 4B beads
(Amersham Biosciences). This fusion protein was used
to pull-down CN A from cell lysates as follows. Beads
containing GST or GST-Rcan1 were washed twice with
binding buffer (20 mM Tris, pH 8.0, 100 mM NaCl,
1.5 mM CaCl2, 6 mM MgCl2, 1 mM phenylmethylsulfonyl
fluoride, 1 lM dithiothreitol, 1 lg/mL aprotinin, 1 lg/mL
pepstatin, 100 lM benzamidine, and 0.2% Triton X-100)
and incubated with lysates of cultured murine cortical
astrocytes, or Jurkat cells, in binding buffer for 30 min
on a rocking platform at 4?C. Samples were then briefly
centrifuged at 4?C and the supernatants discarded. The
beads were washed (53) with freshly made ice-cold bind-
ing buffer, and bound proteins were eluted by boiling in
Laemmli buffer. Eluted proteins were separated by 10%
SDS-PAGE. Calcineurin was detected by immunoblotting
with a mouse monoclonal anti-CN A antibody (clone
RNA Isolation, Reverse Transcription,
and Real-Time PCR Analysis
Total RNA was isolated from cultured rat or mouse
cortical astrocytes as described (Cano et al., 2005). Dif-
ferential gene expression in astrocytes was assessed by
real-time quantitative RT-PCR of target genes on a
microfluidic card assay system developed by Applied
Biosystems (Foster City, CA) (Cano et al., 2005).
Transcripts encoding rat Cox-2 were further analyzed
by semiquantitative RT-PCR. Total RNA (1 lg) was
reverse transcribed to cDNA and used for PCR amplifi-
cation with specific primers for rat Cox-2 or b-actin as
follows: Cox-2 forward primer, 50-ACTTGCTCACTTTGT
TGAGTCATTC-30; reverse primer, 50-TTTGATTAGTACT
GTAGGGTTAATG-30; b-actin forward primer, 50-TGAC
PCR reactions were run over 25–35 cycles of denatur-
ing at 95?C for 1 min, annealing at 58?C for 1 min, and
extension at 72?C for 1 min. Amplified cDNAs were sep-
arated by agarose gel electrophoresis, and bands were
visualized by ethidium bromide staining. The data
shown correspond to the number of cycles where the
amount of amplified product is proportional to the abun-
dance of starting material.
Transcripts encoding rat Rcan 1-4, rat Cox-2, and
mouse Nfatc1, Nfatc2, Nfatc3, and Nfatc4 were quanti-
CN/NFAT TARGET GENES IN CORTICAL ASTROCYTES
fied by TaqMan real-time quantitative RT-PCR. PCR pri-
mers and TaqMan probes were obtained from Applied
Biosystems and the conditions were optimized according
to the manufacturer’s protocol. The 18S rRNA transcript
was used as an internal control gene and was amplified
in the same tube to normalize for variation in input
RNA. The amounts of target mRNA in samples was esti-
mated by the 22DDCTrelative quantification method
(Livak and Schmittgen, 2001). Ratios were calculated
between the amounts of mRNA from stimulated and
Nuclear Extracts and EMSA
Confluent astrocytes grown on 100-mm culture dishes
were exposed to vehicle or CsA for 1 h and then treated
with PIo. Cells were lysed and electrophoretic mobility
shift assays (EMSA) performed as described (Cano et al.,
Plasmid Constructs and Transient
The luciferase reporter plasmid NFAT/AP1 Luc con-
tains three tandem copies of the composite NFAT/AP1-
binding site from the IL-2 gene promoter coupled to the
IL-2 minimal promoter (Durand et al., 1988). The Rcan
(21,664/183) Luc plasmid containing the human Rcan
(Minami et al., 2004). The human full-length (p2-1900)
Cox-2 promoter construct, and deletion mutant versions,
in the pXP2Luc promoter plasmid was as described (Ini-
guez et al., 2000). The expression vector encoding consti-
tutively nuclear human NFATc2 was as described (Oka-
mura et al., 2000). The GST-Rcan1 construct expressing
aminoacids 170–252 of CALP1L/Rcan1 fused to glutathi-
one S-transferase (GST) was as described (Aubareda et
al., 2006). Astrocytes were plated on 35-mm dishes at
50–60% confluence the day before transfection. For tran-
sient transfection, the culture medium was replaced
with 1 mL serum-free Opti-MEM (Invitrogen). Cells
were transfected by the Lipofectamine Plus method
(Invitrogen), with the plasmids specified in each figure.
Cultures were co-transfected with plasmids encoding
GFP and Renilla (Promega) to normalize for transfection
efficiency. Individual transfections were made up to 1 lg
with empty vector. After 4 h, media were made up to
2 mL with complete Dulbecco’s modified Eagle’s medium
containing 20% serum and cultures were incubated for
12 h at 37?C and 5% CO2. At the end of the transfection
period, media were removed and replaced with complete
D-MEM/10% serum. After 24 h, the cells were exposed
to vehicle or inhibitor (CsA or ActD as specified) for 1 h
and then stimulated. At the end of treatments, cells
were lysed according to the instructions of the Lucifer-
ase assay kit, and luciferase activity was measured in a
Berthold luminometer. All the samples were tested in
triplicate, and the results were normalized to a Renilla
luciferase internal control by using the Dual luciferase
assay kit (Promega) as specified by the manufacturer.
Adenovirus Infection of Astrocytes
The adenovirus encoding constitutively active NFATc3
(Adeno ca NFAT) and the control adenovirus (Adeno
Empty) were as described (Minami et al., 2004). Adeno-
viruses were generated and purified following the stand-
ard protocols. Adenovirus infection was performed on
subconfluent primary astrocytes, and expression of the
encoded protein was monitored by immunoblotting.
Data are presented as means6SD of several determi-
nations. Differences between groups were tested for sig-
nificance using one-way analysis of variance (ANOVA)
and the Neumann-Keuls multiple comparison test as a
post-test. (***) indicates P < 0.001, (**) indicates P <
0.01, and (*) indicates P < 0.05. (Motulsky, HJ Prism 4
Statistics Guide; Graph-Pad Software, San Diego CA).
Primary Cortical Astrocytes in Culture
In astrocytes from healthy brain, the phosphatase calci-
neurin (CN) has been reported to be either weakly
expressed or undetectable (Dawson et al., 1994b; Goto
et al., 1986). However, upon activation by transient ische-
mia, astrocytes express a specific CN A isoform (Hashimoto
et al., 1998). Furthermore, overexpression of a constitu-
tively active form of CN appears to trigger the astrocyte
activation phenotype (Norris et al., 2005). To investigate
the Ca21/CN/NFAT pathway in astrocytes, we first ana-
lyzed the presence of CN in cortical astrocyte primary cul-
tures obtained through standard procedures (see Materials
and Methods section). Culture purity was assessed by im-
munofluorescence analysis of the expression of GFAP pro-
tein (Fig. 1A); the percentage of positive-staining cells was
more than 98% in all cultures. Moreover, the proportion
of cells expressing the microglial marker CD11b was less
than 1% (Fig. 1B). Immunoblotting of whole-cell extracts
detected both the CN A catalytic subunit and the CN B
regulatory subunit (Fig. 1C). To further confirm the speci-
ficity of the CN signal, we carried out affinity purification
experiments with a GST fusion protein of the C-terminal
region of Rcan 1 (aminoacids 170–252), which has been
shown to specifically bind CN A (Aubareda et al., 2006;
Fuentes et al., 2000). These experiments identified endog-
enous CN A in total lysates of mouse and rat primary
astrocyte cultures (Supplemental Fig. 1); the amount of
purified protein was comparable with that obtained from
the same number of Jurkat lymphoid cells, which are
known to endogenously express CN.
NFAT Expression and Ca21-Induced Activation of
the Calcineurin/NFAT Pathway in Primary
The nuclear factor of activated T cells (NFAT) family
of transcription factor is one of the best studied targets
CANELLADA ET AL.
of the calcium/CN pathway. Using real-time PCR analy-
sis, we detected the expression of Nfatc1, Nfatc2, Nfatc3,
and Nfatc4 mRNA in mouse cortical astrocyte cultures
(Fig. 2A). In control experiments, we measured Nfat
mRNA expression in the spleen cells, where Nfatc4
mRNA was not found (Fig. 2B). We next examined the
activation of NFAT proteins in astrocytes treated with a
combination of the phorbol ester phorbol 12-myristate
13-acetate (PMA, 20 ng/mL) and the calcium ionophore
A23187 (Io, 1 lM) (PIo), a conventional pharmacological
means of elevating [Ca21]i. EMSA analysis revealed that
nuclear proteins from PIo-stimulated astrocytes formed
a specific DNA-nuclear protein complex with an NFAT/
AP1 consensus sequence (Fig. 2C, Lane 3). Formation of
this complex was abolished by pretreating cells with cy-
closporine A (CsA) (Fig. 2C, Lane 4). CsA is a well-
known inhibitor of CN (Liu et al., 1991), and therefore
prevents the dephosphorylation and subsequent nuclear
localization of NFAT transcription factors. The presence
of NFAT in the DNA-nuclear protein complex was con-
firmed by preincubating with the anti-all NFAT antibody
674, which recognizes a common epitope in the DNA-
binding domain of all NFAT family members (Hernandez
et al., 2001) (data not shown).
Total extracts from nonstimulated and PIo-stimulated
astrocytes were analyzed by immunoblot with antibodies
against individual NFAT family members. Antibodies
against Nfatc1, Nfatc2, Nfatc3, and Nfatc4 each recog-
nized proteins in the range of 97–116 kD (Fig. 2D). In
cells pretreated with CsA, inhibition of NFAT-protein de-
phosphorylation was evident in the retardation of the
protein bands (Fig. 2D, Lanes 2 and 4). It is notable
that the mobility of NFAT proteins was very similar in
nonstimulated and PIo-treated cells (Fig. 2D, Lanes 1
and 3); however, it was only in PIo-stimulated cells that
we observed increased NFAT-dependent transcriptional
activity (Fig. 2E). The calcium signal elicited by PIo was
inhibited by a pretreatment with CsA (Fig. 2E).
Cyclosporine A-Sensitive Gene Expression
in Primary Cortical Astrocytes in Response
We used a low-density array based on quantitative
real-time PCR (RT-PCR) to investigate the CN-depend-
ent gene transcription. The assay included TaqMan
probes and primers for 19 preselected genes plus endog-
enous controls such as 18S ribosomal RNA. We selected
these genes from candidate NFAT targets identified in
other cell systems and from genes known to be induced
by Ca21in the CNS. Using this approach, we identified
genes whose expression was up-regulated by PIo stimu-
lation to higher levels than by stimulation with PMA
alone. These included the early growth response-tran-
scription factor-1 (Egr-1), cyclooxygenase-2 (Cox-2), and
Rcan 1-4 (Table 1). Inhibition of CN by pretreatment
with CsA partially inhibited synthesis of Cox-2 mRNA,
restricting induced expression to levels similar to those
stimulated by PMA alone. In contrast, CsA pretreatment
inhibited expression of the Rcan 1-4 gene to the level
seen in unstimulated cells (1.14 1/20.07) (Table 1).
Other candidate Ca21-activated CN-dependent genes
included tPa (tissue plasminogen activator) and Il 6;
these genes have not been further investigated in this
cytes in culture express calcineurin A
and B. (A) Immunofluorescence of en-
dogenous glial fibrillary acidic protein
(GFAP) (a) staining with anti-GFAP; (b)
DAPI staining of cell nuclei. (B) Repre-
sentative FACS analysis of a mouse pri-
mary astrocyte culture for the expres-
sion of the cell surface CD11b; expres-
sion in Raw 264.7 cells is included as a
CD11b positive control. Upper panels
show labeling with an isotype control
IgG; lower panels show labeling with
Alexa Fluor 647 conjugated anti CD11b
monoclonal Ab. The percentage of live
positive cells in each condition is shown.
(C) Immunoblots showing the endoge-
nous expression of calcineurin A (CN A)
and calcineurin B (CN B). Lanes 1, 2,
and 3 correspond to total lysates of three
independent astrocyte cultures.
Mouse cortical primary astro-
CN/NFAT TARGET GENES IN CORTICAL ASTROCYTES
Calcium Signaling Induces Cox-2 mRNA and
Protein Synthesis in Cultured Astrocytes
To confirm whether calcium/CN signaling induces
Cox-2 expression in astrocytes, we first measured Cox-2
mRNA in astrocytes treated with PMA, PIo, or Io alone.
Semiquantitative and quantitative RT-PCR revealed pro-
nounced increases in Cox-2 mRNA expression after
treatment with PIo for 4 h (Figs. 3A,C); treatment with
PMA or Io alone elicited smaller increases. Cox-2 mRNA
was undetected in nonstimulated cells, and the expres-
sion of actin mRNA and 18S rRNA genes was unaffected
by stimuli (Fig. 3A and included in the quantitative RT-
Pre-exposure of primary astrocyte cultures to CsA or
another CN inhibitor (FK506; data not shown) potently
inhibited accumulation of Cox-2 mRNA induced by the
PIo calcium stimulus (Figs. 3B,D), similar to the situa-
tion reported in other cell types (Hernandez et al., 2001;
Iniguez et al., 2000). Consistent with the array data (Ta-
ble 1), this inhibition reduced Cox-2 mRNA expression
by approximately threefold, to a level similar to that
induced by PMA alone. It thus appears that PIo stimula-
tion includes a dominant calcium-dependent signaling
component which acts via CN.
PIo-induced up-regulation of Cox-2 mRNA expression
was paralleled by increased production of Cox-2 protein,
detected by immunoblot of total extracts of cultured
astrocytes. Cox-2 protein was significantly increased in
cells treated for 4 h with PIo (Fig. 4A, Lane 4), and was
detectable after stimulation for at least 24 h (not
shown). Cox-2 protein accumulation was inhibited by
the transcriptional inhibitor actinomycin D and by the
translational inhibitor anisomycin (Fig. 4A), indicating
de novo synthesis of the protein. As with Cox-2 mRNA,
CsA pretreatment reduced PIo-induced Cox-2 protein
synthesis to PMA-activated levels (Fig. 4B).
To identify the regions of the Cox-2 promoter that
mediate calcium-dependent expression, we transfected
astrocytes with luciferase reporter constructs; these con-
structs were driven by versions of the human Cox-2 pro-
21,900 bp to 12 bp from the TATA box [Fig. 4C and
(Hernandez et al., 2001)]. Constructs containing the 274
in the regionspanning
mary cortical astrocytes. (A) NFAT member mRNAs from cultured mouse
cortical astrocytes (Nfatc1, Nfatc2, Nfatc3, and Nfatc4) were amplified by
TaqMan RT-PCR (see Materials and Methods section). Nfat mRNA was
normalized to the expression of 18 S rRNA, to control for the quantity
and integrity of total RNA. Quantifications are expressed relative to the
values obtained with 10 ng cDNA, which were assigned a value of 1. (B)
Comparison of Nfat mRNA levels in mouse spleen cells and astrocytes.
Total RNA was isolated from mouse spleen cells and astrocytes and each
NFAT member mRNA was amplified as in (A). Quantifications are
expressed relative to the values obtained with astrocyte Nfat cDNA. (C)
EMSA of nuclear extracts from rat astrocytes pretreated (1 h) with vehi-
cle (Lanes 1 and 3) or 200 ng/mL CsA (Lanes 2 and 4) and then exposed
for 1 h to PIo (20 ng/mL PMA plus 1 lM ionophore). ns, nonstimulated
The calcineurin/NFAT signaling pathway is present in pri-
control cells. Nuclear extracts were incubated with a probe containing
the composite NFAT/AP1 site from the human IL-2 promoter. (D) Immu-
noblot showing endogenous expression and activation of NFAT isoforms
in total extracts of astrocytes pretreated with vehicle (Lanes 1 and 3) or
CsA (Lanes 2 and 4) and then stimulated with PIo for 1 h (Lanes 3 and
4). ns, nonstimulated control cells. Molecular weights (kDa) are indicated
to the left. (E) Calcineurin-dependent activation of NFAT-mediated tran-
scription. Astrocytes were transiently transfected with a NFAT/AP1 lucif-
erase reporter construct. After 24 h, cells were pretreated with CsA or
vehicle. Cells were then left nonstimulated (ns) or stimulated with PIo
for further 4 h. Transcriptional activity is expressed as relative luciferase
units (RLU). Representative experiments of a minimum of three are
shown; values are means 6 SD of triplicate luciferase determinations for
each condition. ***P < 0.001; **P < 0.01; *P < 0.05 (ANOVA) versus ns.
CANELLADA ET AL.
bp upstream of the TATA box (21,900/12, 2431/12, and
2274/12) supported PIo-induced luciferase activities
from 3- to 5-fold greater than that of nonstimulated cells
(Fig. 4C). In contrast, the construct containing only the
proximal 150 bp promoter region (2150/12) did not sup-
port increased luciferase activity above nonstimulated
levels, consistent with earlier reports that two NFAT
binding sites located between base pairs 2274 and 2150
of the human Cox-2 promoter are required for induction
by the calcium/CN pathway (Hernandez et al., 2001; Ini-
guez et al., 2000). CsA pretreatment inhibited PIo-de-
pendent activation to PMA-activated levels (Fig. 4D).
The Endogenous Calcineurin Regulatory Protein
Rcan1-4 is Induced by Calcineurin-Dependent
Calcium Signals in Cultured Astrocytes
Rcan 1-4 is a member of the calcipressin family of pro-
teins, which have been identified as endogenous modula-
tors of CN activity [reviewed in (Davies et al., 2007)].
Quantitative real-time PCR showed that Ca21signals
potently induce accumulation of Rcan 1-4 mRNA in
astrocytes (Table 1 and Fig. 5A). PMA alone did not
increase Rcan 1-4 mRNA levels, but was effective only
in combination with Io (Fig. 5C). In contrast, and con-
firming the array data (Table 1), calcium ionophore
induced Rcan 1-4 mRNA expression in the absence of
PMA, suggesting that in astrocytes this gene is induci-
ble by calcium signaling alone. PIo also induced expres-
sion of Rcan 1-4 protein (Figs. 5C,D), and this was
inhibited by pretreatment with ActD (Fig. 5C, Lane 4),
confirming the requirement for gene transcription. The
induction of Rcan 1-4 was inhibited by CsA (Fig 5D,
Lane 4), consistent with an involvement of CN in PIo-
induced Rcan 1-4 protein expression.
The CN/NFAT Signaling Pathway Is Required
for Activation of Rcan 1-4 Promoter Activity
in Cultured Astrocytes
Astrocyte cultures were transfected with luciferase re-
porter constructs driven by deletion versions of the
human Rcan 1 internal promoter. These deletions
TABLE 1. Calcineurin-Dependent PIo-Inducible Genes in Primary Cortical Astrocytes
Relative quantification (mean 6 SD)
RCAN 1-4 (Down syndrome critical region 1)
EGR-1 (early growth factor)
IGF-1 (insulin-like growth factor)
IP3R1 (inositol 3-phosphate receptor-1)
PAI-2 (plasminogen-activator inhibitor-2)
tPA (tissular plasminogen activator)
uPA (urokinase plasminogen activator)
FasL (Fas ligand)
1.1 6 0.05
7.22 6 0.48
1.72 6 0.07
1.32 6 0.01
0.64 6 0.06
53.79 6 1.60
2.35 6 0.04
27.31 6 2.22
6.22 6 0.38
10.34 6 1.87
0.59 6 0.04
17.27 6 1.87
1.88 6 0.49
1.07 6 0.44
2.57 6 0.35
7.8 6 1.4
14.28 6 2.47
2.12 6 0.11
0.29 6 0.02
0.27 6 0.01
67.21 6 3.64
1.20 6 0.14
31.85 6 1.18
8.87 6 0.18
8.56 6 1.42
0.65 6 0.49
72.50 6 8.58
0.66 6 0.06
0.35 V 0.02
2.14 6 0.41
1.14 6 0.07
2.06 6 0.13
1.10 6 0.25
0.60 6 0.07
0.85 6 0.08
2.75 6 0.04
1.02 6 0.02
1.16 6 0.01
1.61 6 0.08
0.98 6 0.05
0.97 6 0.71
3.45 6 0.42
0.82 6 0.01
0.73 6 0.15
2.52 6 0.80
0.17 6 0.04
25.45 6 2.39
1.35 6 0.15
0.93 6 0.05
0.37 6 0.04
31.22 6 4.36
1.39 6 0.18
56.02 6 1.60
4.76 6 0.33
5.41 6 0.76
0.46 6 0.07
24.45 6 4.59
0.79 6 0.02
0.23 6 0.00
3.69 6 1.31
Cortical primary astrocytes were pretreated or not with 200 lg/mL cyclosporine A (CsA) and then left without treatment (ns) or treated with PMA or PMA plus ionophore
(PIo) during 4 h. Target genes mRNAs were amplified from total RNA by TaqMan RT-PCR as described in the Material and Methods section. mRNAs were quantified in
arbitrary units normalized to the expression of endogenous 18S. Data are expressed as the relative quantification as compared with nonstimulated cells, which were
assigned a value of 1. Values are the means 6 SD of triplicate RT-PCR determinations.
in cultured rat cortical astrocytes is blocked by calcineurin inhibitors.
(A, B) Semiquantitative RT-PCR for Cox-2 mRNA, with b-actin as in-
ternal control. (A) Rat cortical astrocytes were exposed to 20 ng/mL
PMA (P), 1 lM calcium ionophore (Io), or a combination of both (PIo)
for 4 h ns, nonstimulated control cells. (B) Cells were pretreated with
vehicle or 200 ng/mL CsA (CsA) and then exposed as indicated to PIo
(4 h). (C, D) Quantitative Real-Time (RT-PCR). Cox-2 mRNA was quan-
tified in arbitrary units normalized to the expression of 18S rRNA, to
control for the quantity and integrity of total RNA. (C) Astrocytes were
exposed to PIo, PMA, or Io for 4 h as indicated. (D) Cells were pre-
treated with vehicle or CsA and then exposed to PIo for another 4 h.
Quantifications are expressed relative to nonstimulated control cells
(ns). Representative experiments of a minimum of three are shown; val-
ues are means 6 SD of triplicate RT-PCR determinations for each con-
dition. ***P < 0.001; **P < 0.01 (ANOVA) versus ns.
Calcium dependent stimulation of Cox-2 mRNA expression
CN/NFAT TARGET GENES IN CORTICAL ASTROCYTES
spanned the region between 21,664 and 2166 bp from
the TATA box [Fig. 6A and (Cano et al., 2005; Minami
et al., 2004)]. Constructs containing the 350 bp upstream
of the TATA box (21,664/183, 2750/183, and 2350/
183) supported PIo-induced luciferase activities 4- to 6-
fold greater than that of nonstimulated cells, whereas
PMA alone did not induce activity of any promoter con-
struct (Fig. 6B and not shown). CsA inhibited transcrip-
expression in cultured rat cortical astrocytes and activate transcription
from the 2274 and 2150 bp Cox-2 promoter region. (A, B) Immuno-
blots showing endogenous protein expression of Cox-2. (A) Cells were
pretreated with vehicle (2), 10 lg/ml anisomycin (An), or 10 lg/ml acti-
nomycin D (Act D), and then exposed as indicated to PIo (Lanes 4–6)
for another 4 h. (B) Cells were pretreated as indicated with vehicle or
CsA and then treated (4 h) with PMA or PIo. Representative experi-
ments are shown of a minimum of three. (C) Rat astrocytes were trans-
fected with luciferase reporter plasmids containing human Cox-2 pro-
moter regions starting from 21900 to 2150 upstream of the transcrip-
Calcium signals induce calcineurin-dependent Cox-2 protein
tion initiation site. After 24 h, cells were treated as indicated for 4 h
with PIo (solid bars). Transcriptional activity is expressed as the fold
increase in luciferase activity above baseline levels from transfected,
nonstimulated cells (ns). Cultures were co-transfected with GFP and
Renilla plasmids to normalize for transfection efficiency. Representative
experiments of a minimum of three are shown; values are the means 6
SD of triplicate luciferase determinations for each condition. (D) Astro-
cytes transfected with the 2431 Cox-2 luciferase reporter construct
were pretreated with vehicle or CsA and treated (4 h) with PIo or
PMA. Data are presented as in (C). ***P < 0.001; **P < 0.01 (ANOVA)
by cultured cortical astrocytes is blocked by inhibition of calcineurin.
(A, B), Rcan 1-4 mRNA was amplified from total RNA by TaqMan RT-
PCR. (A) Rat cortical astrocytes were exposed as indicated (4 h) to PIo,
PMA, or Io. (B) Astrocytes were pretreated with vehicle or with either
ActD or CsA and exposed (4 h) to PIo. Rcan 1-4 mRNA was quantified
in arbitrary units normalized to the expression of 18 S rRNA. Quantifi-
Calcium-inducible expression of Rcan 1-4 mRNA and protein
cations are relative to values obtained with nonstimulated controls (ns).
Representative experiments of a minimum of three are shown; values
are means 6 SD of triplicate RT-PCR determinations for each condi-
tion. ***P < 0.001; **P < 0.01 (ANOVA) versus ns. (C, D) Immunoblots
showing endogenous Rcan 1-4 protein expression, with b-tubulin
expression as loading control. Cells were pretreated as indicated with
ActD or CsA and then treated with PIo (4 h).
CANELLADA ET AL.
tion from the 21,664/183 construct to below the levels
in nonstimulated cells, consistent with involvement of
CN in the promoter activation, and suggesting CN-
dependent promoter activity in unstimulated cells (Fig.
6B). To confirm a positive role of NFAT, we tested the
ability of exogenous NFAT protein to induce transcrip-
tion from the Rcan 1-4 promoter. Astrocytes were co-
transfected with a version of human NFATc2 in which
the NLS is mutated, resulting in constitutive nuclear
localization (Canellada et al., 2006), together with the
2350/183 Rcan 1-4 promoter construct; this promoter
region contains putative NFAT regulatory motifs (Cano
et al., 2005; Minami et al., 2004). Nuclear expression of
NFATc2 strongly induced luciferase activity (Fig. 6C).
Active NFAT Directly Up-Regulates Cox-2 and
Rcan 1-4 mRNA and Protein Expression in
Primary Cultured Cortical Astrocytes
To obtain direct evidence that NFAT mediates the cal-
cium/CN-dependent component of PIo-induced Cox-2
and Rcan 1-4 expression, we infected astrocytes with an
adenovirus encoding constitutively active (ca) NFATc3
(Adeno ca NFAT) (Minami et al., 2004), or with an
empty adenovirus vector (Adeno Empty) as a mock con-
trol. Cells were stimulated with PMA alone, since the
expression of NFAT should substitute the calcium-
dependent response elicited by Io. Cox-2 protein expression
was specifically induced by PMA in astrocytes infected
with Adeno ca NFAT, and reached a level comparable
with that induced by PIo in noninfected cells (Fig. 7A,
upper panel). In contrast, nonstimulated Adeno ca NFAT-
infected cells showed no Cox-2 protein induction (Fig. 7A,
upper panel Lane 1), thus indicating that although active
NFAT is required for the full induction of Cox-2 protein
expression in astrocytes it is not sufficient. Very similar
results were obtained by quantitative RT-PCR analysis
(Fig. 7B, upper panel). These findings are consistent with
the results in Figure 3, which show that stimulation of
astrocytes with calcium mobilizer alone is insufficient to
fully activate Cox-2 gene transcription, and that a combi-
nation of stimuli is needed to fully activate the promoter
(Fig. 3 and Discussion section).
Astrocytes infected with Adeno ca NFAT also expressed
Rcan 1-4 protein, but in this case PMA stimulation was
unnecessary (Fig. 7A, lower panel, Lanes 5 and 6). The
observed increase in Rcan 1-4 protein expression, there-
fore, appears to be because of the exogenous ca NFAT
protein, consistent with the sufficiency of Io stimulation
to induce Rcan 1-4 mRNA synthesis (Fig. 5C). Moreover,
although every care was taken to ensure the same levels
of exogenous protein expression in the different condi-
tions, we found that PMA stimulation increased the
expression of exogenous NFAT in Adeno ca NFAT-infected
astrocytes (Fig. 7A, lower panel, Lane 5 versus Lane 6).
The parallel higher accumulation of Rcan 1-4 protein in
increase in exogenous active NFAT protein, and not to a
direct action of PMA on Rcan 1-4 expression. The same
holds for Rcan 1-4 mRNA accumulation (Fig. 7B, lower
panel). Endogenous expression of NFATc3 was detectable
by longer exposure of these blots (not shown).
Up-Regulation of CN-Dependent Gene Expression
in Primary Astrocytes by Physiological
In the experiments presented so far using PIo, and in
others with thapsigargin (not shown), we used pharma-
cological stimuli to achieve a consistent elevation of
[Ca21]ithat would allow us to dissect the Ca/CN/NFAT
pathway in astrocytes. To confirm that this pathway can
be activated by physiological stimuli, we treated astro-
cytes with four of the many physiological stimuli known
to induce increases of [Ca21]i in this cell type: the L-
type-voltage gated calcium channel (L-VGCC) agonist
BAY K 8644 (Bay K); the purinergic receptor agonist
[reviewed in (Fiacco and McCarthy, 2006; Verkhratsky
et al., 1998)]; and thrombin (D’Ascenzo et al., 2004; Ubl
and Reiser, 1997). Cultured primary astrocytes treated
for 4 h with Bay K showed a clear accumulation of
Rcan1-4 protein (Fig. 8A, upper panel). This up-regula-
tion was inhibited by CsA (Fig. 8A, upper panel, Lane
6), similar to the pattern seen in Io-treated cells (Lanes
3 and 4). To test the specificity of the Bay K effect, we
pretreated astrocytes with the specific L-VGCC inhibi-
tors nifedipine and verapamil. These compounds inhib-
neurin-dependent manner and by constitutively nuclear NFAT. (A)
Schematic of a luciferase reporter plasmid prepared from the proximal
1,664 bp region of the human Rcan 1 internal promoter situated
between exons 3 and 4, showing positions of putative transcription fac-
tor response elements. (B) Twenty-four hours after transfection, astro-
cytes were pretreated with vehicle or CsA and exposed to PMA or PIo
(4 h). Transcriptional activity is expressed as the fold increase above
baseline levels from transfected, nonstimulated cells (ns). (C) Astro-
cytes were co-transfected with a luciferase reporter construct of the
proximal 350 bp region of the human Rcan 1 internal promoter and an
expression plasmid encoding constitutively nuclear NFATc2. Luciferase
activity was determined in cell lysates 36 h after transfection. Tran-
scriptional activity is expressed relative to baseline levels from cells
transfected with empty vector. Representative experiments of a mini-
mum of three are shown; values are means 6 SD of triplicate luciferase
determinations for each condition. . ***P < 0.001; **P < 0.01 (ANOVA)
The Rcan 1-4 internal promoter is activated by PIo in a calci-
CN/NFAT TARGET GENES IN CORTICAL ASTROCYTES
ited the accumulation of Rcan 1-4 protein induced by Bay
K (Fig. 8A, lower panel, Lanes 4–9), but, as expected,
had no effect on Rcan 1-4 accumulation in Io-stimulated
cells (Lanes 10–12). Cox-2 expression in Bay K-stimu-
lated astrocytes required co-stimulation with PMA (not
The multifunctional serine protease thrombin in-
creases the levels of intracellular calcium in astrocytes
(Ubl and Reiser, 1997) and also activates signaling via
PKC (Pindon et al., 1998). Thrombin-treated astrocytes
showed a clear up-regulation of Rcan 1-4 and Cox-2 pro-
teins that was inhibited by CsA; this was clearly observ-
able at a higher thrombin dose of 20 U/mL (Fig. 8B,
Lanes 4 and 7), and could be detected at lower thrombin
concentrations when the blots were exposed for longer
times. The partial inhibition of Cox-2 expression by CsA
was similar to the effect of CsA on PIo-stimulated cells,
seen in Fig. 4B.
Astrocytes are also known to express purinergic and
metabotropic glutamate receptors, and activation of
these receptors triggers Ca21
[(Fiacco and McCarthy, 2006; Verkhratsky et al., 1998)
and references therein]. Furthermore, astrocytes have
been shown to release ATP in response to injury, sug-
gesting that purinergic signals might play an important
entry in these cells
role in the immediate Ca21responses of astrocytes to
brain damage (Ahmed et al., 2000). Treatment with 100 lM
ATP or glutamate up-regulated protein expression of
Astrocyte changes are among the earliest and most
dramatic responses to brain injury (Petito and Babiak,
1982), and astrocyte malfunction is proposed as a key de-
terminant of neuronal death (Nedergaard and Dirnagl,
2005). Increases in intracellular calcium concentration
are a common feature of many neurodegenerative, ische-
mic, and ageing-related processes that likely involve
astrocytes. Here, we demonstrate the existence of an in-
ducible calcium/calcineurin/NFAT signaling pathway in
primary cultured astrocytes. These cells express the
essential components of the pathway, calcineurin CN A
and CN B subunits and NFAT. Moreover, we show that
increases in [Ca21]i in this cell type activate NFAT-
dependent mRNA and protein expression of targets such
as cyclooxygenase 2 and Rcan 1-4. The calcium/calcineurin/
NFAT signaling pathway is thus functional in cortical
astrocytes, and might account for the regulation of some
of the initial responses to calcium in this cell type.
Rcan 1-4 mRNA and protein expression in cultured rat astrocytes. Astro-
cytes were infected with either a constitutively active (ca) NFAT-encod-
ing adenovirus (Adeno ca NFAT) or empty adenovirus (Adeno Empty).
After 48 h, infected and noninfected (N.i) cells were quiesced, and then
stimulated (4 h) with PMA (P) or PIo as indicated; ns, nonstimulated
cells. (A) Immunoblots showing protein expression of Cox-2 (upper panel)
and NFATc3 and Rcan1-4 (lower panel). b-Tubulin was detected as a
Expression of constitutively active NFAT supports Cox-2 and
loading control; molecular weights (kDa) are indicated to the left. (B)
Quantitative RT PCR of mRNA expression of Cox-2 (upper panel) and
Rcan 1-4 (lower panel) quantified as in Figs. 3 and 5. PMA-stimulated
levels are presented as the fold expression above nonstimulated cells
(ns). Representative experiments of three are shown; values are means
6 SD of triplicate RT-PCR determinations for each condition. Identical
concentrations of adenoviral particles were used for each infection in all
experiments. ***P < 0.001; **P < 0.01 (ANOVA) versus ns.
CANELLADA ET AL.
Calcium signaling is critical for activation of astro-
cytes, but although the existence of the calcium/CN/
NFAT pathway in glial cells has been suggested before,
this was not demonstrated in the earlier studies. The
appearance of the reactive astrocyte phenotype has been
linked to the presence of CN (McMillian et al., 1994)
and to its constitutive activation (Norris et al., 2005).
NFATc3 has been described in U373 astrocytoma and C6
glioma cells, and a CsA-sensitive DNA-binding protein
has been detected in astrocyte primary cultures (Jones
et al., 2003; Mosieniak et al., 1998). Nevertheless, the
transcriptional outcome and possible roles of the activa-
tion of this pathway have not been addressed.
Our results show that near-pure primary astrocyte
cultures express mRNA and protein of all four calcium-
regulated members of the NFAT family (see Fig. 2).
Determining the relative amounts of the different mem-
bers is a difficult task, because of the different sensitiv-
ities of the antibodies for their corresponding proteins.
However, by comparing identical amounts of total RNA
from primary astrocytes and spleen cells, we were able
to show that Nfatc2 mRNA is relatively less abundant
in glial cells than in spleen, and that while Nfatc4 is
absent from spleen cells it is expressed in astrocytes.
The absence of Nfatc4 from lymphocytes has been
described before (Lyakh et al., 1997).
CN is reported to be expressed mainly in neuronal
cells, with no expression reported in astrocytes from
normal tissue (Dawson et al., 1994b; Goto et al., 1986).
There are two possible explanations for the discrep-
ancy between these reports and our results. It may be
that resting astrocytes do express CN, but at relatively
low levels compared with neurons. CN was first puri-
fied from brain, where it is highly expressed (Klee et
al., 1979), and in the brain the vast majority of CN is
expressed in neurons (Dawson et al., 1994b; Goto
et al., 1986); it is thus possible that the signal from
neuronal CN would obscure a weaker signal from
astrocytes by immunohistochemistry of brain sections.
The other possibility is that astrocytes in culture are
not fully resting, and that the cells initiate some
aspects of the activated phenotype without progressing
to hypertrophy. This interesting question will require
Whatever the significance of CN expression in resting
astrocytes, astrocyte activation is associated with up-
regulation of CN expression (McMillian et al., 1994).
Furthermore, expression of constitutively active CN in
astrocyte cultures has been shown to mimic the pheno-
type and much of the gene transcription output associ-
ated with astrocyte activation (Norris et al., 2005).
Immunosuppressant inhibitors of CN such as CsA and
FK506 protect astrocytes from apoptosis (Matsuda et al.,
1998), althoughvery high
increase apoptosis in activated astrocytes (Pyrzynska
et al., 2001). More recently, Fernandez et al. (2007) have
reported that targeted overexpression of constitutively
active CN A in astrocytes is able to protect against brain
inflammatory injury, as measured by negative regulation
of hallmarks of inflammation such as LPS-inducible
Cox-2 andiNOS enzymes;
authors could not detect NFAT-dependent gene tran-
scription when constitutively active CN A was trans-
fected into astrocytes. Our finding that Cox-2 expression
in astrocytes is induced by calcium/CN signals in the ab-
sence of proinflammatory challenge may have implica-
tions for the study of calcium-mediated neurological
processes associated with brain ischemia, trauma, and
neurodegenerative diseases, all of which are character-
ized by increases in [Ca21]iin glial cells.
expression in primary astrocytes. (A) Immunoblots showing endogenous
expression of Rcan 1-4 protein in astrocytes treated with the L-type volt-
age-gated calcium channel agonist Bay K 8644 (Bay K). Upper panel:
Cells were pretreated with vehicle (2) or 200 ng/mL CsA, and then
exposed to 1 lM ionophore (Io,) or 5 lM Bay K (4 h). Lower panel: Cells
were pretreated with vehicle, 1 lM nifedipine (Nf) or 5 lM verapamil
(Vp) stimulated (4 h) as indicated with Bay K, 20 ng/mL PMA plus
Physiological Ca21mobilizers up-regulate CN-dependent gene
BayK (PBayK), or Io. (B) Immunoblots showing endogenous expression
of Rcan 1-4 and Cox-2 protein in astrocytes treated with thrombin. Cells
were pretreated with vehicle or CsA, and then exposed (4 h) to 0.2–20 U/
mL thrombin or PIo. (C) Immunoblots showing Rcan 1-4 protein expres-
sion in astrocytes treated with the purinergic agonist ATP or glutamate.
Vehicle of CsA pretreated cells were stimulated (4 h) with 100 lM ATP
or glutamate for 4 h. b-Tubulin staining was used as loading control,
and results are from representative experiments of three.
CN/NFAT TARGET GENES IN CORTICAL ASTROCYTES
The CsA-sensitive transcriptional induction of Cox-2
by calcium signals in primary astrocytes is similar to
what happens in other cell types (Hernandez et al.,
2001; Iniguez et al., 2000). Three lines of evidence show
that the observed induction of Cox-2 mRNA and protein
is only partially dependent on NFAT transcription fac-
tors. First, CN inhibitors such as CsA (see Fig. 3) and
FK506 (data not shown) only partially inhibited the PIo-
dependent induction of Cox-2. Second, pharmacological
(Ionophore) and physiological stimuli that only augment
[Ca21]i are not sufficient to induce Cox-2 expression
(Fig. 3 and not shown). Third, the PMA-induced signals
are required for full induction (Figs. 3A,C and 7). These
last two observations are consistent with the known
tendency of NFATs to cooperate with other transcription
factors, and thereby integrate diverse signaling path-
ways (Crabtree and Olson, 2002; Hogan et al., 2003)
These results are consistent with a previous study
(Koyama et al., 1999), which showed that a combination
of protein kinase C (PKC) and calcium-dependent signal-
ing pathways is required for full activation of astrocyte
Cox-2 expression. The induction of CsA-sensitive Cox-2
expression by thrombin (see Fig. 8), which activates
PKC in addition to mobilizing calcium, suggests that PIo
mimics a physiological pathway of CN/NFAT activation
The expression of Cox-2 protein in neuroinflammation
and neurodegenerative disease is well established (Con-
silvio et al., 2004). In cultured cells and in brain, Cox-2
induction in astrocytes has been reported in response to
many cytokines and insults (See Introduction). In vivo,
Cox-2 co-localizes with GFAP in infarcted human brains
(Sairanen et al., 1998). The role of this Cox-2 induction
and of the prostaglandin metabolites it produces is
uncertain, since some downstream products of the Cox-2
enzyme seem to be proapoptotic, while others are neuro-
protective. The use of Cox-2 inhibitors has been pro-
posed for the treatment of neurodegenerative diseases
(O’Banion, 1999) and as a means of delaying neuronal
death after ischemic insults (Nakayama et al., 1998).
But since the effects of some Cox-2 metabolites may be
positive and others negative, depending on the cellular
context, it may be more appropriate to inhibit Cox-2
expression specifically in those cells in which its activity
will lead to deleterious effects. The challenge is there-
fore to understand the beneficial and damaging effects
of neuroinflammation and dissect the spatial and tempo-
ral regulation that takes place.
We also examined NFAT-dependent regulation of the
Rcan 1-4 gene. Our earlier work showed that transcrip-
tional regulation of this gene is highly dependent on
NFAT (Cano et al., 2005; Minami et al., 2004); here we
show that the presence of NFAT protein in astrocyte
nuclei, achieved either by stimulation with calcium-
inducing agents alone (see Fig. 5) or transient over-
expression of a constitutively nuclear NFAT (see Fig. 7),
is sufficient to increase the levels of Rcan 1-4 mRNA
and protein. Rcan 1-4 protein is an efficient inhibitor of
CN (Fuentes et al., 2000; Gorlach et al., 2000; Kings-
bury and Cunningham, 2000; Rothermel et al., 2000),
and therefore of NFAT-transcriptional activity (Cano et
al., 2005; Minami et al., 2004). The Rcan 1-4 gene, also
known as Adapt 78, DSCR1-4, and MCIP 1-4, is
expressed in many human tissues (Davies et al., 2007).
In the brain, Rcan 1 mRNA expression has been
detected mainly in neurons within the cerebral cortex,
hippocampus, substantia nigra, thalamus, and medulla
oblongata; and the same study also reported an increase
in the amount of Rcan 1 in the brains of patients with
Alzheimer’s disease (Ermak et al., 2001). This study did
not report expression of Rcan 1 mRNA in astrocytes or
microglia. Two reasons for this are that the probe used
recognized Rcan exons 1, 5, 6, and 7, and that only nor-
mal, healthy human and rat brains were examined. This
data has been corroborated by Porta et al. (2007a), who
analyzed the expression pattern in the mouse brain of
the three family members, Rcan 1, Rcan 2, and Rcan 3.
In resting adult brain, only Rcan 2 was observed in glial
cells, though only in oligodendrocytes. Our results indi-
cate that the calcium-regulated isoform of the Rcan pro-
tein (Rcan 1-4) is either not present or is weakly
expressed in quiescent or nonstimulated cells, and that
Rcan 1-4 mRNA expression is induced upon stimulation
with agents that increase [Ca21]i. Furthermore, Rcan
1-4 protein induction is achieved by four physiological
agents that mobilize calcium via distinct mechanisms.
Increases in [Ca21]i are moreover sufficient to induce
astrocyte expression of Rcan mRNA (Fig. 5A) and
Rcan1-4 protein (Fig. 8A, upper panel). Similar up-regu-
lation of this protein may occur in other brain cell types.
Indeed, preliminary results in primary cortical neuron
cultures show that this cell type also responds to
increases in [Ca21]iwith an up-regulation of Rcan 1-4
protein (data not shown).
The identification of Rcan 1-4 as an NFAT target gene
in these cells suggests an important role for calcium/cal-
astrocytes. Rcan 1-4 has been described as a dual regu-
lator of calcineurin activity, able to either inhibit or pro-
mote CN activity depending on the cellular context
(Fuentes et al., 2000; Gorlach et al., 2000; Kingsbury
and Cunningham, 2000; Lee et al., 2003; Sanna et al.,
2006; Vega et al., 2003). Two recent studies have exam-
ined the role of Rcan 1 in neurons. Porta et al. (2007b)
showed that neurons deficient in Rcan 1 are more resist-
ant to damage by oxidative stress, and suggest a possi-
ble neuroprotective role for CN-NFAT dependent signals.
The second study suggests a role for Rcan1 in normal
brain function, since Rcan1 deficiency impairs long-term
potentiation (LTP) and memory (Hoeffer et al., 2007). It
will be of great interest to examine the role of Rcan 1-4
in glial biology, and its influence on the actions of the
calcium/CN/NFAT pathway in the different neuropatho-
logical processes in which calcium is implicated.
The mechanisms involved in CN up-regulation in
astrocytes remain to be elucidated, and it remains
unclear what role the activation of CN, and therefore
NFAT-regulated gene transcription, plays in glial cells at
the different stages in their activation. Our findings,
showing functional CN expression in glial cells and CN/
CANELLADA ET AL.
NFAT-dependent gene activation in response to pharma-
cological and physiological calcium mobilizers, support
an important role for this pathway in the onset of an
activated astrocyte phenotype. Control of the transcrip-
tional output of glial cells via the calcium/CN/NFAT
pathway might become a prime target for neuroprotec-
tive therapies aimed at preventing or regressing pathol-
ogies associated with activated astrocytes, such as Alz-
heimer’s disease, stroke, and other age-related illnesses.
We thank Dr. Simon Bartlett for excellent editorial as-
sistance and help in the preparation of the manuscript.
We also thank members of the lab and Dr. Marta Nieto
for helpful discussions. We are grateful to Drs. Mar? ıa
Monsalve and Javier Diez-Guerra for technical assis-
tance in adenoviral handling and primary astrocyte cul-
turing, respectively. We also thank Dr. Pilar Gonzalo
and the CNIC Cytometry Unit for their assistance with
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