 |
INTRODUCTION |
Large amounts of ATP and other nucleotides can be rapidly released
from different cellular sources such as nerve terminals, antigen-stimulated T cells, activated platelets, endothelial cells, and
other cell types under either physiological and pathological conditions
such as hypoxia, stress, and tissue damage. Particularly in the immune
and nervous system, extracellular ATP serves as a mediator of
cell-to-cell communication by triggering a variety of biological
responses including excitatory transmitter function, mitogenic
stimulation, or induction of cell death (reviewed in Refs. 1-3). These
effects are not the result of nonspecific membrane alterations but
rather are mediated through the activation of specific surface
molecules called P2 purinoreceptors (reviewed in Refs. 1, 4, and 5). At
least two mechanistically distinct subclasses of P2 purinoreceptors are
currently known. The metabotrophic P2Y receptors (formerly P2u, P2t,
and P2y), which bind either UTP or ATP, initiate their biological
effects through the G-protein-coupled activation of phospholipase C and
subsequent Ca2+ mobilization from intracellular stores. The
P2X receptors are a distinct subfamily of receptors that are related to
glutamate receptors and function as ligand-gated ion channels.
Engagement of P2X receptors by ATP causes an increase in
Ca2+ permeability that is entirely dependent upon
extracellular Ca2+ ions.
Recently, among the P2X subfamily, the molecular structure of the P2Z
receptor, also called P2X7, has been elucidated (6, 7). The
P2Z receptor contains two transmembrane domains and a large
extracellular loop, structural features that are characteristic of
members of the P2X family. Unlike other P2X receptors, the P2Z receptor
has an unusually long C-terminal domain that does not contain any known
signaling motifs. P2Z receptor expression appears to be rather
restricted to myeloic cells such as dendritic cells, mature
macrophages, mast cells, and microglial cells (reviewed in Refs. 8 and
9). The receptor is not expressed on monocytes but is induced by
-interferon and during monocyte differentiation (10, 11). A unique
response of the P2Z receptor is the formation of a large transmembrane
pore permeable to hydrophilic molecules of up to 900 Da in size, which
is formed by the ATP-induced aggregation of receptor subunits.
There is increasing evidence for a functional role of the P2Z receptor
in immune reactions (8, 9). Continuous activation of the receptor and
pore formation cause perturbations of ion homeostasis that may finally
lead to cell death. Interestingly, it has been found that, in contrast
to several other cell death inducers, only P2Z receptor ligation killed
mycobacteria within BCG-infected macrophages (12, 13). It has been also
postulated that P2Z receptor ligation is responsible for the formation
of multinucleated giant cells during inflammatory granulomatous
reactions (14). In addition, in lipopolysaccharide-primed macrophages and microglial cells, stimulation of the P2Z receptor induces the
immediate release of interleukin
(IL)1 1
, which is probably
mediated by the activation of IL-1
-converting enzyme (15-18).
Despite this accumulating evidence for an important function of the P2Z
receptor in inflammatory processes, signaling events underlying these
biological effects are almost entirely unknown.
We have recently shown that P2Z receptor ligation causes a rather
unusual delayed and sustained activation of the transcription factor
NF
B (19). Important transcriptional regulators, which weakly
resemble NF
B in their DNA binding domain, comprise proteins of the
nuclear factor of activated T cells (NFAT) family (reviewed in Refs.
20-22). NFAT proteins play an important role in inducible gene
transcription by controlling the expression of several cytokines, such
as IL-2, IL-4, granulocyte macrophage colony-stimulating factor, tumor
necrosis factor, CD40 ligand, and CD95 ligand (21, 22). To date, the
cDNAs of four different genes belonging to the NFAT family have
been described encoding NFAT-1 (NFATp) (23), NFAT-2 (NFATc) (24),
NFAT-3 (25), and NFAT-4 (NFATx) (25-27). NFAT proteins show different
tissue distribution and inducibility upon cell stimulation, raising the
possibility that their functions may be distinct. In addition, some
NFAT members are synthesized as multiple isoforms due to alternative
splicing, translation initiation, and polyadenylation events
(28-30).
The signaling mechanisms of NFAT have been most intensively studied in
T- and B-lymphocytes, but there is a paucity of information on their
role in other cell types. The NFAT complex is composed of a cytoplasmic
subunit whose subcellular localization and DNA binding activity is
controlled by Ca2+ mobilization. Nuclear translocation of
NFAT is regulated by the Ca2+-dependent
serine/threonine phosphatase calcineurin, which is the target for the
immunosuppressive drugs cyclosporin A and FK506 (31-34). After nuclear
translocation, NFAT may couple to activator protein 1 (AP-1) or other
transcription factors, resulting in the coordinate induction of
proinflammatory cytokine expression (35, 36).
In the present study, we investigated the expression and mechanism of
NFAT activation in microglial cells, which are considered to be
important immune effector cells of the brain (37). It is shown that
extracellular ATP instantly induced the nuclear translocation of both
NFAT-1 and NFAT-2 by a calcineurin-dependent mechanism. This
effect required the influx of extracellular Ca2+ and was
exclusively mediated by the P2Z and not by other P2 purinoreceptors. P2Z receptor-mediated NFAT activation may therefore represent a
heretofore unappreciated mechanism by which extracellular ATP can
modulate inflammatory processes within the nervous and other cellular systems.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Reagents--
The mouse microglial cell line N9
has been described previously (38) and was kindly provided by Dr. P. Ricciardi-Castagnoli (University of Milan, Milan, Italy). Cells were
grown in RPMI 1640 medium supplemented with 10% fetal calf serum and 2 mM glutamine and routinely passaged by trypsinization. ATP
and other nucleotides were purchased from Roche Molecular Biochemicals.
Periodate-oxidized ATP was a kind gift of Drs. S. Hanau and F. Di
Virgilio (University of Ferrara, Ferrara, Italy) and was synthesized as
described previously (39). FK506 and cyclosporin A (CsA) were obtained
from the clinical pharmacy (Medical Clinics, Tübingen, Germany).
The anti-mouse NFAT-1 antibody was purchased from Upstate Biotechnology
(Lake Placid, NY), and the anti-NFAT-2 antibody (clone 7A6) was
obtained from Alexis Biochemicals (Grünberg, Germany). For
detection of NFAT-3, different rabbit antisera raised against residues
886-902 or residues 614-632 of human NFAT-3 (Ref. 29; kindly provided by Dr. N. R. Rice (National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD)) and a serum directed against residues 387-406 of human NFAT-3 (Santa Cruz, Heidelberg, Germany) were used. For NFAT-4, an antiserum raised against residues 130-149 of human NFAT-4 (Ref. 29; a kind gift from Dr. N. R. Rice) and a serum directed against mouse NFAT-4 (kindly provided by
Drs. A. Avots and E. Serfling (University of Würzburg,
Würzburg, Germany)) were used. An anti-c-Fos antiserum was
obtained from Dianova (Hamburg, Germany).
Preparation of Nuclear Extracts--
N9 cells (2 × 106) were plated in 6-well plates and allowed to adhere
overnight. Cells were then treated with the indicated stimuli.
Pretreatment with CsA was performed for 15 min, and pretreatment with
FK506 was performed for 30 min. Nuclear extracts were prepared by
resuspending phosphate-buffered saline-washed cells in 150 µl of
Buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 2 µg/ml each of aprotinin and leupeptin). After a 20-min incubation on
ice, cells were lysed by passing them three times through a G20 needle.
The samples were then centrifuged, and the nuclear pellet was
resuspended in 70 µl of Buffer C (20 mM Hepes, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA,
1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 2 µg/ml each of aprotinin and leupeptin). After centrifugation at 4 °C for 20 min, nuclear extracts were used for
electrophoretic mobility shift assays (EMSAs) and Western blot analysis.
Electrophoretic Mobility Shift Assay--
Equal amounts of the
nuclear extracts (4 µg of protein) were incubated with the
32P-labeled NFAT-specific oligonucleotide. Binding
reactions were performed in a 24 µl volume containing 4 µl of
extract, 4 µl of 5× binding buffer (20 mM Hepes, pH 7.5, 50 mM KCl, 1 mM dithiothreitol, 2.5 mM MgCl2, and 20% Ficoll), 1 µg poly(dI-dC)
as nonspecific competitor DNA, 2 µg of bovine serum albumin, and
40,000 cpm (Cerenkov) of the labeled oligonucleotide. After a 20-min
binding reaction at 4 °C, samples were loaded on a 4% nondenaturing
polyacrylamide gel that was pre-run for 4 h at 4 °C in 0.5×
TBE. After electrophoresis, gels were dried under a gel dryer and
exposed to an x-ray film. The NFAT-binding oligonucleotide
corresponding to the distal NFAT motif from the murine IL-2 promoter
(5'-TCGACAAAGAGGAAAATTTGTTTCATACAGAAG-3') was end-labeled using
[
-32P]ATP (3,000 Ci/mmol; Amersham-Buchler) and T4
polynucleotide kinase (Roche Molecular Biochemicals), followed by P-10
gel filtration (Bio-Rad) to remove nonincorporated radioactivity. A
cold oligonucleotide mutated in the core NFAT recognition sequence
(5'-TCGACAAAGAGGAAAATTTGTTTATATCAGAAG-3') was used for competition
experiments (35). When supershift analysis was performed, nuclear
extracts were preincubated with the antibodies for 30 min on ice.
Western Blotting--
After the indicated treatments, nuclear
extracts were prepared from 2 × 106 cells and loaded
on an 8% SDS-PAGE under reducing conditions. Subsequently, separated
proteins were electroblotted to a polyvinylidene difluoride membrane
(Amersham-Buchler). The membranes were blocked for 1 h with 5%
nonfat dry milk powder in Tris-buffered saline and incubated with
different NFAT-specific antibodies. Membranes were washed four times
with Tris-buffered saline and 0.05% Tween-20 and incubated with the
respective peroxidase-conjugated affinity-purified secondary antibody
for 1 h. After extensive washing, the reaction was developed by
enhanced chemiluminescence staining using ECL reagents
(Amersham-Buchler).
 |
RESULTS |
NFAT Is Activated upon Stimulation with Extracellular
ATP--
Incubation of N9 mouse microglial cells with extracellular
ATP elicits a rapid increase in the intracellular Ca2+
concentration mediated by both the P2Z and P2Y receptors expressed in
these cells (17). Whereas stimulation of P2Y receptors causes a
transient Ca2+ increase mainly from intracellular stores,
activation of the P2Z receptor involves a long-lasting Ca2+
influx across the plasma membrane. Because an increase in intracellular Ca2+ concentrations is required to activate the
Ca2+-calmodulin-dependent phosphatase
calcineurin that binds and dephosphorylates NFAT, we investigated the
effect of extracellular ATP on NFAT activation in N9 cells. Until now,
NFAT expression and activation have not been studied in microglial
cells. In Fig. 1, we compared the effects
of different agents leading to the elevation of intracellular Ca2+ ions. N9 cells were stimulated with ATP, the
Ca2+ ionophore A23187, and thapsigargin, an inhibitor of
the endoplasmic Ca2+-ATPase. After stimulation, nuclear
extracts were prepared and analyzed for NFAT activation by EMSA.
Treating cells with ATP potently induced NFAT activation, which was
similar to the effect elicited by A23187 and thapsigargin.

View larger version (57K):
[in this window]
[in a new window]
|
Fig. 1.
Extracellular ATP induces NFAT activation in
microglial cells. N9 cells were either left untreated or incubated
for 15 min with 3 mM ATP, the Ca2+ ionophore
A23187 (40 µM), or thapsigargin (2 µM), an
inhibitor of the endoplasmic Ca2+-ATPase. Cells were then
lysed, and nuclear extracts were prepared, incubated with a
32P-labeled NFAT-specific oligonucleotide, and analyzed by
EMSA. The NFAT-specific DNA complex is indicated by an
arrowhead.
|
|
ATP-induced Activations of NF
B and NFAT Show Different
Kinetics--
We have previously demonstrated that ATP stimulation of
the P2Z receptor can activate transcription factor NF
B (19). In Fig.
2, microglial cells were stimulated with
3 mM ATP for different times, and then identical nuclear
extracts were analyzed for the activation of NF
B and NFAT. The
activation of NFAT was already detectable after 1 min of ATP
stimulation, reached a maximum after 15 min, and strongly decreased
after 60 min. In contrast, activation of NF
B was much delayed and
sustained. Strong activation of NF
B was not detectable before 3 h of stimulation, but it was still maintained after 6 h. These
results indicate that ATP stimulation of N9 cells activates both
transcription factors, but with a strikingly opposite time
dependence.

View larger version (57K):
[in this window]
[in a new window]
|
Fig. 2.
Time course of ATP-induced activation of NFAT
and NF B. N9 cells were stimulated for the indicated times with
3 mM ATP. Nuclear extracts were then prepared, incubated
with two different 32P-labeled oligonucleotides containing
a NFAT or NF B binding motif, and analyzed by EMSA. An
arrowhead indicates the position of the NFAT and NF B DNA
complexes. A faster-migrating nonspecific complex is indicated
( ).
|
|
NFAT Activation Is Selectively Mediated by the P2Z
Receptor--
Because different members of the purinoreceptor
subfamilies exhibit distinct affinities for ATP, we determined the dose
dependence of ATP-induced NFAT activation. As shown in Fig.
3, NFAT activation became visible after
cells were incubated with 1 mM ATP. Maximal NFAT activation
was obtained with 3 mM ATP, whereas a higher concentration of 5 mM was less effective. This dose dependence
corresponds to other effects of P2Z purinoreceptor signal transduction
in N9 cells (17) and mouse macrophages (18).

View larger version (88K):
[in this window]
[in a new window]
|
Fig. 3.
Dose dependence of NFAT activation in
response to ATP. N9 cells were incubated with different ATP
concentrations for 15 min. Nuclear extracts were then prepared and
analyzed for NFAT DNA binding by EMSA.
|
|
Because N9 cells express both P2Y and P2Z purinoreceptors, we further
tried to specify the receptor subtype involved by using different
nucleotides and pharmacological ATP analogues. In addition to ATP,
benzoylbenzoic ATP, which is a more potent agonist for the P2Z receptor
than ATP, was able to induce NFAT activation, even at a concentration
of 1 mM (Fig. 4A).
In contrast, other nucleotides such as ADP, GTP, or CTP, which are not
agonists of the P2Z receptor, did not induce NFAT activation.
Remarkably, UTP, which also ligates P2Y receptors, failed to activate
NFAT, suggesting that NFAT activation was specifically mediated by the P2Z receptor.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of different nucleotides and ATP
analogues on NFAT activation. A, N9 cells were stimulated
with 3 mM of the indicated nucleotides or 1 mM
benzoylbenzoic ATP (BzATP). Nuclear extracts were prepared
after 15 min and analyzed by EMSA. B, ATP and benzoylbenzoic
ATP induce the nuclear translocation of NFAT-1. Cells were treated as
described in A, and nuclear extracts were analyzed by an
anti-NFAT-1-specific antibody. The position of NFAT-1 is indicated by
an arrowhead.
|
|
Results similar to those of EMSAs were obtained by Western blot
analysis of nuclear extracts using an anti-NFAT-1 antibody. Fig.
4B demonstrates that ATP as well as benzoylbenzoic ATP
induced the translocation of a protein of approximately 120 kDa,
corresponding to the molecular size of NFAT-1, into the cell nucleus.
NFAT-1, in contrast, was not detectable in nuclear extracts of cells
treated with UTP and other nucleotides.
The selective involvement of the P2Z receptor in ATP-induced NFAT
activation was further supported by additional experiments. P2Z
receptor-mediated effects have been shown to be antagonized by oxidized
ATP that covalently binds and inhibits the receptor (39). Pretreatment
of cells with 300 µM oxidized ATP completely abolished
ATP-induced NFAT activation (Fig.
5A). In contrast, oxidized ATP
did not inhibit NFAT activation in response to the Ca2+
ionophore A23187, demonstrating that it did not interfere with the
intracellular signaling of NFAT dephosphorylation and translocation. We
also investigated the effect of NFAT activation in N9 derivative cells
that lack the P2Z receptor but still express P2Y purinoreceptors (17,
41). As shown in Fig. 5B, NFAT activation was almost not
inducible by ATP in clone N9R17, whereas a Ca2+ ionophore
strongly activated the transcription factor.

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 5.
Specificity of ATP-induced NFAT activation.
A, inhibition of ATP-induced NFAT activation by the P2X
inhibitor oxidized ATP. N9 cells were pretreated with 300 µM oxidized ATP for 1 h and then treated with 3 mM ATP or 40 µM A23187 for 15 min, and then
nuclear extracts were analyzed by EMSA. B, lack of NFAT
activation in response to ATP in the P2Z-deficient cell clone N9R17.
N9R17 cells were stimulated with 3 mM ATP or 40 µM A23187 for 15 min, and nuclear extracts were then
examined by EMSA.
|
|
Cyclosporin A and FK506 Inhibit P2Z Receptor-mediated NFAT
Activation which Requires a Sustained Increase of Extracellular
Calcium--
The immunosuppressive drugs CsA and FK506 prevent NFAT
activation by inhibiting the
Ca2+-calmodulin-dependent phosphatase calcineurin
that dephosphorylates NFAT and allows the nuclear translocation of
NFAT. As shown in Fig. 6A,
pretreatment of N9 cells with CsA or FK506 was able to prevent NFAT
binding to DNA in ATP-stimulated cells. Essentially the same results
were demonstrated by the absence of an anti-NFAT-1 immunoreactive
protein in the nuclear extracts of cells pretreated with
immunosuppressive drugs (Fig. 6B). The results therefore indicate that ATP-triggered NFAT translocation and DNA binding are
calcineurin-dependent processes.

View larger version (71K):
[in this window]
[in a new window]
|
Fig. 6.
Inhibition of calcineurin and
Ca2+ chelation prevent ATP-induced NFAT activation and
nuclear translocation. N9 cells were preincubated with 1 mM CsA or 100 nM FK506 for 15 and 30 min,
respectively, and then challenged for 15 min with 3 mM ATP.
Ca2+ chelation was performed by adding 2 mM
EGTA to the medium. Nuclear extracts were analyzed either by EMSA for
NFAT DNA binding activity (A) or by Western blot analysis
for nuclear translocation of NFAT-1 (B).
|
|
A common intracellular event triggered by antigen-stimulated receptors
such as the T- and B-cell receptors is an increase in the cytoplasmic
free Ca2+ concentration, leading to calcineurin and
subsequent NFAT activation. To investigate whether Ca2+ was
essential for ATP-induced NFAT activation, N9 cells were stimulated
with the nucleotide in the presence of the Ca2+ chelator
EGTA. Pretreatment of cells with 2 mM EGTA completely prevented NFAT DNA binding as well as the nuclear translocation of
NFAT-1, indicating that activation of the transcription factor required
extracellular Ca2+. These data concur with the above
experiments, demonstrating that P2Y receptors, which mediate a
transient Ca2+ increase from intracellular stores, were
unable to activate NFAT. Therefore, in microglial cells, ATP-induced
activation of NFAT required a sustained increase in the intracellular
Ca2+ concentration, which is exclusively triggered by the
P2Z receptor.
Microglial Cells Express and Activate NFAT-1 and NFAT-2--
Among
the NFAT family, four different genes encoding NFAT-1, NFAT-2, NFAT-3,
and NFAT-4 have been identified (reviewed in Ref. 22). Although all
members exhibit similar DNA binding specificity, they differ in their
tissue distribution and inducibility upon cell stimulation, suggesting
that each protein may serve specific functions. Whereas NFAT-1 is
constitutively expressed in lymphocytes and in several non-lymphoid
cells, NFAT-2 expression has been reported to be induced in activated T
and B cells (24, 42). To analyze specificity and investigate which NFAT
member contributes to the ATP-induced DNA complex, we performed
competition and supershift analyses. In these experiments, higher
resolution gels were used that could separate two ATP-induced DNA
complexes. Both DNA complexes were efficiently competed by an excess of
the NFAT-binding oligonucleotide, whereas an oligonucleotide mutated in
the NFAT recognition sequence did not markedly affect NFAT DNA binding
(Fig. 7A). As shown in Fig.
7B, an antibody against NFAT-1 reduced and supershifted the ATP-triggered DNA-protein complexes. Formation of the NFAT-specific complex, in particular, the lower DNA complex, was also inhibited by
anti-NFAT-2, whereas no immunoreactivity was observed using a panel of
antisera directed against different epitopes of either NFAT-3 or
NFAT-4. In addition, most of the induced DNA complexes were inhibited
and supershifted by a combination of anti-NFAT-1 and anti-NFAT-2.
Because transcription factor AP-1 can associate with NFAT, we also used
an anti-c-Fos antibody that supershifted the upper ATP-induced DNA
complex (Fig. 7B).

View larger version (59K):
[in this window]
[in a new window]
|
Fig. 7.
ATP induces the activation of NFAT-1 and
NFAT-2 in microglial cells. A, competition analysis. Nuclear
extracts of nonstimulated (lane 1) or ATP-stimulated N9
cells (lanes 2-8) were incubated with and without a 25-, 50-, or 100-fold excess of the unlabeled NFAT-binding oligonucleotide
(NFAT-wt, lanes 3-5) or an oligonucleotide with a mutation
in the NFAT recognition sequence (NFAT-mut, lanes 6-8).
B, supershift analysis. Nuclear extracts of ATP-stimulated
cells were either left untreated (lane 1) or incubated with
specific antibodies against NFAT-1 (lane 2), NFAT-2
(lane 3), NFAT-1 plus NFAT-2 (lane 4), NFAT-3
(lane 5), NFAT-4 (lane 6), or c-Fos (lane
7). The open arrow indicates the supershifted complex,
and the closed arrows indicate the position of the
NFAT-specific complexes. Whereas anti-NFAT-1, anti-NFAT-2, and
anti-c-Fos supershifted or competed with DNA complex formation, no
effect was observed using different antisera against NFAT-3 and NFAT-4
or control IgG (data not shown). C, ATP-induced nuclear
translocation of NFAT-1 and NFAT-2. Nuclear extracts of untreated or
ATP-stimulated N9 cells were separated by SDS-polyacrylamide gel
electrophoresis and analyzed by immunoblot analysis using antibodies
directed against NFAT-1 (left panel) or NFAT-2 (right
panel).
|
|
The activation of NFAT-1 and NFAT-2 by ATP was confirmed by Western
blot analysis of nuclear extracts from ATP-stimulated cells. After ATP
stimulation, anti-NFAT-1 detected a nuclear protein of approximately
120 kDa that was absent in nuclear extracts from control cells (Fig.
7C). Anti-NFAT-2 recognized a prominent protein doublet band
of approximately 90 kDa, similar in size to the short NFAT-2 isoform
described previously (24, 29, 30). In contrast, we could not detect the
expression and nuclear translocation of NFAT-3 or NFAT-4 in immunoblot
analyses using different antisera (data not shown). Together, these
results suggest that microglial cells express and activate NFAT by the
P2Z purinoreceptor that consists mainly of NFAT-1 and NFAT-2.
 |
DISCUSSION |
In the present study, we demonstrate that extracellular ATP is a
potent activator of the proinflammatory transcription factor NFAT in
microglial cells. Using different pharmacological approaches and a
receptor-deficient cell clone, evidence is provided that ATP-induced
NFAT activation is selectively triggered by the P2Z receptor and not by
other purinoreceptor subtypes. Because the P2Z receptor, but not P2Y
receptors, induces a long-lasting transmembrane Ca2+
influx, the data concur with our observation that ATP-induced NFAT
activation is dependent on the cytoplasmic increase of extracellular Ca2+. The results therefore demonstrate a novel pathway by
which extracellular ATP can modulate the activation of NFAT and
subsequent proinflammatory processes.
The P2Z receptor is attracting increasing interest due to its role in
inflammatory reactions and the induction of cell death. Several reports
have recently demonstrated that the P2Z receptor is a potent mediator
of IL-1
release, most likely by the activation of IL-1
-converting
enzyme that cleaves the IL-1
precursor to the mature cytokine
(15-18, 43). We have recently described that the P2Z receptor may also
be involved in gene-regulatory events by activating transcription
factor NF
B (19). In addition, it has been found that extracellular
ATP is involved in mitogenic stimulation of T-lymphocytes (44).
Although the purinoreceptor subtype involved in this effect has not
been clearly defined, its pharmacological profile resembles that of the
P2Z receptor. It is therefore tempting to speculate that ATP-triggered
T-cell mitogenicity may involve NFAT activation and subsequent IL-2 expression.
Both the NFAT and NF
B transcription factors are
Ca2+-sensitive. Interestingly, activation of the two
transcription factors occurred with strikingly different kinetics. NFAT
DNA binding was visible within 1 min after ATP stimulation, whereas
significant NF
B activation was not detectable before 3 h of
stimulation, when NFAT activation had already declined. It can be
presumed that the required amplitude and duration of Ca2+
signals differ for the activation of NF
B and NFAT and may therefore contribute to this temporal transcriptional specificity of ATP stimulation. In this respect, it has been demonstrated that in B cells,
a low sustained Ca2+ increase is sufficient for NFAT
activation upon B-cell receptor ligation, whereas high levels of
Ca2+ are required to activate NF
B (45, 46). Another
reason for the different kinetic of activation of the two transcription
factors could be that NF
B may not be activated by Ca2+
alone but may require additional second messenger systems. Likely candidates in this respect are reactive oxygen intermediates, which are
generated upon Ca2+ overload and other stress conditions
(47, 48). Indeed, ATP-induced NF
B activation could be abolished by
antioxidants in microglial cells (19).
Currently, there is very little information about the expression and
role of NFAT within the brain. Our study shows for the first time that
microglial cells, which are regarded as the principal immune effector
cells of the brain (37), may express both NFAT-1 and NFAT-2. Initially,
it was reported that NFAT DNA binding activity could not be detected in
crude brain extracts (49). More recently, however, NFAT has been found
in PC12 pheochromocytoma cells as well as in neurons within the
olfactory bulb (50). It will be interesting to investigate whether
extracellular ATP, which exerts excitatory transmitter function,
activates NFAT in these cells.
In lymphocytes, the expression of NFAT proteins is differentially
regulated. Resting cells express only NFAT-1, whereas expression of
NFAT-2 is induced to significant levels upon stimulation by Ca2+-dependent signaling (24, 42). In addition,
in normal lymphocytes, NFAT-3 is not expressed, and NFAT-4 is only
detectable at low levels (29). In microglial cells, we observed that
NFAT-1 and NFAT-2 were activated and translocated to the nucleus with a
similar rapid kinetic, suggesting that both proteins are constitutively expressed in these cells. Because we demonstrated NFAT activation in
the N9 cell line, future experiments will have to investigate the
regulation of NFAT proteins in primary microglial cells. We could not
detect expression and activation of NFAT-3 or NFAT-4, whereas, similar
to previous reports (32, 35, 36), part of the ATP-induced NFAT complex
was obviously associated with AP-1. The antiserum against murine NFAT-4
used in this study recognizes NFATx, the longest isoform of NFAT-4, and
detects all isoforms of the protein. In addition, we used several
antisera raised against different epitopes of human NFAT-3. Although we
cannot completely exclude that the failure to detect NFAT-3 may be due
to a lack of cross-reactivity with the murine protein, we consider it
rather unlikely that NFAT-3 was activated in N9 cells. A combination of
anti-NFAT-1 and anti-NFAT-2 strongly prevented DNA binding, indicating
that the ATP-induced DNA complex predominantly consisted of NFAT-1 and
NFAT-2.
It can be presumed that microglial NFAT is involved in the inducible
expression of proinflammatory cytokines. In this respect, it is known
that activated microglia in inflammatory processes, Alzheimer's
disease, and other forms of neurodegeneration are capable of producing
high amounts of cytokines that have been implicated in disease
progression (reviewed in Ref. 37). Recent data further suggested that
certain NFAT proteins play an important role exceeding their
established immunoregulatory function in cytokine secretion. NFAT-2 and
NFAT-3 have been implicated in heart development and cardiac
hypertrophy (51, 52), indicating a morphogenetic and developmental role
of NFAT in some tissues. Future studies are required to investigate the
functional role of NFAT expressed in microglial cells or other cells
within the brain.
In this study, we used microglial cells that have been previously
characterized in detail for purinoreceptor expression (18, 40). These
cells are a good model, because they may be stimulated to produce
cytokines by ATP from adjacent neurons. ATP has been convincingly shown
to be physiologically released from neurons, where it is co-accumulated
with acetylcholine and noradrenalin in adrenergic and cholinergic nerve
terminals (53, 54). Collectively, the specific activation of NFAT
through P2Z receptor ligation may represent a novel mechanism of how
extracellular ATP may trigger inflammatory processes and exert
neuroimmunomodulatory functions.