From the
Department of Pathology and
Graduate Program in Genetics and Molecular
Biology, Emory University School of Medicine, Atlanta, Georgia 30322
Received for publication, January 30, 2003 , and in revised form, May 7, 2003.
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ABSTRACT |
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INTRODUCTION |
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The motifs that regulate the subcellular localization of NFAT, a latent
cytoplasmic factor, are located within a conserved amino-terminal regulatory
region, the NFAT-homology region. Upon cellular activation, a sustained
Ca2+ flux activates the
Ca2+/calmodulin-dependent phosphatase, calcineurin (Cn).
Cn directly associates with NFAT molecules through recognition/docking sites,
including the SPRIEIT sequence (NFAT14) and CnBP-B (NFAT24)
(6,
7). Activated Cn
dephosphorylates a conserved set of serine-proline repeats (SP boxes), which
induces NFAT translocation to the nucleus
(8). Kinases, including
glycogen synthase kinase re-phosphorylate nuclear NFAT and promote its export
back to the cytoplasm, thus regulating its access to DNA
(9). NFAT molecules associate
with DNA as monomers via a highly conserved domain, the Rel-homology domain
(related to Rel-family transcription factors NFB and Dorsal)
(10). The Rel-homology domain
consists of
300 amino acids and binds the consensus sequence:
5'-(A/T)GGAAAA-3'. NFAT activity often depends on association with
AP-1 molecules. In these cases, AP-1 and NFAT bind co-operatively at composite
DNA elements and activate transcription
(11,
12). The requirement for
co-operative binding of NFAT and AP-1 family members has been one way to
account for the dependence on both PMA (through the activation of a kinase
cascade that includes MAP kinases and PKC family members) and ionomycin
signals (that activate Cn) for expression of some NFAT-regulated genes
(3).
Regions of the NFAT molecule that contribute to transcriptional transactivation have been identified within NFAT1, -2, and -4. NFAT1 transactivation activity is localized to amino acids 1415 (13), a region that includes the SPRIEIT sequence. Two transactivation domains (TADs) have been identified within the human NFAT2 molecule: TAD-A (amino acids 113205) and TAD-B (amino acids 690930 within the carboxyl terminus of NFAT2.C) (4). In NFAT4, a carboxyl-terminal, isoform-specific region (amino acids 10301044) also contributes to transcriptional activation (14).
Despite the conservation of DNA-binding and regulatory domains, the phenotypes of NFAT-null mice illustrate that some NFAT family members have specialized functions. For example, deletion of the NFAT1 gene results in dysregulated lymphoproliferation and a Th2-dominant cytokine response (15). In contrast, NFAT2 / lymphocytes are defective in proliferation and production of Th2 cytokines (16, 17). Such phenotypes suggest that NFAT2 activity drives Th2 cytokine production and proliferation, and is modulated by the repressive action of NFAT1. NFAT2 also has an essential role in cardiac valve formation (18, 19). These studies illustrate the conserved and essential role of NFAT gene products in development and expression of immunologic effector molecules, but also suggest functional specialization among family members. The mechanisms that underlie the ability of NFAT family members to regulate unique programs of gene expression remain undefined.
The existence of multiple isoforms of NFAT1, -2, and -4 suggests that there
is even more complexity in the regulation and function of NFAT family members.
Strategies used to target NFAT genes for deletion result in the loss of
expression of all isoforms derived from the targeted locus. Thus, the
contribution of individual NFAT isoforms to unique patterns of gene expression
has not yet been evaluated in vivo. We previously cloned two isoforms
of NFAT2 from a murine mast cell cDNA library that differ only at their amino
termini (5). NFAT2. and
NFAT2.
contain 42 and 28 unique amino acids, respectively. These two
murine isoforms do not contain the alternative carboxyl termini that are
described for human isoforms: NFAT2.A/B/C
(20). However, the amino
termini are conserved: hNFAT2.A/B corresponds to mNFAT2.
, whereas
hNFAT2.C has the mNFAT2.
amino terminus. Murine NFAT2.
is
expressed predominantly in the spleen, whereas NFAT2.
is also expressed
in the liver and kidney. In both T and mast cell lines, the expression of
NFAT2.
is inducible, whereas NFAT2.
is constitutively expressed
at low levels in T cells, and is up-regulated only in mast cells upon cell
activation.
Because the sequences that regulate DNA-binding and nuclear transport are
identical in murine NFAT2. and NFAT2.
, we hypothesized that the
isoform-specific regions confer distinct abilities to transactivate
transcription, perhaps allowing for cell- and signal-specific NFAT activities.
In this study we used a genetic one-hybrid assay to identify transactivation
domains (TADs) within NFAT2.
and NFAT2.
. Two regions act
independently to confer transcriptional activation in response to PMA and
ionomycin. Both conventional and novel PKCs regulate this activity. In
addition, a novel acidic activation domain (Glu5-Asp19)
within the
-specific amino terminus imparts NFAT2.
with greater
ability to transactivate in response to a broad range of PMA and ionomycin
concentrations. Significantly, it also provides NFAT2.
with the unique
ability to respond to Fc
RI cross-linking. These data demonstrate that
there is functional specialization among NFAT2 isoforms that is regulated at
the level of transactivation in response to specific activation signals.
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EXPERIMENTAL PROCEDURES |
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Transfections5.0 x 106 CFTL-15 cells were electroporated in 0.5 ml of serum-free RPMI at 400 V and 425 µF in 0.4-cm gap cuvettes with a Gene-Pulser II (Bio-Rad). All CFTL-15 shocks contained 50 µg of salmon sperm DNA (Invitrogen). Samples were incubated for 10 min with DNA at room temperature prior to electroporation, and were allowed to recover on ice for 10 min post-electroporation.
One-hybrid Constructs and CAT AssaysPCR-generated fragments
of murine NFAT2. and NFAT2.
were cloned in-frame into the Gal4
DBD expression construct, pM (Clontech). The isoform-specific regions are
represented by
144 and
130. Single amino acid
mutations were constructed with the QuikChangeTM mutagenesis kit
(Stratagene, La Jolla, CA). CFTL-15 assays were performed with 0.00110
µg of effector construct and 5 µg of G5CAT reporter construct
(Clontech). Within an experiment, equal molar quantities of effector
constructs were used. PKC
expression constructs were the kind gift of
A. Altmon (La Jolla Institute for Allergy and Immunology).
For overexpression analysis, NFAT2. and NFAT2.
were cloned
into the expression vector pcDNA3 (Invitrogen); 20 µg of the expression
constructs were transiently transfected into CFTL-15 cells with 20 µgof
either NFAT- or IL-4-CAT reporter constructs. Twenty-four hours
post-electroporation, CFTL-15 cells were split into two to three samples and
treated with PMA/ionomycin, or IgE/
-IgE. Forty-eight hours
post-electroporation, CAT extracts were harvested by the Tris/EDTA/NaCl
(TEN)/Triton X-100 method
(22). CAT activity was
measured in a scintillation:diffusion assay as previously described
(23). All CAT results are
representative of at least three independent experiments. The NFAT
reporter contains 3 tandem repeats of the IL-2 NFAT site upstream of the IL-2
promoter (72 to +25)
(24). The NFAT sites each
contain the composite NFAT:AP-1 "NFAT-responsive element"
(Fig. 4A). The NFAT
reporter was the kind gift of T. J. Murphy (Emory University). The IL-4
reporter construct contains sequences corresponding to 797 to +5 base
pairs of the murine IL-4 promoter and has been previously described
(23).
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Cell Extracts and ImmunodetectionExtracts from COS-1 and
CFTL-15 cells were prepared by collecting and washing approximately 1.0
x 106 cells in cold phosphate-buffered saline. Cells were
resuspended in 200 µl of 1.2x Laemmli sample buffer
(25) on ice. Extracts were
heated to 95 °C for 5 min, then either sonicated or passaged through a
22-gauge needle to disrupt chromatin. Extracts were then centrifuged at 10,000
x g for 10 min. After gel electrophoresis, proteins were
electroblotted to nitrocellulose for 20 min at 15 V in transfer buffer (48
mM Tris, 39 mM glycine, 20% methanol). Blots were
blocked in 5% dry milk/TBST (0.05% Tween 20) for 1 h at room temperature.
Primary antibodies specific for either Gal4 DBD (clone RK5C1, Santa Cruz
Biotechnology, Santa Cruz, CA) or PKC (clone E-7, Santa Cruz
Biotechnology) were added to the same solution for an additional hour. After 3
washes in TBST (5 min each), blots were treated with 1:10,000 dilutions of
secondary antibodies conjugated to horseradish peroxidase in 5% dry milk/TBST
(0.05% Tween 20) for 1 h at room temperature.
Reverse Transcriptase-PCR AnalysisRNA was harvested from
1.0 x 107 cells with RNA STAT-60 reagent (Tel-Test Inc.,
Friendswood, TX). 1.0 µg of DNase I-treated RNA was used as a template in a
Superscript IITM (Invitrogen) first-strand synthesis reaction. PCR
primers to detect PKC transcripts were: PKC
forward,
5'-CTCGTCAAAGAGTATGTCGAATCA-3' and PKC
reverse,
5'-AATTCATTCAGTCCTTTGTGTCACTCA-3'.
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RESULTS |
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As shown in Fig.
1B, the "full-length" amino-terminal
constructs, 1429 and
1415, as well as 31415
(which excludes the isoform-specific domains) were active in this assay.
130 and 91415 represent the smallest independent
constructs that can transactivate. All active constructs possess both basal
and stimulation responsive (20 ng/ml PMA and 1 µg/ml ionomycin) activity.
The common region encompassing amino acids 91415 includes the
previously defined human NFAT2 transactivation domain (TAD-A, amino acids
113205), whereas
130 represents a novel TA domain. Amino
acids 3090 and the carboxyl terminus (amino acids 550704),
regions shared by both NFAT2.
and NFAT2.
, as well as the
isoform-specific region of NFAT2.
(amino acids
144), have
no independent transactivation ability.
144 is inactive over a
broad range of effector concentrations, conditions under which
130 demonstrates a concentration-dependent ability to
transactivate (Fig.
1C).
Regions that have no independent ability to transactivate can contribute to
the activity of adjacent domains. For example, the basal and inducible
activity of 190 is greater than that of
130, yet
constructs containing amino acids 3090 alone are inactive. Deletion of
3090 from the 31415 construct (represented by 91415) has
only a minimal affect on activity, indicating that this modulating influence
is exerted primarily on the
130 transactivation domain.
These differences in transactivation are not the result of significant
variations in the inherent stability of the hybrid effector molecules or in
their ability to be expressed. Whole cell lysates were isolated from COS-1
cells transiently transfected with one-hybrid effector constructs and
subjected to Western blot analysis using Gal4-specific antibodies. A
representative experiment is shown in Fig.
1D. Chimeric Gal4 proteins of the expected molecular
weight are expressed in all transfectants. In addition, the expression levels
appear uniform with the exception of 130 and
190.
Whereas differences in NFAT2 isoform stability have not been reported, the
acidic
-specific amino terminus may confer a shorter protein half-life
on these constructs. This result also indicates that the levels of
transactivation for these two constructs
(Fig. 1B) may be
underestimated by this assay. Therefore, we are unable to determine whether
the overall ability of NFAT2.
to transactivate represents additive or
synergistic contributions from the two distinct transactivation domains
(91415 and the
-specific domain).
130 Contains a Novel Acidic Activation Domain:
Glu5-Asp19The
-specific amino terminus
is highly charged (pI = 2.9) and contains 7 acidic residues (Asp/Glu)
interspersed by a number of hydrophobic residues including phenylalanine, a
pattern conserved in many acidic activation domains (AAD)
(28). An alignment of the
amino termini of human NFAT2.C and murine NFAT2.
(Fig. 2B) reveals a
conserved unit of acidic/hydrophobic residues. We tested whether the acidic
"core" of this domain, Glu5-Asp19 is able to
transactivate transcription in mast cells. The results shown in
Fig. 2A demonstrate
that the activity of Glu5-Asp19 fully complements the
ability of
130 to drive transcription, and respond to
PMA/ionomycin. Thus, a novel 15-amino acid acidic activation domain exists
within the
-specific amino terminus of NFAT2.
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NFAT2./
Exhibit Quantitative Differences in Their
Basal and Inducible Transactivation in Response to PMA and
IonomycinAlthough it is well established that
Ca2+-dependent activation of Cn regulates NFAT nuclear
translocation, and PMA-induced signals regulate the activity of AP-1
(3), the influence of these
signals on transactivation has not been well characterized. We examined the
ability of either PMA or ionomycin treatment alone to induce transactivation
of NFAT2.
and NFAT2.
. As shown in
Fig. 3A, PMA treatment
alone results in a modest increase in transactivation, suggesting that
PMA-induced signals do contribute to transactivation. However, signals induced
by ionomycin or PMA/ionomycin have the most significant affects on inducible
activity. The addition of cyclosporin A inhibits PMA/ionomycin-induced
transactivation (Fig.
3A). Because one-hybrid effector proteins are targeted to
the nucleus via the Gal4 nuclear localization signal, this finding suggests
that calcineurin participates in the regulation of both NFAT2 subcellular
localization and transactivation.
At the fixed doses of PMA and ionomycin used in these assays,
1415 consistently transactivates better than
1429
(Figs. 1B and
3A). It is possible
that the isoform-specific regions confer differential ability to activate
transcription in response to varying activation signal strength. For example,
NFAT2.
may respond equivalently to NFAT2.
only at high signal
strengths. To test this possibility, transfected cells were treated with
increasing concentrations of PMA and ionomycin. As shown in
Fig. 3B, the absolute
level of transactivation differs between the two isoforms over the entire
activator concentration range. However, both isoforms demonstrate inducible
transactivation at the same PMA/ionomycin concentrations, indicating that
there is no qualitative transactivation difference in response to
signal strength.
Endogenous Mast Cell NFAT Activity Is Influenced by Both Novel and
Conventional PKCsSeveral Ser/Thr kinases have been demonstrated to
regulate NFAT transactivation, including: CaMK IV, Cot-1, PIM-1, and PKC
(2932).
The role of PKC family members in the transduction of immunoreceptor signals
is clearly established (for review see Ref.
33). In T cells, T cell
receptor-proximal kinases activate PLC
, leading to the generation of
diacylglycerol and inositol triphosphate. Both of these signaling
intermediates are necessary to activate conventional PKCs (including
,
I, and
II isoforms). Only diacylglycerol-responsive signals
(mimicked by PMA treatment) are required for the activation of novel PKCs
(
,
, µ, and
). To further characterize the molecular
mechanisms that regulate NFAT activity, we used pharmacologic inhibitors
Gö6976 (specific for conventional PKCs)
(34) and rottlerin (specific
for novel PKCs
and
)
(35). We first examined the
effect of these inhibitors on endogenous NFAT activity using an NFAT reporter
construct (Fig. 4A).
Both Gö6976 and rottlerin inhibit NFAT reporter activity induced in
response to PMA/ionomycin treatment and IgE receptor cross-linking, indicating
that both conventional and novel PKCs regulate physiologic mast cell NFAT
activity (Fig.
4B).
The novel PKC, PKC, has been implicated in T cell receptor signaling
pathways. In activated T cells, PKC
is rapidly recruited to the T cell
receptor (36). PKC
also
associates with the actin cytoskeleton
(37). Both PKC
mRNA and
protein are detectable in CFTL-15 mast cells
(Fig. 4C). In
addition, overexpression of dominant active PKC
(A148E) increases basal
and ionomycin-induced NFAT reporter activity
(Fig. 4D).
PKC Induces NFAT2 TransactivationBecause
endogenous NFAT activity depends on Fc
RI- or ionomycin-induced
Ca2+ flux to drive nuclear localization, our PKC
experiments using NFAT reporter constructs do not distinguish between effects
on translocation versus transcriptional activation. Furthermore, the
NFAT reporter contains composite NFAT:AP-1 elements, and does not discriminate
between NFAT- and AP-1-dependent activity. Thus, one-hybrid constructs were
used in transfection experiments to examine affects of PKC inhibitors on NFAT2
transactivation in isolation. As shown in
Fig. 5, PMA/ionomycin-induced
transactivation of both
1429 and
1415 is inhibited
by treatment with either Gö6976 or rottlerin. These results demonstrate
that both novel and conventional PKCs participate in NFAT2
transactivation.
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We next asked whether PKC regulates NFAT2 transactivation. As shown
in Fig. 6A,
transactivation of both NFAT2.
and NFAT2.
is induced by
co-expression of PKC
(A148E). This response is mediated by both the
-specific transactivation domain, as well as the amino-terminal common
region (31415) (Fig.
6B). Transactivation of p53 and VP-16 one-hybrid
constructs is not induced by PKC
A/E overexpression (nor by
PMA/ionomycin treatment) (Fig.
6C), indicating that the PKC
effect is specific
for NFAT2.
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To determine whether NFAT2. and NFAT2.
are equally sensitive
to PKC
signals, we performed a dose-response experiment. Like
PMA/ionomycin treatment, overexpression of dominant active PKC
equally
affects both NFAT2 isoforms (Fig.
6D). PKC enzymes phosphorylate the motif:
S/T-X-K/R. Several of these consensus motifs are present throughout
91415, but are absent in the
130 sequence. However, two
residues, Thr2 and Ser21 exist within
130
that could be non-consensus targets of phosphorylation. To evaluate this
possibility, alanine substitutions of Thr2 and Ser21
were introduced into
130 and
1415, and the
consequences on transactivation ability were assessed. Transactivation of the
non-phosphorylatable, doubly mutated effectors,
130(T2A/S21A) and
1415(T2A/S21A) (data not shown) is unaffected. The activity of
130(T2A/S21A) remains similar to the wild type construct over a
range of PKC
A/E input concentrations
(Fig. 6E). Thus
PKC
appears to influence
130 transactivation through an
indirect mechanism.
The -Specific Transactivation Domain Confers Unique
Responsiveness to IgE Receptor Cross-linkingMast cells are
activated by a variety of agonists including neuropeptides, proteases,
bacterial products, and specific antigen
(38,
39). Antigen cross-linkage of
the high affinity IgE receptor (Fc
RI) expressed on mast cells results in
the activation of signaling cascades mediated by Ca2+
and diacylglycerol and is perhaps the best characterized mode of mast cell
activation (40). The use of
PMA and ionomycin to activate mast cells can also provide these intracellular
signals and bypasses the need for cell surface receptor engagement. However,
these agents cannot faithfully mimic early receptor-proximal events that may
affect signal strength and outcome of the response. Thus, we considered the
possibility that physiological activators (that deliver quantitatively or
qualitatively different signals) would lead to differences in the ability of
NFAT2 isoforms to transactivate.
Cross-linking of FcRI by treatment of transfected cells with
IgE/
-IgE results in inducible transactivation of
130 and
1415 but not
144,
1429, or
31415 (Fig.
7A). Even under conditions where cells were treated with
a range of IgE/
-IgE concentrations,
1429 and 31415
are unable to respond (Fig.
7B). We repeated this experiment in a second
IgE-responsive, non-transformed mast cell line, MC9.
Fig. 7C demonstrates
that NFAT2.
is selectively responsive to IgE-mediated signals, and that
the
130 TAD is required for significant inducible
transactivation.
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This selectivity appears to be specific for IgE-mediated signals. For
example, LPS induces signals through toll-like receptor 4, a receptor
expressed on mast cells (data not shown and Ref.
41). Although LPS is known to
transduce signals through the activation of NFB
(42), we show in
Fig. 7D that both
1429 and
1415 transactivate in response to a range
of concentrations. However, as we observed with PMA/ionomycin treatment, there
were no qualitative differences in ability of either NFAT2 isoform to
transactivate in response to this physiologic stimuli.
Overexpression of NFAT2. Induces More NFAT- and IL-4
Reporter Activity Than NFAT2.
Demonstration of
functional differences among NFAT2 isoforms in vivo will require the
use of technologies that eliminate expression of specific isoforms. In the
absence of such conclusive experiments, we tested whether overexpression of
either NFAT2 isoform would result in differential activation of an exogenous
reporter construct in response to Fc
RI signaling. CFTL-15 mast cells
were co-transfected with an NFAT2.
or NFAT2.
expression construct
and an NFAT-dependent promoter/reporter construct. CAT reporter activity was
measured 24 h after activation by Fc
RI cross-linking.
Fig. 8A demonstrates
that overexpression of NFAT2.
results in greater NFAT reporter activity
in response to Fc
RI signal than either NFAT2.
or the vector
control, pcDNA3. A reporter construct containing 797 bp of the proximal IL-4
promoter is also more active in cells overexpressing NFAT2.
(Fig. 8B). The modest
increase in Fc
RI-induced reporter activity observed in NFAT2.
transfectants appears to conflict with our data demonstrating that
NFAT2.
is unable to mediate significant transactivation in response to
Fc
RI signals (Fig. 7,
AC). At least two possible mechanisms may
reconcile these findings: 1) NFAT2.
can still associate with AP-1
family members, and thus recruit the AP-1-dependent transactivation ability to
the promoter, and/or 2) overexpression of NFAT2.
may titrate out
molecules that regulate NFAT subcellular localization (calcineurin and
glycogen synthase kinase for example), resulting in an increased ability of
endogenous NFAT2.
to enter the nucleus and activate transcription.
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DISCUSSION |
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We demonstrate that the -specific domain is necessary for optimal
responsiveness to all these signals. Deletion of amino acids 130 from
1415 (represented by the 31415 construct) ablates
Fc
RI responsiveness (Fig. 7,
B and C) and significantly reduces PMA/ionomycin
inducible activity (Fig.
1B). A 15-amino acid sequence,
Glu5-Asp19, located within
130 is
responsible for this activity and has the hallmarks of an AAD. AADs are
comprised of acidic amino acids interspersed with hydrophobic residues (Phe in
particular) and are among the most potent transcriptional activators
(28). It has been shown that
AADs of p53 and VP16 make contacts with the RNA polymerase II holoenzyme
through the co-activator, hTAFII31
(43,
44). These protein-protein
contacts depend on a motif: F-X-X-
-
, that occurs
in two orientations within Glu5-Asp19 (forward, amino
acids 1216, and reverse, amino acids 1418). Studies to determine
whether the
-specific TAD also mediates its transactivation function
through this co-activator are underway.
Proteins containing acidic activation domains such as VP16 and p53 are also
characterized by a short half-life
(45,
46). Molinari et al.
(47) have used multimers of
acidic activation domains to show that protein half-life is inversely
correlated with transactivation activity; they and others speculate that this
is one mechanism used to regulate the activity of strong transactivators. The
observation that 130 has the lowest steady-state protein level
among our effector constructs may be a reflection of this phenomenon.
The varied abilities of NFAT2 isoforms to transactivate transcription in
response to cell type-specific signals likely contributes to unique patterns
of gene expression. How might this be accomplished? At the level of signal
transduction, distinct signals can elicit differential co-activator activation
and recruitment and lead to varying levels of transcriptional activation. We
speculate that IgE receptor cross-linking results in the activation of one or
more co-activators recruited by the -specific, but not the common
(91415) transactivation domain, leading to selective transactivation.
Signals downstream of the IgE receptor on mast cells may also facilitate
increased association of co-factors such as CBP/p300 (which can contribute
enzymatic activities (histone acetyltransferase activity in this case)
(48)) with NFAT2.
(via
the
130 and 91415 domains). These co-activators could act
to increase local acetylation and enhance transcriptional activation relative
to NFAT2.
by reducing the "chromosomal barrier" to
transcription.
In contrast to our findings, Monticelli and Rao
(49) recently reported that
overexpression of "constitutively active" NFAT1 and NFAT2
(including isoforms corresponding to and
) did not result in
differential IL-4 expression in response to PMA/ionomycin treatment in primary
T cells. These apparently contradictory observations may reflect either
signal- or cell-specific differences in the ability of NFAT2 isoforms to
induce transcription. Alternatively, the IL-4 gene may be representative of a
subset of NFAT-target genes that have low energetic barriers to transcription
(because of their chromosomal context), and thus may not be differentially
regulated by NFAT2 isoforms of distinct transactivation capabilities.
The presence of consensus PKC sites (S/T-X-K/R) throughout the
31415 "common region," and within 144 led us
to examine the role of PKC family members in NFAT2 transactivation. The novel
PKC, PKC
, has received particular attention for its role in
transduction of T cell receptor signals upstream of c-Jun
NH2-terminal kinase and NF
B
(50). Our demonstration that
PKC
message and protein are detectable in CFTL-15 mast cells
(Fig. 4C), taken
together with its well characterized role in immunoreceptor signaling,
suggests that PKC
may be implicated in Fc
RI-dependent NFAT
activation. These data are consistent with recent evidence showing that 1)
Nef-dependent NFAT activation in T cells requires PKC
(51), 2) PKC
(but not
or
) specifically synergizes with calcineurin to induce
endogenous NFAT activity in Jurkat T cells
(52), and finally 3) FasL
promoter activity in Jurkat cells requires an NFAT cis-regulatory element to
mediate responsiveness to dominant active PKC
and calcineurin
(35). Of note, studies of
PKC
/ thymocytes did not indicate a defect in
NFAT activity (53). Our
observation may represent a cell-specific phenomenon. Alternatively, PKC
family members may be redundant with respect to this function. The inhibitor
studies presented here demonstrate that both novel and conventional PKCs
regulate NFAT activity, thus PKC
is not likely to be the
exclusive mediator of Fc
RI signals to NFAT. Both NFAT2.
and NFAT2.
are responsive to PKC
overexpression, yet only the
isoform responds to IgE receptor cross-linking. Therefore it is likely
that additional Fc
RI-dependent signals are required to induce
transactivation of the
-specific domain under physiologic
conditions.
PKC responsiveness of the non-phosphorylatable construct
130(T2A/S21A) demonstrates that at least part of the PKC
effect on NFAT2 TA is indirect. PKC
may act in concert with
calcineurin to induce a co-activator required for NFAT2 transactivation. We
and others have shown that NFAT transactivation is cyclosporin A-sensitive,
and the requirement for active calcineurin in other models of
PKC
-responsive NFAT activity has been demonstrated
(54). Cyclosporin A treatment
also inhibits transactivation from the NFAT1 amino-terminal region in Jurkat T
cells (32).
We also made the unexpected observation that LPS treatment of mast cells
induced transactivation from both NFAT2. and NFAT2.
. Toll-like
receptor signaling is known to induce degradation of I
Bs and NF
B
activation (42), but has not
previously been shown to influence NFAT activity. Endogenous NFAT activity (as
measured by an NFAT reporter assay, data not shown) was not detectable,
indicating that LPS signals regulate NFAT activity after nuclear localization
(toll-like receptor 4 has not been reported to induce the sustained
Ca2+ flux necessary to drive NFAT nuclear localization).
These data suggest that a convergence of signals is required for NFAT function
in response to LPS. Our finding may represent a clinically important example
of signaling cross-talk. The inflammation associated with asthma, which
depends, in part, on IgE-dependent activation of mast cells, is regulated by
NFAT-dependent gene expression
(55) and can be altered by LPS
exposure (56). A likely
candidate to mediate cross-talk is the atypical PKC, PKC
. PKC
induces NFAT1 transactivation, can induce NFAT1/2-dependent NFAT reporter
activity, and physically associates with NFAT1/2
(32). PKC
also plays a
critical role in LPS-induced activation of c-Jun NH2-terminal
kinase, mitogen-activated protein kinase, and extracellular signal-regulated
kinase in macrophages
(57).
As our demonstration that LPS induces NFAT2 transactivation suggests, the range of signals that converge on NFAT is broad. The confluence of other signaling cascades is especially relevant in mast cells, which are able to respond to a variety of signals, and are in physiologic locations where combinatorial activation is likely. We propose that the expression of selected NFAT isoforms, of varying ability to transactivate transcription, allows a cell to "tune" its responsiveness to an array of cellular signals. Signal-specific transactivation is one mechanism through which functionally distinct NFAT isoforms can differentially regulate gene expression.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Northwestern University School of Medicine, Dept. of Microbiology and Immunology, Tarry Medical Research Bldg., Rm. 7-711, mail code S213, 320 East Superior St., Chicago, IL 60611-3010. Tel.: 312-503-0108; Fax: 312-503-1339; E-mail: m-brown12{at}northwestern.edu.
1 The abbreviations used are: NFAT, nuclear factor of activated T cells; PKC,
protein kinase C; TA, transactivation; DBD, DNA-binding domain; PMA, phorbol
12-myristate 13-acetate; Cn, calcineurin; IL, interleukin; LPS,
lipopolysaccharide; CAT, chloramphenicol acetyltransferase; TA, transactivate;
AAD, acidic activation domains.
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