NF-ATc Isoforms Are Differentially

Expressed and Regulated in Murine T and

This information is current as
of June 30, 2016.
Mast Cells
Melanie A. Sherman, Doris R. Powell, Deborah L. Weiss and
Melissa A. Brown
J Immunol 1999; 162:2820-2828; ;
http://www.jimmunol.org/content/162/5/2820

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NF-ATc Isoforms Are Differentially Expressed and Regulated
in Murine T and Mast Cells1,2

Melanie A. Sherman,* Doris R. Powell,* Deborah L. Weiss,‡ and Melissa A. Brown3*†

NF of activated T cells (NF-AT) denotes a family of transcription factors that regulate the activation-dependent expression of many
immunologically important proteins. At least four distinct genes encode the various family members, and several isoforms of these
have been identified as well. The overlapping expression patterns and similar in vitro binding and trans-activation activities on
various promoter elements of NF-AT-regulated genes suggest some redundancy in the function of these proteins. However, the
phenotypic analysis of NF-AT-deficient mice supports the idea that there are tissue- and gene-specific functions as well. In this
study we have characterized the expression of NF-AT cDNAs in murine mast cells. The majority of clones identified correspond
to two NF-ATc isoforms that differ only in their amino-terminal sequence. Despite minimal discrepancies in the coding region,
there are striking tissue- and cell type-specific differences in isoform expression patterns. Detection of NF-ATc.a mRNA is strictly
dependent on cell activation signals in both T and mast cell lines. In contrast, the b isoform is expressed at very low constitutive

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levels in both cell types but is only up-regulated in response to mast cell activation signals delivered through the FceRI or via
calcium ionophores. These results demonstrate another level of regulation within the NF-AT family that can contribute to cell
type-specific gene expression. The Journal of Immunology, 1999, 162: 2820 –2828.

N uclear factor of activated T cells (NF-AT)4 was origi-
nally described as a T cell-specific DNA binding protein
that regulates the activation-dependent transcription of
the IL-2 gene (1). It is now known to denote a family of transcrip-
tion factors that are expressed in a wide range of tissues and cell
types, including mast cells (2), B cells (3–5), macrophages (6), NK
cells (7), and neuronal cells (8). Although most studies have fo-
cused on characterizing its role in the transcription of genes that
regulate the immune system (9), NF-AT also influences other pro-
cesses such as cardiac development (10, 11). Four family mem-
bers, NF-ATp (NF-AT1) (12), NF-ATc (NF-AT2) (13), NF-AT3
(14), and NF-AT4/x/c3 (14 –16), have been described. Three of the
four NF-AT genes also give rise to multiple isoforms. Family
members are defined by related DNA binding domains and con-
served sequence motifs, including several serine/proline-rich re-
gions that appear to regulate the nuclear localization of these fac-
tors (17–19). All NF-AT proteins also appear to be a molecular
target of the immunosuppressive drugs cyclosporin A and FK506
(for a review, see Ref. 9). These agents interfere with the ability of
*Department of Experimental Pathology, and †Graduate Program in Immunology and
Molecular Pathogenesis and Genetics and Molecular Biology, Emory University
School of Medicine, Atlanta, GA 30322; and ‡Department of Chemistry, Williams
College, Williamstown, MA 02167
Received for publication September 1, 1998. Accepted for publication December
3, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the Multiple Sclerosis Society and the
National Institutes of Health (CA47992), a scholarship from the Leukemia Society of
America (to M.A.B.), and a fellowship from the Cancer Research Institute (to
M.A.S.).
2
The sequences of the murine NF-ATc.a (accession no. AF087434) and NF-ATc.b
(accession no. AF049606) were deposited in the GenBank database.
3
Address correspondence and reprint requests to Dr. Melissa A. Brown, Department
of Experimental Pathology, Emory University, 1639 Pierce Dr., Atlanta, GA 30322.
E-mail address: mbrow18@emory.edu
4
Abbreviations used in this paper: NF-AT, NF of activated T cells; FceR1, high
affinity Ig E receptor; RSD, rel similarity domain; BMMC, bone marrow-derived mast
cells; UTR, untranslated region.
calcineurin, a serine-threonine phosphatase, to dephosphorylate
cytoplasmic NF-AT and thus prevent its shuttling to the nucleus in
activated cells.
The existence of multiple NF-AT proteins that can recognize the
same DNA element raises questions regarding their in vivo func-
tion. The overlapping expression patterns of some of these proteins
and their similar in vitro binding and trans-activation activities on
promoter elements of NF-AT-regulated genes argue that these fac-
tors are largely redundant. However, several lines of evidence sup-
port the idea that family members may also have cell-specific
and/or gene-specific activities. 1) Outside the regions of high ho-
mology, the NF-AT family members have quite diverse sequences,
providing the opportunity for interaction with unique cofactors re-
quired for the transcription of a subset of NF-AT-regulated genes.
2) Tissue-restricted expression of some individual family members
and their isoforms has also been observed. For example, NF-AT3
is expressed at very low levels in the thymus, whereas NF-AT4 is
strongly expressed at this site (14 –16). Likewise, isoforms of NF-
AT3 show distinct expression patterns: the 3-kb form is expressed
predominantly in the placenta, lung, kidney, testis, and ovary, and
the 4.5-kb form is expressed in the heart and colon (14). 3) Recent
studies using antisera that can distinguish family members reveal
the existence of NF-AT binding specificity. Timmerman et al. showed
that while NF-AT1 and NF-AT2 can bind equally well to either the
IL-2 or IL-4 promoter in in vitro DNA binding assays, NF-AT3 and
NF-AT4 exhibit at least a 10-fold lower affinity (20). Similarly, NF-
ATx was shown to be the major component of IL-2 promoter DNA-
protein complexes in double-positive thymocytes (21).
The most compelling argument for gene-specific activities of
NF-AT comes from the phenotypic analysis of NF-ATp- and NF-
ATc-deficient mice. Stimulated T lymphocytes from NF-ATc2/2
mice have an impaired ability to produce IL-4 (22, 23). The pro-
duction of Ig isotypes associated with Th2 responses is also di-
minished (22). Expression of other NF-AT-regulated genes in T
cells, such as IL-2, was only slightly affected. These data demon-
strate that IL-4 gene expression is a specific target of regulation by
NF-ATc (22, 23). In contrast, although the kinetics of IL-4 ex-
pression by activated T cells are delayed in NF-ATp-deficient

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00

The Journal of Immunology
mice, IL-4 production is significantly enhanced overall compared
with that observed in wild-type animals. NF-AT42/2 animals pro-
duce relatively normal amounts of Th1 and Th2 cytokines such as
IL-4, but positive selection of T cells is defective (22). These re-
sults suggest the possibility that IL-4 transcription is positively
regulated by NF-ATc and is repressed by NF-ATp (24, 25).
In this study we provide evidence for cell-type specific expres-
sion of NF-ATc isoforms. These studies were prompted by our
earlier observation that the NF-AT-mediated regulation of IL-4
gene transcription in mast cells exhibits striking differences com-
pared with that of T cell IL-4 transcription (2). Although the tran-
scription of IL-4 in both cell types is dependent on an NF-AT site
between 288 and 260 in the murine IL-4 gene, the protein-DNA
complexes that form at this site are distinct. Unlike the T cell IL-4
complex that also contains activating protein-1 (AP-1), these Jun/
Fos family members are not present in the mast cell NF-AT com-
plex. DNA affinity purification and Western blot experiments also
revealed differences in the size and regulation of the mast cell
NF-AT protein associated with this site. These data suggested the
possibility that this NF-AT was either a unique isoform or the
product of a previously undescribed gene. Our efforts to address
this issue led to the isolation of several NF-AT cDNA clones from
a murine mast cell library. Here we report the full sequence of two
murine NF-ATc isoforms, designated NF-ATc.a and -b. Each
form shows unique features in sequence and expression pattern
compared with the previously described human NF-ATc genes
(26). In addition, murine T and mast cells exhibit striking differ-
ences in the relative expression of NF-ATc.a and NF-ATc.b. This
is due in part to the different inducibilities of these isoforms in
response to TCR- and FceRI-mediated signaling. The selective ex-
pression of NF-ATc isoforms may contribute to cell- and/or gene-
specific transcription of molecules that function in inflammation
and that have not been previously appreciated in knockout exper-
iments.
Materials and Methods
Nucleotide sequence accession number
The sequences of the murine NF-ATc.a (accession no. AF087434) and
NF-ATc.b (accession no. AF049606) were deposited in the GenBank
database.
cDNA cloning
A murine mast cell cDNA library was constructed in the lgt11 (Clontech,
Palo Alto, CA.) vector. Poly(A)1 mRNA from stimulated (ionomycin for
90 min) CFTL15 mast cells (27) was used as the template for first-strand
cDNA synthesis using both oligo(dT) and random hexamer primers. The
library was screened at low stringency (23 SSC/0.5% SDS at 55°C) with
a murine NF-ATp probe obtained by RT-PCR amplification of murine
EL-4 T cell RNA. The probe contained sequences corresponding to nucle-
otides 1038 –1517 within the rel similarity domain (RSD) of NF-ATp (28).
Positive clones were purified, and inserts were subcloned into pBluescript
plasmids for further analysis. Sequencing of both strands of the cDNA
inserts was performed using the dideoxynucleotide chain termination
method with reagents from U.S. Biochemical Corp. (Cleveland, OH).
RNase protection analysis
Total RNA samples from cell lines and murine tissues (from unimmunized
C3H/HeNCr mammary tumor virus (MTV)2 mice) were isolated using the
RNA-STAT reagent according to the manufacturer’s instructions (Tel-
Test, Friendswood, TX). RNase protection assays were performed using
NF-ATc.a (nucleotides 10 –196 in the a sequence) and NF-ATc.b (nucle-
otides 15–161 in b sequence) cDNA fragments as templates for the syn-
thesis of antisense RNA labeled to high sp. act. with [32P]UTP (Riboprobe
kit, Promega, Madison, WI). Full-length RNA probes were gel-purified and
hybridized (5 3 105 cpm) to 10 mg of total RNA overnight at 45°C in 40
mM PIPES (pH 6.4), 400 mM NaCl, 1 mM EDTA, and 80% formamide in
a total volume of 30 ml. Samples were incubated for 1 h at 30°C after
addition of 350 ml of digestion buffer (10 mM Tris (pH 7.5), 5 mM EDTA,
300 mM NaCl, 0.14 mg of RNase T1, and 1 mg of RNase A). Proteinase
2821
K (5 ml of 10 mg/ml stock) and SDS (10 ml of 20% solution) were added
and incubated for an additional 30 min at 37°C. Samples were extracted
with phenol/chloroform, precipitated, resuspended in gel loading buffer,
and analyzed by 6% denaturing PAGE. Densitometric quantitation of rel-
ative mRNA expression levels was performed using the National Institutes
of Health Image program.
Northern blot analysis
The murine tissue Northern blots were purchased from Clontech. For anal-
ysis of expression in cell lines, 10 mg of total RNA or 2 mg of poly(A)1
RNA was electrophoresed on a 1% formaldehyde gel and transferred to
nitrocellulose. DNA probes were labeled by random hexamer priming and
hybridized as described previously (29). The NF-ATc-specific probe cor-
responds to nucleotides 1162–1727 based on the NF-ATc.b sequence
shown in Fig. 1 and contains a significant portion of the RSD. The murine
b-actin probe includes nucleotides 147–332 of the published sequence
(30). The IL-4 cDNA probe corresponds to nucleotides 40 – 412 (31).
Cells
CFTL15 is an IL-3-dependent murine mast cell line derived from fetal liver
cells and was previously described (27). Bone marrow-derived mast cells
(BMMC) were isolated from BALB/c bone marrow cultured in IL-3 and
stem cell factor. After 4 wk the cells were analyzed by flow cytometry for
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the surface expression of c-Kit and FceRI. The c-Kit levels were deter-
mined using directly conjugated anti-CD117-phycoerythrin (PharMingen,
San Diego, CA). Expression of the high affinity Fce was assessed using a
two-step staining procedure. Cells were first incubated with purified mouse
IgE (Sigma). After three washes, a second IgE bound to FceR was detected
using a rat anti-mouse IgE-FITC Ab (PharMingen). Data were acquired on
a FACS caliber flow cytometer (Becton Dickinson, San Jose, CA) gating
on propidium iodide-negative cells. Data were analyzed using CellQuest
software (BDIS; Becton Dickinson). P815 and ABFTL3 are transformed
mast cells that express IL-4 mRNA constitutively (29). EL-4 is a murine
thymoma line (32) that was obtained from American Type Culture Col-
lection (Manassas, VA). D011.10 Th1 and Th2 cell lines were derived from
the OVA-specific TCR transgenic mouse (33). M12.4.1 B cells were a gift
from Dr. Paul Rothman (34); L929 fibroblast cells were obtained from
American Type Culture Collection and have been previously described
(35). Mast cells were activated by culturing with the calcium ionophore,
ionomycin (1 mg/ml; Calbiochem, La Jolla, CA) or by cross-linking the
high affinity FceR by priming the cells with 1 mg/ml purified anti-DNP IgE
(Sigma) for 2 h followed by activation with 5 mg/ml DNP-keyhole limpet
hemocyanin (Calbiochem). In some experiments mast cells were cultured
for 2 days with IgE before stimulation. T cells were stimulated with 1
mg/ml ionomycin and 20 ng/ml PMA (Sigma) or with Ag (2.5 3 105/ml
BALB/c spleen cells, 104 U/ml IL-2, and 0.5 mM OVA323–339 peptide) for
the indicated periods of time.
Results
Murine NF-ATc cDNAs encode a and b isoforms that differ
only at their N-termini
To identify NF-AT genes expressed in mast cells, a cDNA probe
derived from the murine NF-ATp RSD was used to screen both
oligo(dT)- and random hexamer-primed CFTL15 murine mast cell
libraries at low stringency. Approximately 1.6 3 105 plaques were
screened. Several clones were characterized, including one that
corresponded to NF-ATp. The majority of the cDNAs analyzed
were homologous to human NF-ATc.b, an isoform identified in
Raji B cells (26). The longest clone is comprised of 3435 nucle-
otides, which includes an approximately 1.3-kb 39 untranslated
region (39UTR) that is unrelated to the 39UTR of human NF-ATc
(Fig. 1). Within the coding region, this cDNA exhibits .83% iden-
tity overall at the DNA level with the human gene (Fig. 2A). The
cDNA encodes a protein of 704 amino acids (aa) with a predicted
molecular mass of 70 kDa. A comparison of amino acid sequences
reveals that NF-ATc is highly conserved in human and mouse.
There is 96.7% identity and 98% similarity within the RSD (en-
coded by aa 408 – 684), which contains the DNA binding domain.
The amino-terminal region (aa 1– 407) contains three serine/pro-
line (SP) motifs and is 83% identical and 91% similar to its human
NF-ATc.b homologue. A short C-terminal region (aa 685–704)
2822
FIGURE 1. Nucleotide and deduced amino acid sequence of murine NF-ATc.b. Nucleotide sequences are numbered on the left, and amino acid
sequences are numbered on the right. The nucleotides unique to the b isoform are overlined.
does not show similarity to the reported sequence of human NF-
ATc.
In addition to the b isoform of NF-ATc, cDNAs encoding the
murine NF-ATc.a homologue (13) were also isolated. It is notable
that unlike the human cDNAs that encode isoform-specific amino
acids at both the N- and C-terminal portions of the proteins (26),
the differences between murine NF-ATc.a and -b were limited to
the 59 end. In NF-ATc.a cDNAs, sequences specifying the first 27
aa of NF-ATc.b are replaced by nucleotides that can potentially
encode 39 a isoform-specific amino acids (Fig. 2B). The remain-
der of the cDNA sequence is identical with the b isoform. While
there is an in-frame AUG codon within this sequence, we were
unable to obtain additional 59 sequence encoding an upstream me-
thionine using several approaches. These include rapid amplifica-
tion of 59 cDNA ends (RACE), direct screening of the cDNA
library with a murine NF-ATc.a probe, and RT-PCR using murine
T and mast cell cDNA and primers derived from the murine NF-
ATc common region and the human NF-ATc.a 59UTR. However,
NF-ATc ISOFORMS IN MAST CELLS AND T CELLS
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it is likely that these sequences exist. A recent report indicates that,
like the human a isoform, a murine homologue isolated from T
cells contains nucleotides that specify an additional 3 aa (36).
These sequences include an in-frame AUG codon in a context that
is suboptimal for protein translation initiation (cggAUGc) (37).
This upstream AUG is used for the translation initiation of a minor
NF-ATc protein species in human T cells. Our clones contain the
downstream AUG, which lies in an optimal context (accAUGa)
and initiates the major form of human NF-ATc.a translation (37,
38). A comparison of the murine and human a-specific sequences
shows that they share 80% identity and 80% similarity (Fig. 2C).
NF-ATc expression differs in human and murine tissues
Previous Northern blot analysis with human tissues, using probes
that distinguish the a and b isoforms, reveal that NF-ATc.b
mRNA is approximately 4.5 kb in size and is preferentially ex-
pressed in spleen, testis, and ovary; the a isoform is encoded by a
2.7-kb mRNA and is detected predominantly in the thymus and
The Journal of Immunology 2823

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FIGURE 2. A, Comparison of murine and human NF-ATc.b cDNAs. The murine sequence is shown on the upper strand, and the human sequence is
shown on the lower strand. The sequences were aligned using a blosum62 matrix (45). The RSD is boxed, and the SP boxes are shaded. B, NF-ATc isoforms
differ only at the amino-terminal end. The unique amino acids that define the NF-ATc.a and -b isoforms are shown. Within the N-terminal portion of
NF-ATc.a, the methionines encoded by AUG codon within an optimal context for translation initiation (37) are underlined. NF-ATc.a cDNA isolated from
murine T cells is reported to contain sequences encoding 3 additional aa (shown in brackets) including a potential ATG translation initiation codon (36).
C, A comparison of the amino acid sequences that define the murine (top strand) and human (bottom strand) NF-ATc.a proteins. The sequences were
aligned as described in A.

peripheral blood leukocytes (26). In murine tissues, probes derived
from both the 59 region (not shown) and the RSD (Fig. 3A) hy-
bridize with two closely migrating mRNA species of about 4.5 kb,
suggesting that both the a and b isoforms mRNAs are of similar
size. Levels of murine NF-ATc RNA are highest in spleen, lung,
and skeletal muscle. Detectable, but much lower, expression was
observed in heart, brain, liver, and kidney. There was no detectable
expression in mouse testis, unlike results reported for human
NF-ATc.
A variety of T and mast cell lines were also analyzed for ex-
pression under resting and activated conditions. As shown in Fig.
3B, mRNAs of several sizes were observed. An approximately
4.5-kb band is the predominant species in both cell types and ap-
pears as an apparent doublet in mast cell lines. Consistent with
studies in human cells demonstrating that NF-ATc expression is
dependent on cell activation, no mRNA was detected in unstimu-
lated T cells (13). Several minor species appear to be expressed
exclusively in mast cells. At least two mRNAs of about 3.0 kb
were detected in activated mast cell lines. An additional 2.0 kb
inducible mRNA was present in BMMC and P815 mast cells. Un-
stimulated L929 fibroblast cells and M12.4.1 B cells do not ex-
press detectable NF-ATc mRNA (data not shown).
NF-ATc.a and -b mRNA are differentially expressed
Results from Northern analyses are consistent with the idea that
there is cell- and tissue-specific expression of NF-ATc isoforms.
RNase protection assays were performed using probes that can
distinguish between the a and b isoforms of NF-ATc to explore
this possibility. The integrity of the RNA in the samples was ver-
ified in parallel reactions using a b-actin probe (data not shown).
As shown in Fig. 4, the b isoform is strongly expressed in heart,
spleen, and kidney, whereas a-specific mRNA is present only in
spleen. Neither isoform is present at detectable levels in liver or
brain. However, RNA samples from these tissues did protect a
2824
FIGURE 3. A, Northern blot analysis of NF-ATc expression in murine
tissues. The filter was hybridized with a probe corresponding to a portion
of the RSD of murine NF-ATc. A murine actin probe was used as a loading
control. The relative migration of the RNA size standards are indicated on
the left. Arrows denote specific hybridizing bands. B, Northern blot anal-
ysis of NF-ATc expression in murine T and mast cell lines. Cells were left
untreated (2) or were stimulated with ionomycin (mast cells), PMA and
ionomycin (EL-4 T cells), or Ag (D011.10 T cells) for 3 h. Ten micrograms
of total RNA were used for the analysis of most cell lines. Poly(A)1 RNA
(2 mg) from CFTL15 mast cells was also included in this analysis. Hy-
bridization of the blot was performed as described above.
band of 70 bp corresponding to the region of the b probe that is
shared with a, indicating the presence of an additional isoform(s)
of NF-ATc in the sample.
Differential expression of NF-ATc isoforms is also observed
using RNA from cell lines. Significant expression of the a isoform
is strictly dependent on cell activation in a variety of both T and
mast cell lines (Fig. 5A). Stimulation through the Ag receptor or
with PMA/ionomycin induces expression in DO11.10 Th1 and
Th2 cells. EL-4 T cells also show significant expression in re-
sponse to PMA/ionomycin activation. Cross-linkage of the high
affinity Fce receptor or activation with calcium ionophore induces
NF-ATc.a mRNA in BMMC and CFTL15 mast cells (Fig. 5, A
and B). NF-ATc.a mRNA is only weakly expressed in P815 and
ABFTL3 transformed mast cell lines.
In contrast, the expression of significant NF-ATc.b mRNA ap-
pears to be limited to normal mast cells; mast cells express low
basal levels of steady state mRNA, which are significantly in-
creased by stimulation with ionomycin or through IgE receptor
NF-ATc ISOFORMS IN MAST CELLS AND T CELLS
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FIGURE 4. Selective expression of NF-ATc.a and NF-ATc.b in murine
tissues. Ten-microgram aliquots of total RNA from the indicated tissues
from unimmunized mice were used in RNase protection assays, using
probes derived from the unique sequences of the NF-ATc.a and NF-ATc.b
cDNAs.
cross-linking (Fig. 5, A and B). T cells also express detectable
basal levels of the b isoform mRNA. However, activation through
either the Ag receptor or with PMA/ionomycin has little effect on
the expression of NF-ATc.b in these cells. The transformed mast
cell lines P815 and ABFTL3 as well as L929 and M12 B cells
express very low levels of NF-ATc.b mRNA. Detection of signals
in these cells requires long autoradiograph exposure times. These
results indicate that induction of RNA encoding the two NF-ATc
isoforms is differentially responsive to T and mast cell activation
signals.
Steady state levels of NF-ATc isoforms are differentially
regulated in activated T and mast cells
The apparent differences in NF-ATc inducibility between these
distinct cell types could be explained simply by cell-specific dif-
ferences in the kinetics of activation. To assess this possibility, T
and mast cell lines were stimulated for various times, and total
RNA was isolated for use in RNase protection assays. As shown in
Fig. 6, maximal expression of NF-ATc.a was observed between 1
and 2.5 h, and then rapidly fell to undetectable levels in CFTL15
mast cells. In the T cell lines tested, the maximal expression of the
a isoform message was observed between 2 and 3 h. There were
also notable time-dependent differences in the expression of NF-
ATc.a in the T cell lines; by 6 h poststimulation, little a-specific
RNA was detected in Th1 cells, whereas detectable amounts per-
sisted for at least 24 h after stimulation in Th2 cells and for 48 h
in EL-4 cells.
NF-ATc.b mRNA was significantly increased within 30 min of cell
activation and remained high through 6 h poststimulation in CFTL15
mast cells. The levels of NF-ATc.b mRNA remained unaffected by
activation in all T cells. These data confirm that the apparent differ-
ences in the inducible expression of NF-ATc.b in T and mast cell
lines are not due to differences in the kinetics of NF-ATc mRNA
The Journal of Immunology 2825

FIGURE 5. A and B, NF-ATc isoform expression is regulated by cell type-specific activation signals. Ten-microgram aliquots of RNA from the indicated
cell lines were used in RNase protection assays as described in Fig. 4. CFTL15 and BMMC mast cell lines were stimulated with ionomycin or IgE/DNP
for 3 h before RNA harvest. IgE 3 2 indicates cultures that were preincubated for 2 days with IgE before subsequent IgE/DNP activation. EL-4 and D011.10

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Th2 T cells were stimulated with PMA and ionomycin for 3 h. Ag-activated D011.10 Th1 and Th2 cells were cultured for 3 h with OVA peptide and splenic
APCs before RNA harvest. Unstimulated cells were cultured under similar conditions but without Ag.

expression poststimulation, but rather to an inability of T cell activa-
tion signals to up-regulate NF-ATc.b expression.
Cell type-specific differences in mRNA stability exist
To investigate the contribution of differences in mRNA stability to the
cell-specific expression patterns we observed, CFTL15 mast cells and
EL-4 T cells were stimulated for 2 h before treatment with actino-
mycin D to block ongoing transcription. Total mRNA was then iso-
lated at 0.5-h intervals, and steady state levels were assessed by
RNase protection. As shown in Fig. 7A, both NF-ATc.a and -b
mRNA persisted in CFTL15 mast cells at peak levels for at least 2 h
after actinomycin D treatment. In fact, NF-ATc.a mRNA persisted
much longer in actinomycin D-treated cells (Fig. 7B), suggesting that
an mRNA-destabilizing product is transcribed concomitantly with
NF-ATc in mast cells. NF-ATc.a mRNA expression in EL-4 cells is
much more labile. Within 30 min of blocking transcription, levels of
steady state mRNA are reduced by more than half. These data indicate
that the persistence of NF-ATc.a expression in T cells is dependent in
part on ongoing transcription.
Discussion
In this report we describe the cloning and characterization of mu-
rine NF-ATc. Two variations of cDNA clones that are homologues

FIGURE 6. Kinetics of NF-ATc.a

and -b expression in response to acti-
vation signals. Cells were stimulated
with ionomycin (CFTL15 cells), iono-
mycin and PMA (EL4 T cells), or OVA
peptide and splenic APC (DO11.10
Th1 and Th2 cells). The last lanes in
the DO11.10 panels (p) represent RNA
after PMA/ionomycin stimulation for
3 h. RNA was isolated at the indicated
times poststimulation and was used in
RNase protection assays with a and b
isoform-specific probes as described in
Fig. 4. Relative mRNA expression was
quantitated by densitometry using the
National Institutes of Health Image pro-
gram. The mRNA levels in activated
cells are expressed relative to unstimu-
lated control values.
2826 NF-ATc ISOFORMS IN MAST CELLS AND T CELLS

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FIGURE 7. A, The mRNA stability of NF-ATc isoforms. CFTL15 mast cells and EL-4 T cells were stimulated as described above. At 2 h poststimu-
lation, actinomycin D (Act D) was added to the cultures. Total RNA was harvested at 0.5-h intervals, and NF-ATc.a and b expression was assessed by
RNase protection assays. Relative expression levels were quantitated by densitometry as described in Fig. 6 and are presented as a percentage of the mRNA
observed at 2 h poststimulation. B, Comparison of NF-ATc.a mRNA persistence in untreated (M) and actinomycin D-treated mast cells (added at 2 h
poststimulation; F). Relative mRNA levels from actinomycin D-treated CFTL15 mast cells (Fig. 7) and untreated cells (Fig. 6) were determined by
densitometry as described above. Values at each time point are expressed as a percentage of the maximum inducible level observed at 2 h poststimulation,
the time of addition of actinomycin D.

of the previously described human NF-ATc.a and NF-ATc.b iso-
forms were isolated from a murine mast cell library (13, 26). Like
the human isoforms, the murine cDNAs are distinguished by iso-
form-specific sequences at the 59 end of the coding region. These
sequences are highly related to the human sequences. Murine NF-
ATc.a and NF-ATc.b cDNAs are otherwise identical and do not
contain the isoform-specific differences in sequences 39 of the RSD
that characterize the human sequences. In fact, the murine se-
quences located 39 of the RSD are unique and share no homology
at the DNA or protein level with human NF-ATc.a and -b. The
murine NF-ATc.a DNA sequence reported here is similar, but not
identical, with cDNAs derived from activated T cells reported by
Pan et al. (36).
In addition to the variants described in this report, it is likely that
other NF-ATc isoforms exist. Although RNase protection assays
demonstrate that brain and liver do not express appreciable
amounts of either form, a protected band corresponding to the
sequences encoding the common region was observed (Fig. 4).
Similarly, several smaller mRNAs ranging in size from about 4.0
to ,2.0 kb were easily detected in P815 cells by Northern blot
analysis (Fig. 3B) despite the lack of an a- or b-specific signal in
these cells (Fig. 5, A and B). A subset of these small mRNA spe-
cies was also observed in normal mast cells, suggesting that they
are not unique to transformed mast cells. Western blot data like-
wise provide evidence for the existence of multiple protein iso-
forms of NF-ATc. We previously described a single anti-NF-ATc-
reactive species of approximately 56 kDa in unstimulated mast
cells and two additional proteins between 120 and 160 kDa whose
expression is activation dependent (2). In studies by Lyach et al.,
at least three proteins of distinct m.w. were observed in activated
T cells using NF-ATc-specific antisera (38). Whether these are
generated by alternative splicing, the use of alternative promoters,
or the selective utilization of translation start sites remains to be
determined.
Why do multiple NF-ATc isoforms exist? We speculate that the
unique regions of the NF-ATc isoforms confer distinct functions to
these proteins. Structure-function studies have demonstrated that
the N-terminal domain of NF-ATp (aa 1– 415), human NF-ATc.a
(aa 1– 418), and the N- and C-terminal domains of NF-ATx func-
tion as trans-activation domains (39 – 41). It is possible that unique
domains act to attract unique coactivators. In support of this theory
is the finding that p300/CREB binding protein can be recruited by
NF-ATp (39). In addition, we showed that AP-1 associates exclu-
sively with the IL-4 promoter NF-AT complex in T cells but not in
mast cells despite equivalent expression of these proteins in both
cell types (2). Proteins such as c-Maf, NF-AT-interacting protein-
45, and GATA-3, which have been described as essential cofactors
for IL-4 gene expression in T cells, are also candidates for a role
in cell type-specific coactivation (42– 44). The existence of mul-
tiple isoforms that contain unique functional domains expands the
potential repertoire of coactivators that can cooperate with NF-
ATc to achieve selective gene expression in a given cell type.
Previous studies demonstrate that the regulation of NF-AT ac-
tivity occurs at several levels (for a review, see Ref. 9). All family
members appear to undergo activation-dependent trans-location
from the cytoplasm to the nucleus. These events require the cal-
cium-dependent phosphatase, calcineurin, which acts on cytoplas-
mic NF-AT through direct protein-protein interactions and allows
the shuttling of NF-AT to the nucleus. A nuclear kinase, glycogen
The Journal of Immunology
synthase kinase-3, regulates the export of NF-AT back to the cy-
toplasm (19). Tissue-specific expression patterns of NF-AT family
members and their isoforms also regulate the range of NF-AT-
responsive genes that can be influenced in any given cell type (9).
NF-ATp, NF-AT3, and NF-AT4/x are constitutively expressed in
target tissues and are not subject to control by cell activation sig-
nals. In contrast, NF-ATc mRNA and protein expression are de-
pendent on stimulation in human T cells (12, 13, 15, 16, 38).
The results presented here demonstrate that additional levels of
NF-AT regulation exist. In murine T cells and mast cells, activa-
tion through the TCR or high affinity FceR is absolutely required
for the expression of NF-ATc.a. In contrast, although the b iso-
form is expressed at low constitutive levels in most of the cell lines
we analyzed, only the mast cells demonstrate a significant increase
in steady state mRNA levels upon stimulation. The clear-cut dif-
ferences in expressions of the a and b isoform mRNAs in response
to activation signals support the hypothesis that distinct promoters
responsive to T cell and/or mast cell activation signals contribute
to the selective expression of these factors. We also provide evi-
dence that mechanisms exist to differentially regulate mRNA half-
life. Blocking transcription with actinomycin D prolongs the ex-
pression of NF-ATc.a, but not -b, in mast cells, indicating that an
active destabilizing agent functions to down-regulate steady state
mRNA levels.
The analysis of cytokine responses in NF-ATc-deficient mice
demonstrates that this factor is responsible for IL-4 production in
activated T cells (10, 11). Our data support the idea that NF-
ATc.a, but not -b, is involved in IL-4 transcription in Th2 cells.
This conclusion is based on the observation that NF-ATc.b is only
minimally expressed in T cells and that the kinetics of NF-ATc.a
closely parallel the expression of IL-4 in these cells (data not
shown). Furthermore, cloning of NF-ATc from both human and
murine T cells resulted in the isolation of cDNAs corresponding
only to NF-ATc.a, indicating that this is the predominant NF-AT
isoform expressed in this cell lineage (13, 36).
Because the mast cell population was not affected by NF-ATc
gene targeting in NF-ATc2/2 mice, its in vivo role in mast cell
IL-4 gene transcription has not been addressed. The current studies
were initiated to identify the NF-AT factor(s) associated with the
IL-4 promoter in mast cells. However, the identity of this factor is
still elusive. DNA affinity purification experiments demonstrate
that the major protein associated with the IL-4 NF-AT site between
288 and 260 is 41 kDa, a size not observed in analysis of anti-
NF-ATc-reactive proteins by Western blot. Furthermore, this pro-
tein was reactive with NF-ATp antisera, although the specificity/
cross-reactivity of both NF-ATp and NF-ATc antisera have not
been rigorously tested. Given the prevalence of both isoforms in
mast cells as well as activation kinetics that correlate with IL-4
expression, it is likely that NF-ATc plays a role. We are currently
exploring the possibility that the shorter NF-ATc mRNAs encode
this smaller protein associated with the IL-4 promoter in mast
cells. A better understanding of the NF-ATc chromosomal gene
will allow the targeting of isoform-specific sequences to assess
their roles in vivo.
Acknowledgments
We thank Tammy Nachman, who provided invaluable technical assis-
tance with the experiments involving RNA isolation and Northern blot
analysis and the maintenance of Ag-specific T cell lines. We also ap-
preciate the assistance of Dr. Richard Lopez, who performed flow cy-
tometric analyses to analyze the BMMC phenotype, Don Drake, who
provided murine tissue samples for RNase protection analysis, Scott
Sammons for help with the sequence analysis, and John Hural for com-
puter graphics assistance.
2827
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