From the
Nuclear factor of activated T-cells (NFAT) is a transcriptional
activator that binds to the interleukin-2 promoter and is believed to
be responsible for T-cell-specific interleukin-2 gene expression. Here
we demonstrate using electrophoretic mobility shift assays that nuclear
NFAT can be induced in the rat basophilic leukemia (RBL-2H3) mast cell
line and rat bone marrow-derived mast cells upon cross-linkage of the
high affinity receptor (Fc
Nuclear factor of activated T-cells (NFAT)
Although the
exact molecular make-up of the NFAT complex is unknown, there is
evidence that it is composed of a lymphocyte-specific cytoplasmic
component (or components) present in unactivated cells, designated
NFATp/c, and a ubiquitous, inducible nuclear component containing Fos
and Jun proteins (Jain et al., 1992; Northrop et al.,
1993). Cross-linkage of the T-cell receptor results in two
intracellular signals required for the assembly of active NFAT in the
nucleus: activation of protein kinase C and mobilization of cytosolic
calcium. The activation of PKC is necessary for the transcription of
Fos and Jun genes (Jain et al., 1992; Northrop et
al., 1993), and an increase in intracellular calcium is required
for the translocation of NFATp into the nucleus (Flanagan et
al., 1991). The translocation of NFATp from the cytoplasm to the
nucleus is thought to be controlled either directly or indirectly by
the calcium-dependent protein phosphatase calcineurin (Clipstone and
Crabtree, 1992). The role of calcineurin in this signaling pathway has
been further substantiated by the fact that a constitutively active
form of calcineurin can bypass the calcium requirement for IL-2
transcriptional activation (O'Keefe et al., 1992). The
immunosuppressive drugs cyclosporin A (CsA) and FK506 block
transcription of the IL-2 gene by inhibiting the phosphatase activity
of calcineurin and preventing the intracellular translocation of NFATp
(McCaffrey et al., 1993a; Clipstone and Crabtree 1992; Jain et al., 1993; Mattila et al., 1990). Once assembled
in the nucleus, the NFAT complex initiates transcription by binding to
two sequence-specific binding sites located in the enhancer region of
the IL-2 promoter (Shaw et al., 1988).
The signal
transduction pathway emanating from the Fc
Here we show that antigenic stimulation through the Fc
For activation experiments,
4-day-old confluent monolayer cultures were used. When IgE-dependent
activation was required, the cells were sensitized 12 h before the
experiment by replacing the culture medium with medium containing
approximately 3 µg/ml anti-2,4,6-trinitrophenyl (TNP) IgE produced
from the IGEL a2 hybridoma (ATCC TIB 142) (Rudolph et al.,
1981). On the day of the experiment, cells were removed from the
culture flasks by rinsing the monolayers twice with phosphate-buffered
saline containing 0.66 mM EDTA and then incubating the cells
in the same solution for 4 min at 37 °C. The cells were dislodged
from the flasks, transferred to a centrifuge tube, and diluted 2:1 with
Eagle's minimum essential medium supplemented with 10 mM
HEPES, 0.5 mg/ml BSA, 100 units/ml penicillin G, and 100 µg/ml
streptomycin. The cells were pelleted, resuspended in the same medium,
and incubated for 90 min before stimulation. This incubation permitted
decay of the initial NFAT activity induced by the cell isolation
protocol itself (see ``Discussion'').
Cells were activated
by adding concentrated stock solutions of the triggering agents
directly to the cell suspensions (1
Using specific DNA binding as a criterion, activation of RBL
cells by cross-linkage of the Fc
As
in previous studies with lymphocytes (see, e.g., Shaw et
al.(1988)), modest NFAT activity sometimes was detected in nuclear
extracts from unstimulated RBL cells. This activity appeared within
15-30 min after cells were removed from the culture flasks and
was dependent upon the presence of extracellular calcium. Nuclear
extracts prepared from cells eluted with EDTA and not restored to
medium that contained mM Ca
Two findings
indicate directly that the induction of NFAT activity by cross-linkage
of the Fc
What role
does calcium play in the induction of mast cell NFAT activity? The
ability of CsA, a potent calcineurin inhibitor, to block NFAT induction
suggests that as with T-cells, calcium-dependent activation of this
serine/threonine phosphatase is a key step in activation of NFAT via
the Fc
AP-1
and NFAT activity appeared in the nucleus with a similar time course
following antigenic stimulation of RBL cells (Fig. 4, A and B). An NFAT nuclear complex in T-cells is thought to
contain AP-1, based in part upon the finding that AP-1 oligonucleotides
reverse binding of this putative complex to the NFAT recognition
sequence, whereas the latter has no effect on binding of AP-1 to the
AP-1 recognition motif (Jain et al., 1993a; Northrop, et
al., 1993). The identical observations with RBL cells (Fig. 3A) suggest that the NFAT complex therein also
contains AP-1, although AP-1 in isolation cannot bind tightly to the
NFAT recognition motif (data not shown) (Northrop, et al.,
1993; Jain et al., 1992). Here, we show that antibodies to the
AP-1 proteins c-Fos and c-Jun, in combination, completely block NFAT
DNA binding, thus providing independent evidence that, as in T-cells,
these proteins contribute to the mast cell NFAT complex (Fig. 3B).
Of a variety of tissues tested so far
(Verweij et al., 1990), transcription mediated by the distal
NFAT site in the IL-2 promoter appears restricted to T- and
B-lymphocytes, and to a subpopulation of cells in the dermis, where
skin mast cells are concentrated. DNA binding activity specific for
this site in B-cells also was confirmed in EMSA studies of Verweij et al.(1990) and Yaseen et al.(1993), although in the
latter study transcriptional activity was not demonstrated. Given that
unstimulated lymphocytes generally do not exhibit substantial NFAT
activity, and that other tissues tested were not stimulated as were the
T-cells used for comparison, the conclusion on tissue specificity
remains tentative. Future examination of other cell types may reveal,
as found here, that stimulation via relevant receptors or with PMA and
ionomycin, induces NFAT activity.
Two recent studies indicate that
assembly of the NFAT complex(es) is likely to involve more components
and more intricate mechanisms than originally envisaged. McCaffrey et al. (1993b) cloned a cDNA corresponding to the preexisting
cytoplasmic component NFATp from murine T-cells. A truncated
recombinant protein expressed from this cDNA was shown to bind directly
the murine distal NFAT recognition motif from the IL-2 promoter, to
combine with Fos and Jun proteins to form another DNA-binding complex,
and to be constitutively expressed in T-cells but not L-cells
(McCaffrey et al., 1993b). There appear to be three
alternatively spliced products of this gene. A similar study with human
T-cells adds a new twist to the shuttle model for NFAT assembly.
Northrop et al.(1994) cloned a human gene encoding another
cytosolic protein, NFATc, which was transcribed differentially from
NFATp in different tissues. Unexpectedly, NFATc transcripts were absent
or barely discernible in unstimulated T-cells and lymphoid tissue,
although NFATp transcripts were present constitutively. PMA and
ionomycin induced transcription of NFATc but had no effect on NFATp
(Northrop et al. 1994). It will be interesting to determine
which if any of the constitutive and inducible cytosolic components
participate in nuclear NFAT assembly in mast cells.
The present
results indicate that at least two types of mast cells, RBL-2H3 cells
and rat bone marrow cultured mast cells, in addition to lymphocytes,
express latent NFAT DNA binding activity. As with lymphocytes, the role
of AP-1 in, and the precise subunit composition of the mast cell NFAT
complex are key questions for ongoing research. Mast cells provide a
unique opportunity for studies of NFAT assembly, in that they can be
stimulated much more readily through a physiologically relevant
pathway, antigen binding, than can T-cells. Future studies may soon
reveal whether mast cell NFAT mediates the transcription of one or more
cytokine genes elaborated in response to Fc
RI) for immunoglobulin E (IgE).
Receptor-dependent activation of NFAT was mimicked by the combination
of the protein kinase C activator phorbol myristate acetate and the
calcium ionophore ionomycin. The induced binding activity was specific
for the NFAT recognition motif because competition with nonradioactive
NFAT oligonucleotide abolished the DNA binding activity, whereas
nonradioactive oligonucleotides recognized by the transcription factors
NF
B, glucocorticoid receptors, and TFIID did not. An
oligonucleotide representing the AP-1 recognition sequence also blocked
the NFAT DNA binding activity, as did a combination of anti-Fos and
anti-Jun antibodies. Using electrophoretic mobility shift assays,
AP-1-binding proteins were found to be induced in RBL-2H3 cells under
the same conditions as was the NFAT binding activity. Together these
data suggest that the NFAT complex in mast cells contains Fos and Jun
proteins as does NFAT in T-cells. The appearance of nuclear NFAT
binding activity was dependent in part upon calcium mobilization, as
buffering the antigen-induced calcium rise with intracellular BAPTA
strongly inhibited NFAT activation. Prevention of calcium influx with
external EGTA also inhibited NFAT activation, indicating that release
of calcium from internal stores was insufficient for sustained
activation of mast cell NFAT. Cyclosporin A, a potent inhibitor of the
calmodulin-dependent phosphatase calcineurin, blocked the induction of
NFAT-DNA binding activity, implicating calcineurin as a key signaling
enzyme in this pathway. These results suggest that NFAT is present in
the mast cell line RBL-2H3 and in primary bone marrow-derived mast
cells, is similar in subunit composition to the T-cell NFAT, and may
play a role in calcium-dependent signal transduction in mast cells.
(
)is a transcription complex believed to mediate the
final step in the signal transduction pathway linking T-cell receptor
engagement with the expression of the interleukin-2 (IL-2) gene (for
reviews, see Ullman et al.(1990) and Rao (1994)). Although
selective expression of NFAT is thought to account for the T-cell
specificity of IL-2 gene expression, recent evidence suggests the
possibility that NFAT may be one in a family of related transcription
factors that regulate the transcription of cytokine genes in other
leukocytes. The present work was undertaken to determine whether
NFAT-related activity can be induced in mast cells via cross-linkage of
the receptor with high affinity for IgE (Fc
RI).
RI in mast cells shares
many relevant features with the T cell receptor-initiated pathway,
including an increase in intracellular calcium (Beaven et al., 1984)
and activation of PKC, which in turn modulates the expression of AP-1
binding proteins such as Jun and Fos (Lewin et al., 1993;
Baranes and Razin, 1991). Antigen-activated mast cells produce
cytokines such as IL-3, GM-CSF, TNF-
, IL-4, IL-5, IL-6, and IL-1
(Kaye et al., 1992; Wodnar-Filipowicz et al., 1989;
Plaut et al., 1989), and NFAT binding sites have been
identified in the regulatory regions of several of these genes
(reviewed in Rao, 1994). The NFAT binding site located in the
intergenic enhancer region of the IL-3/GM-CSF genes has been shown to
bind NFAT from activated T-cells (Cockerill et al., 1993), and
CsA inhibits the induction of DNA binding activity directed to this
site in Jurkat cells treated with PMA and ionomycin. CsA blocks the
Fc
RI-dependent induction of IL-1
, TNF-
, and IL-6 mRNA in
mouse bone marrow-derived mast cells, apparently through the mediation
of some of the same immunophilin proteins present in T-cells (Hultsch et al., 1991; Kaye et al., 1992). This CsA
sensitivity suggests a possible role for calcineurin in the
Fc
RI-mediated expression of cytokine genes in mast cells. Given
the presence of NFAT binding sites in the regulatory regions of
cytokines induced by Fc
RI cross-linkage, a key question is, does
NFAT mediate transduction of signals from the Fc
RI to the nucleus?
receptor
induces NFAT DNA binding activity in two types of rat mast cells. The
presumed NFAT complex in mast cells, like that in T-cells, contains
AP-1 proteins in association with (an)other species conferring
specificity for the NFAT recognition motif. Induction of NFAT binding
activity by the Fc
RI was mimicked by simultaneous treatment with
the PKC activator PMA and the calcium ionophore ionomycin, required
calcium mobilization, and was blocked by the calcineurin inhibitor,
CsA. The presence of NFAT in mast cells and its activation via the
Fc
RI suggest that it may mediate the terminal steps of signal
transduction via the Fc
RI, namely the transcription of one or more
cytokine genes.
Cell Culture and Stimulation
The RBL-2H3 (RBL)
cell line was maintained in 75-cm flasks as monolayers in
Eagle's minimum essential medium (Earle's salts)
supplemented with 20% fetal bovine serum (Life Technologies, Inc.), 100
units/ml penicillin G, and 100 µg/ml streptomycin as described
previously (Barsumian et al., 1981). The cells were cultured
at 37 °C in a 5% CO
atmosphere in a humidified
incubator. Every 4 days the cells were removed by trypsinization and
passed to new flasks at a cell density of 2
10
cells/20 ml of medium. Rat bone marrow-derived mast cells (RBMMC)
were cultured from bone marrow of 3-5-week-old Fisher 344 rats in
the presence of recombinant murine stem cell factor and rat
interleukin-3. Details of the culture methods and functional and
molecular properties of this cell population are described
elsewhere.(
)
10
cells/ml).
Trinitrophenylated bovine serum albumin (TNP-BSA) was used at a final
concentration of 50 ng/ml, and PMA and ionomycin were used at 32 nM and 2 µM, respectively. CsA (Sandoz Research
Institute, Hanover, NJ) stock solutions were prepared by first
dissolving 1 mg of CsA in 100 µl of 100% ethanol containing 20
µl of Tween 20. The volume was adjusted to 1 ml with culture
medium, and appropriate dilutions of this stock solution were made
directly into the cell suspensions immediately following the 90-min
incubation. The cells were pretreated with CsA 15 min before
activation. In treatments where cells were triggered in the absence of
calcium, the cells were incubated for 30 min in medium containing the
calcium chelators before cell activation. Solid EGTA was added at a
concentration of 3.2 mM to basal medium (1.8 mM
Ca
, 0.8 mM Mg
) and the pH
adjusted to 7.40 with NaOH. This yields an estimated free
[Ca
] of 80 nM. For chelation of
extracellular calcium, cells were pelleted and resuspended in fresh
medium containing EGTA. BAPTA-AM was added directly to the cell
suspension as a concentrated stock solution in methyl sulfoxide to
attain a final concentration of 20 µM.
Nuclear Extraction
Nuclear extracts were prepared
essentially as described (Fiering et al., 1990) with the
following modifications. Between 1-5 10
cells
were used per time point. To begin the extractions the cells were
pelleted and washed once in ice-cold phosphate-buffered saline. All
subsequent steps were performed at 4 °C in a 1.5-ml microcentrifuge
tube with the following protease inhibitors added to all buffers
immediately before use: 1 mM phenylmethylsulfonyl fluoride and
2 µg/ml antipain, leupeptin, pepstatin A, and aprotinin. After
washing, the cells were pelleted by centrifugation and resuspended in 1
ml of buffer A (10 mM HEPES, pH 7.8, 15 mM KCl, 2
mM MgCl
, 1 mM dithiothreitol, 0.1 mM EDTA). The cells were pelleted again, resuspended in 1 ml of
buffer B (buffer A plus 0.05% Nonidet P-40), and allowed to incubate on
ice for 1 min. The cell suspension was then pipetted up and down once.
At this point >90% of the cells were lysed, yielding intact nuclei
as determined by trypan blue staining and light microscopy. Intact
nuclei were pelleted and resuspended in 315 µl of buffer C (50
mM HEPES, pH 7.8, 50 mM KCl, 0.1 mM EDTA, 1
mM dithiothreitol, 10% glycerol) and 35 µl of 3 M
(NH
)
SO
, pH 7.8. The resultant
solution was mixed at 4 °C for 30 min to extract nuclear proteins
and then centrifuged at 150,000
g for 15 min. An equal
volume of 3 M (NH
)
SO
, pH
7.8, was added to the supernatant. Precipitated proteins were pelleted
by centrifugation at 100,000
g for 10 min and
resuspended in 50-100 µl of buffer C. The protein samples
were desalted on P6DG columns (Bio-Rad), and protein concentrations
were determined using a dye binding assay (Bradford, 1976). The samples
were stored in aliquots at -80 °C.
Electrophoretic Mobility Shift
Assays
Electrophoretic mobility shift assays were done as
described previously (Fiering et al., 1990). Binding reactions
for NFAT were carried out in a 20-µl volume containing 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 5%
glycerol, 1.7 µg of poly(dI-dC), and 10-30 µg of nuclear
protein. Binding reactions for AP-1 were carried out in a 20-µl
volume consisting of 50 mM Tris-HCl, pH 7.5, 13 mM MgCl, 1 mM EDTA, 5% glycerol, 1.7 µg of
poly(dI-dC), and 10 µg of nuclear proteins (Mattila et
al., 1990). Competitor oligonucleotides and anti-Fos and anti-Jun
antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, catalog nos.
sc-253X (Fos) and sc-44X (Jun)) were added at concentrations of 25
ng/20 µl and 1 µg/20 µl, respectively. Reaction mixtures
containing competitor oligonucleotides or antibodies were incubated on
ice for 10 min before the addition of 0.1-0.5 ng of
double-stranded oligonucleotides end-labeled with
[
-
P]dATP. The probe for NFAT was based upon
the distal NFAT recognition site in the promoter for human IL-2 (Emmel et al., 1989). It was prepared for the binding assay by
annealing the single-stranded oligonucleotides
5`-GGAGGAAAAACTGTTTCATACAGAAGGCGT-3` and
5`-ACGCCT-TCTGTATGAAACAGTTTTTCCTCC-3` (Midland Certified Reagent
Company, Midland, TX) in the presence of 50 mM NaCl. The
probes for AP-1, NF
B, TFIID, and GRE were purchased as
double-stranded oligonucleotides with the sequences
5`-CGCTTGATGAGTCAGCCGGAA-3`, 5`-AGTTGAGGGGACTTTCCCAGGC-3`,
5`-GCAGAGCATATAAGGTGAGGTAGGA-3`, and 5`-TCGACTGTACAGGATGTTCTAGCTACT-3`,
respectively. These oligonucleotides were end-labeled with
[
-
P]dATP as needed (Promega). After a
40-min incubation on ice, the binding reactions were electrophoresed on
prerun 5% polyacrylamide gels in 0.5
TBE (0.5 times TBE
= 45 mM Tris borate, 1 mM EDTA) for 2.5 h at
150 V. The gels were dried on Whatman No. 3MM filter paper and
autoradiographed.
Induction of NFAT Binding Activity in RBL-2H3 Cells and
Rat Bone Marrow-derived Mast Cells
Because NFAT activity in the
Jurkat human T-cell line has been extensively characterized (Durand et al., 1988; Shaw et al., 1988; Emmel et
al., 1989, Clipstone and Crabtree, 1992), we used this cell line
as a positive control in the present experiments. To test whether NFAT
binding activity is present in activated mast cells, we stimulated
RBL-2H3 cells and RBMMC either through the FcRI or with PMA and
ionomycin. Fig. 1and Fig. 2show that in fact, NFAT
binding activity was induced in RBL cells and RBMMC after a 2-h
incubation with PMA and ionomycin (lanes 5 and 3,
respectively), and also after a 2-h stimulation via the Fc
receptor with the multivalent antigen TNP-BSA (lanes6 and 2, respectively). Both stimulation protocols caused
the appearance of a band that migrated to a position similar to that of
the band induced in Jurkat cells treated with PMA and ionomycin.
Triggering RBMMC through the Fc
receptor induced only modest NFAT
activity, compared with that induced by activating the cells with PMA
and ionomycin (Fig. 2).
Figure 1:
EMSA showing
inducibility of NFAT in RBL cells by either PMA and ionomycin or by
cross-linkage of the Fc receptor. Nuclear extracts were prepared
from RBL and Jurkat cells that were unstimulated or stimulated with PMA
(32 nM) and ionomycin (2 µM) (P/I) for 2
h and RBL cells stimulated with 50 ng/ml TNP-BSA (Ag) for 2 h
as indicated. Equivalent amounts (20 µg) of nuclear proteins from
each treatment were incubated with double-stranded
P-labeled oligonucleotide containing the NFAT binding
site. Retarded bands representing NFAT complexes are indicated by an arrow. The first lane (fp) shows free probe without
nuclear extract. Data are representative of five independent
experiments.
Figure 2:
EMSA showing inducibility of NFAT in RBMMC
by either PMA and ionomycin or by cross-linkage of the Fc
receptor. Nuclear extracts were prepared from RBMMC that were
unstimulated, stimulated with PMA (32 nM) and ionomycin (1
µM) (P/I), or stimulated with 50 ng/ml
TNP-BSA (Ag) for 2 h as indicated. Equivalent amounts (20
µg) of nuclear proteins from each treatment were incubated with
double-stranded
P-labeled oligonucleotide containing the
NFAT binding site. Retarded bands representing NFAT complexes are
indicated by an arrow. Data are representative of three
independent experiments.
Specificity and Subunit Composition of NFAT Binding
Activity in RBL Cells
To determine the specificity of the NFAT
DNA binding activity induced in RBL cells, unlabeled oligonucleotides
containing various recognition sequences were added in excess to the
EMSA reaction. As expected, excess unlabeled NFAT oligonucleotide
completely reversed the NFAT DNA binding activity present in the
nuclear extracts from RBL and Jurkat cells (Fig. 3A). In
contrast, the presence of excess unlabeled NFB, TFIID, and GRE
oligonucleotides had no effect on the NFAT binding activity in either
RBL or Jurkat nuclear extracts. Hence, the NFAT DNA binding activity
induced in RBL cells by stimulation via the Fc
RI was highly
specific for the NFAT recognition motif. As stimulation of RBL cells
via the Fc
RI induces expression of the AP-1 transcription complex
(Baranes and Razin, 1991; Lewin et al., 1984), which in
T-cells assembles with NFATc/p to form the NFAT transcription complex,
we tested the hypothesis that AP-1 forms part of this complex in mast
cells. As shown in Fig. 3A, unlabeled AP-1
oligonucleotides indeed removed the NFAT band both in RBL and in Jurkat
cells at least as completely as the unlabeled NFAT oligonucleotides did
when present at the same concentration. This is consistent with the
idea that, as with T-cells, AP-1 forms part of the NFAT complex in RBL
cells. To further explore this possibility, we performed EMSA in the
presence of antibodies to the AP-1 proteins Fos and Jun. Using nuclear
proteins from RBL cells stimulated through the Fc
RI receptor, Fig. 3B shows that either the Fos or the Jun antibody
alone slightly reduced the NFAT binding activity and caused the
appearance of a faint band of decreased mobility (lanes2 and 3). When used in combination, the Fos and Jun
antibodies completely abrogated the NFAT DNA binding activity induced
by Fc
RI cross-linkage (lane4).
Figure 3:
The NFAT complex in RBL and Jurkat cells
contains AP-1 proteins. A, EMSA was performed using a P-labeled NFAT probe and nuclear extracts (20 µg) from
Jurkat cells stimulated with PMA and ionomycin for 2 h, or RBL cells
triggered with antigen for 60 min. As indicated, either no competitor
DNA or a 200-fold excess of unlabeled NFAT, AP-1, NF
B, TFIID, or
GRE oligonucleotide was added. B, EMSA was performed using a
P-labeled NFAT probe and nuclear extracts (20 µg) from
RBL cells triggered with antigen for 2 h. As indicated, either no
antibodies or 1 µg of anti-Fos antibody (F), 1 µg of
anti-Jun antibody (J), or 1 µg each of anti-Fos and
anti-Jun antibodies (F/J) were added to the binding reaction.
Data are representative of three independent
experiments.
Kinetics of Appearance of Nuclear NFAT and AP-1
Activity
When triggered through the FcRI, NFAT binding
activity was detectable within 10 min after exposure to antigen, and
the activity steadily increased for up to 120 min (Fig. 4A). A low background level of NFAT binding
activity could be detected in unstimulated cells. This background
activity varied between experiments but did not mask the increase in
NFAT induced by cell activation.
Figure 4:
Time course of appearance of NFAT and AP-1
activity in RBL cells in response to cross-linkage of the Fc
receptor. Nuclear extracts were prepared from RBL cells at the
indicated times (in min) after the addition of 50 ng/ml TNP-BSA.
Equivalent amounts of nuclear proteins (20 µg (A) or 10
µg (B)) from each time point were incubated with
double-stranded
P-labeled oligonucleotide containing the
NFAT or AP-1 binding sites as indicated. Retarded bands representing
NFAT (A) and AP-1 (B) complexes are indicated by arrows. Data are representative of three independent
experiments.
Having evidence that AP-1 proteins
contribute to the NFAT transcriptional complex present in RBL cells, we
examined the kinetics of appearance of AP-1 activity following cell
activation through the FcRI. Fig. 4B shows that
AP-1 activity appeared in RBL nuclear extracts in a similar kinetic
pattern as did NFAT, although virtually no AP-1 binding activity was
present prior to activation. A slight induction was detected at 10 min,
followed by a gradual increase in AP-1 activity up to 120 min. The
absolute amount of binding-competent AP-1 appears greater at all time
points than that of the NFAT complex, given the smaller amount of
extract used in the AP-1 assays; this suggests that the rate of
appearance of AP-1 does not limit the rate of appearance of NFAT DNA
binding activity.
Calcium Dependence of NFAT Induction by Fc
To determine whether the appearance of NFAT in the
nucleus of RBL cells is calcium-dependent, cells were activated in the
presence of extracellular EGTA sufficient to buffer extracellular
calcium to RI
Cross-linkage
80 nM. Alternatively, cells were incubated
with the cell permeant BAPTA-AM (20 µM) for 30 min at 37
°C, conditions previously determined to block the calcium rise
produced by Fc
RI cross-linkage.(
)
The 30-min
pretreatment with EGTA did not by itself induce NFAT binding activity
above the level present in the cells incubated in medium alone (Fig. 5, lanes1 and 6). Similarly,
incubating RBL cells in the presence of BAPTA-AM for 30 min prior to
beginning the experiment and for 60 min after beginning the experiment
did not induce the appearance of NFAT above the level of the control (Fig. 5, lanes1, 3, and 4).
However, preincubation of cells with BAPTA-AM did inhibit completely
the induction of NFAT binding activity by antigen (Fig. 5, lanes2 and 5). Triggering RBL cells with
antigen in the presence of extracellular EGTA induced very modest NFAT
binding activity, considerably less than that induced in the presence
of external millimolar free [Ca
] (Fig. 5, lanes2 and 7). Nevertheless,
prevention of calcium influx with EGTA did not inhibit NFAT induction
as completely as did intracellular BAPTA, which quenches the rise in
intracellular calcium due to both release from internal stores and
influx across the plasma membrane. Hence, intracellular release
apparently can support a modest NFAT induction, but a large, sustained
induction requires influx of extracellular calcium.
Figure 5:
The induction of NFAT in RBL cells is
dependent on the availability of calcium. EMSA was performed using a P-labeled NFAT probe and nuclear proteins (20 µg) from
RBL cells unstimulated or stimulated with antigen for 60 min as shown:
basal medium, after incubation with 20 µM BAPTA-AM or in
the presence of excess EGTA (extracellular free
[Ca
]
80 nM). Data are
representative of three independent
experiments.
CsA Sensitivity of NFAT Induction by Fc
To determine whether the calcium-dependent protein
phophatase calcineurin is involved in the induction of NFAT binding
activity in mast cells, we activated RBL cells with antigen in the
presence of the calcineurin inhibitor CsA at concentrations from 1 to
1000 ng/ml. CsA at 1 and 10 ng/ml had no effect on antigen-induced NFAT
binding activity, whereas 100 ng/ml CsA reduced the activity markedly
and 1000 ng/ml completely blocked nuclear NFAT induction (Fig. 6).
RI
Cross-linkage
Figure 6:
The induction of NFAT in RBL cells is
inhibited by CsA. EMSA was performed using a P-labeled
NFAT probe and nuclear proteins (20 µg) from RBL cells unstimulated
or stimulated with antigen for 2 h in the presence of 1, 10, 100, and
1000 ng/ml CsA. Data are representative of three independent
experiments.
receptor induced NFAT activity
quantitatively similar to that seen in Jurkat cells stimulated with PMA
and ionomycin (Fig. 1). NFAT was detected in RBL nuclear extracts
within 10 min following antigenic stimulation, with the activity
steadily increasing for at least 2 h (Fig. 4A). This is
similar to the kinetics of appearance of nuclear NFAT activity in
Jurkat human T-cells stimulated with PMA and ionomycin (Shaw et
al., 1988), although somewhat faster than the appearance of
AP-1-dependent NFAT activity in murine Ar-5 T-cells stimulated with
immobilized anti-CD3
antibodies (Jain et al., 1993b).
Because RBL cells are a transformed analog of mast cells, the
possibility exists that the presence of NFAT activity in these cells is
an artifact of the transformation process. To address this possibility,
we also assayed a non-transformed mast cell population derived from rat
bone marrow. These cells showed nuclear NFAT activity inducible under
the same activation regimens used with RBL cells (Fig. 2).
did not possess
NFAT DNA binding activity. To reduce basal NFAT activity to a low,
albeit detectable level, cells were incubated in calcium-replete medium
for 90 min prior to antigenic stimulation. We have observed large,
transient rises in intracellular [Ca
] in
RBL cells exposed to an air-water interface,
suggesting
that adventitious calcium influx during cell preparation may be
responsible for basal NFAT activity (see below).
RI is likely to require an elevation of cytosolic
[Ca
]. First, removal of the steep
[Ca
] gradient across the plasma membrane by
buffering external free [Ca
] at
80
nM with extracellular EGTA markedly, although not completely,
reduced the NFAT activity induced by 60 min (Fig. 5). Second,
pretreatment of cells with BAPTA-AM under conditions found to block the
antigen-induced calcium signal
completely inhibited
induction of NFAT activity by antigen (Fig. 5). Thus, the
appearance of NFAT DNA binding activity in the nucleus of RBL cells
could be part of a Ca
-dependent signaling pathway
like that postulated to mediate the translocation of NFATp/c in
T-cells. That some nuclear NFAT activity appeared when influx of
extracellular Ca
alone was prevented suggests that
sufficient Ca
may be mobilized from intracellular
stores to initiate formation of the active NFAT complex but that
sustained activity requires Ca
influx.
RI. Antigen-induced NFAT activity in RBL cells was not
affected by CsA at a concentration of 1 and 10 ng/ml but was
significantly reduced by CsA at 100 ng/ml and was completely blocked at
1000 ng/ml (Fig. 6). This is similar, although not identical, to
the dose-response curve for CsA inhibition of NFAT activity obtained
with Jurkat cells stimulated with PMA and ionomycin. Emmel et
al.(1989) found that CsA at concentrations of 1 and 10 ng/ml
inhibited only slightly the induction of NFAT activity in Jurkat cells,
and detectable activity was still present even at 1 µg/ml CsA. Also
in Jurkat cells, Mattila et al.(1990) found no inhibition of
NFAT binding activity with 1 ng/ml CsA, but a significant reduction in
NFAT binding with 10 and 100 ng/ml CsA. Thus, the effective
concentration of CsA for inhibiting NFAT induction in RBL cells is
similar to that found in Jurkat cells. In mouse bone marrow-derived
mast cells cultured in IL-3, CsA has been shown to inhibit
antigen-induced production of IL1-
, tumor necrosis factor-
,
and IL-6 mRNA at IC
values of 4.4, 72, and 143 ng/ml,
respectively (Kaye, et al., 1992). Our results showing that
CsA inhibits substantially the induction of NFAT activity at a similar
effective concentration (100 ng/ml) suggests that NFAT may play a role
in the transcriptional activation of these genes in mast cells.
RI cross-linkage.
RI, receptor with high affinity for IgE; IL,
interleukin; EMSA, electrophorectic mobility shift assay; RBL, rat
basophilic leukemia; RBMMC, rat bone marrow-derived mast cells; PKC,
protein kinase C; PMA, phorbol 12-myristate 13-acetate; BSA, bovine
serum albumin; TNP-BSA, trinitrophenylated bovine serum albumin;
BAPTA-AM, 1,2-bis(2-aminophenoxy)eth-ane-N,N,N`,N`-tetraacetic
acid; TBE, Tris-borate-EDTA buffer; CsA, cyclosporin A.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.