(Received for publication, May 3, 1995; and in revised form, June 13, 1995)
From the
Signals transduced by the T cell antigen receptor (TCR) regulate developmental transitions in the thymus and also mediate the immunologic activation of mature, peripheral T cells. In both cases TCR stimulation leads to the assembly of the NFAT transcription complex as a result of the calcium-dependent nuclear translocation of cytosolic subunits, NFATc, and the Ras/protein kinase C-dependent induction of a nuclear subunit, NFATn. To further understand the diverse roles of antigen receptor signaling throughout T cell development, we have identified a new NFATc family member, NFATc3, that is expressed at highest levels in the thymus. NFATc3 is the product of a gene on murine chromosome 8 that is not linked to the other NFATc genes. NFATc3, like other NFATc family members, contains a conserved rel similarity domain, and also defines a region conserved among NFATc family members, the SP repeat region, characterized by the repeated motif SPxxSPxxSPrxsxx(D/E)(D/E)swl. NFATc3 activates NFAT site-dependent transcription when overexpressed, yet exhibits a pattern of DNA site specificity distinct from other NFATc proteins. Additionally, thymic NFATc3 undergoes modifications in response to agents that mimic T cell receptor signaling, including a decrease in apparent molecular mass upon elevation of intracellular calcium that is inhibited by the immunosuppressant FK506. Given the preferential expression of NFATc3 in the thymus, NFATc family members may regulate distinct subsets of genes during T cell development.
The antigen receptor of T lymphocytes subserves diverse
functions during development. In the thymus, signals from the antigen
receptor rescue from death those cells that have low avidity receptors
for self-antigens bound to major histocompatability complex molecules
(positive selection), whereas high avidity self-antigens induce
programmed cell death (negative selection)(1) . In mature,
peripheral T cells, interaction with foreign antigen leads to
immunologic activation. In each case characteristic sets of genes are
activated or repressed by signals emanating from the antigen receptor.
For example, antigen receptor-induced repression of the RAG-1 and RAG-2
genes (2) and activation of the CD69 gene (3) are
hallmarks of thymic selection. In mature T cells, antigen
receptor-induced expression of growth factor genes such as IL-2 ()and genes that encode cell-cell interaction molecules,
such as the CD40 ligand, are essential for immunologic function and
proliferation. How these diverse cell fates and functions are initiated
by the T cell antigen receptor (TCR) is not understood, but likely
involve the use of distinct antigen receptor response elements to
activate genes essential for specific developmental transitions.
T
cell antigen receptor response elements (ARREs) were initially
described in the IL-2 gene of mature T cells(4) . The protein
complex that binds to one of these elements, designated nuclear factor
of activated T cells (NFAT; (5) ), appears to integrate Ras-
and calcium-dependent signals initiated by the antigen receptor through
the assembly of cytosolic (NFATc) and nuclear (NFATn) components. NFATc
is present in the cytosol of resting lymphocytes in a transcriptionally
active form and translocates to the nucleus within 5 min of T cell
activation(6) . This translocation event, as well as NFAT
complex formation and NFAT site-dependent transcription, is calcium-
and calcineurin-dependent and completely blocked by the
immunosuppressive drugs FK506 and cyclosporin
A(6, 7, 8, 9) . NFATn is synthesized
within 20 min of T cell activation by a Ras/protein kinase C-dependent
pathway and can be replaced by high levels of
AP1(6, 10, 11) . In addition to the NFAT site
within the IL-2 gene promoter, putative NFAT DNA binding sites have
been identified in the promoters of several genes that are
transcriptionally induced upon antigen receptor activation in a
CsA/FK506-sensitive manner, including IL-3/GM-CSF(12) ,
IL-4(13, 14, 15) , TNF(16) ,
CD40L(17, 18) , and granzyme B(19) . Thus,
NFAT represents a multicomponent transcription factor complex that
integrates signals transduced by the TCR through the use of
constituents that are the targets of distinct signaling pathways.
In addition to its role in the transcriptional induction of cytokine genes in mature T lymphocytes, NFAT may also be involved in T cell ontogeny in the thymus. Although ARREs have not been defined for TCR signaling in the thymus, the ability of thymocytes to induce NFAT DNA binding activity to the IL-2 ARRE appears to be developmentally regulated. This conclusion is based upon results demonstrating that in short term thymocyte cultures, NFAT is inducible in CD4-CD8- cells, noninducible in CD4+CD8+ thymocytes, and inducible in the single-positive populations(20, 21) . A role for NFAT in thymic maturation is also suggested by the observations that cyclosporin A or FK506, which inhibits calcineurin and completely blocks transcription directed by the NFAT site(6, 8, 9) , blocks the development of the CD4+CD8- and CD4-CD8+ subpopulations of thymocytes(22, 23) . Thus, although a variety of studies suggest a role for the NFAT transcription factor complex in regulating development in the thymus, there is no clear understanding of the specific mechanism by which this occurs. This is due, in part, to a lack of understanding of the molecular characteristics of the NFAT complex in developing thymocytes and the lack of definition of ARREs for intrathymic signaling by the T lymphocyte receptor.
The
purification and molecular cloning of the preexisting or cytosolic
component of NFAT resulted in the identification of two distinct genes,
NFATp (24) and NFATc(25) , both of which encode
proteins that are capable of binding to NFAT DNA sites and are present
in the NFAT gel shift complex. These proteins share a conserved region
of limited similarity (20% amino acid identity) to the rel
homology domain of dorsal/rel/NF
B transcription factors, and
therefore appear to define a distinct family or subfamily of
transcription factors(25) . Whether this family will also be
characterized by other features shared by NFATc and NFATp, such as
their function as targets of calcium-dependent signal transduction, is
uncertain. (
)To further investigate the role of the NFATc
family of transcription factors in signal transduction and T cell
development, a cDNA library derived from thymocytes induced to undergo
negative selection (26) was screened for additional NFATc
family members. An additional cDNA was isolated that encodes a novel
NFATc family member, NFATc3. Characterization of this cDNA indicates
that NFATc3 RNA is preferentially expressed in thymus, spleen, and
lymph node and that NFATc3 exhibits DNA binding specificity distinct
from that of other NFATc family members. Furthermore, thymic NFATc3
undergoes alterations in apparent molecular mass in response to
increases in intracellular calcium that are reversed by the
immunosuppressant FK506, suggesting that NFATc3 is regulated by
calcineurin in a manner similar to the other NFATc family members.
These results extend our understanding of the molecular characteristics
of NFATc proteins and should permit a more rigorous analysis of the
role of the NFATc family of transcription factors in thymic development
and signal transduction.
Figure 2: NFATc family members are defined by the rel similarity domain and a region containing three SP repeat motifs. A, schematic comparison of NFATc1, NFATc2, and NFATc3. The rel similarity domain is shadedblack, and the SP repeat motifs are shaded gray. The schematic representations of the NFATc proteins are drawn to scale, relative to the length of the primary amino acid sequence. Thus, the relative differences in distances between different regions are accurate. The region of NFATc3 used to generate antisera is indicated by a thick line, and the region corresponding to the location of the ribonuclease protection probe is indicated by a thin line. The designations ``h'' and ``m'' refer to the human and murine cDNAs. B, sequence comparison of the SP repeat region and rel similarity domain of NFATc family members. The amino acid sequences of the NFATc proteins were aligned using the PileUp program and displayed using the Pretty program (Genetics Computer Group, Inc.). A dash indicates identity with the human NFATc1 sequence, and periods indicate inserted gaps. C, SP repeat motif consensus sequence. Uppercase letters represent residues conserved in each of the nine SP repeat motifs (three motifs present in each of the three NFATc proteins); lowercase letters represent residues conserved in at least five of the nine SP repeat motifs.
Figure 1: Nucleotide and predicted amino acid sequence of murine NFATc3. The SP repeat motifs are underlined, and the rel similarity domain is represented in italics.
The region of
amino acid sequence similarity encompassed the RSD (Fig. 2, A and B), over which NFATc3 is 69% identical to
NFATc1 and 65% identical to NFATc2 (NFATc1 is 70% identical to NFATc2
in this region). All three sequences exhibit greater similarity over a
170-amino acid region within the RSD (79-89% identical),
consistent with this more conserved portion of the RSD functioning as
the minimal DNA binding domain (36) . ()Although the
COOH-terminal portion of the RSD is less conserved, a putative nuclear
localization signal at residue 676 stands out as a stretch of amino
acids that are conserved, suggesting that this may in fact represent a
functional sequence. Furthermore, the location of the RSD within NFATc
proteins differs. The RSD is located at the carboxyl terminus of
NFATc1, whereas it is more centrally located within the linear amino
acid sequence of NFATc2 and NFATc3. This is in contrast to proteins
containing the rel homology domain, which is uniformly located at or
near to the amino terminus of the protein(37) .
In addition
to the RSD, NFATc3 also exhibits sequence similarity to NFATc1 and
NFATc2 within a region extending 300 amino acids toward the
NH
terminus (Fig. 2, A and B). The
murine NFATc2 cDNA clone, being a partial cDNA, encompasses only
approximately 220 amino acids of this domain. This region of similarity
is 35% identical between NFATc3 and NFATc1, but is additionally unique
in that it contains a conserved motif characterized by a serine/proline
repeat consensus sequence
SPxxSPxxSPrxsxt[D/E][D/E]swl,
which is itself repeated three times (Fig. 2, A and C). Interestingly, the location of the three SP repeat motifs
relative to each other and to the RSD is conserved among NFATc
proteins, suggesting a functional relationship between these domains (Fig. 2A). The third repeat (SP repeat C) contains an
additional, conserved four amino acid NH
-terminal extension
consisting of another SPxx repeat, where x is
histidine in NFATc2 and NFATc3 (Fig. 2C). Additionally, the SP
repeat motifs in NFATc2 lack the conserved tryptophan that is present
near the COOH terminus of the motif in both NFATc1 and NFATc3. No other
proteins with this motif have been identified in searches of sequence
data banks. Although the function of this domain remains unknown, the
fact that it is conserved among NFATc family members suggests that it
may have a function that is characteristic of this family.
Finally, the carboxyl-terminal portion of NFATc3 extending past the rel similarity region is relatively rich in glutamine, which represents 10% of the COOH-terminal 384 amino acids. This feature is also seen in the COOH-terminal portion of NFATc2. In contrast, the NFATc1 protein does not extend past the rel similarity region and contains no glutamine-rich regions. Additionally, NFATc3 is rich in serine and threonine (20%), contains two potential glycosylation sites (N-linked), and also contains a consensus sequence for a single tyrosine phosphorylation site (amino acids 133-140).
Figure 3: Chromosomal mapping of Nfatc3 gene by fluorescence in situ hybridization. Left: arrows indicate specific hybridization signals is on both chromosome 8 homologs at band D. Right: G-banding ideogram of mouse chromosome 8 (42) with localization of Nfatc3. Comparison of the cytological map of human chromosome 16, bands q13-q24, and the linkage map of mouse chromosome 8, that corresponds to bands C-E, depicts several genes mapped to the conserved syntenic regions(43) . It is highly likely, therefore, that the human NFATc3 gene is located in that region as well.
Figure 4:
NFATc3
is preferentially expressed in lymphoid tissues. Ribonuclease
protection assays were performed using both NFATc3 and -actin RNA
probes in the same protection assay. A, each sample represents
protected RNA from 10 µg of total cellular RNA isolated from the
indicated tissues. B, the indicated cell lines were either
unstimulated(-) or stimulated with ionomycin (1 µM)
and PMA (20 ng/ml) for 3 h. PD31 represents a pre-B cell line,
BalI7 and 38C13 represent mature B cell lines, and MOPC represents a plasma cell line. The separated lanes are all from
the same gel and exposure.
Figure 5:
NFATc family members bind DNA with
distinct specificities. Nuclear extracts used in gel shift assays were
obtained from COS cells transfected with the indicated NFATc expression
construct or with the plasmid vector as a negative control. A,
titration of the nonspecific competitor poly(dI-dC) reveals that
NFATc3, unlike NFATc1 or NFATc2, binds to the IL-2 NFAT site only at
low poly(dI-dC) concentrations. Concentrations of poly(dI-dC) were 1,
0.5, and 0.25 µg/reaction (left-to-right), as shown schematically. B, binding of NFATc family members to the murine distal IL-2
NFAT site is inhibited by an excess of unlabeled NFAT or AP1 site DNA.
Binding reactions were performed as described, using 0.25 µg of
poly(dI-dC)/reaction. Competitor DNA was added at 50-fold excess over
probe DNA. C, identical extracts were used in gel shift assays
with either the murine IL-2 NFAT site, the murine IL-4 NFAT site, the
murine TNF
3 NFAT site, or the IL-3/GM-CSF NFAT site, GM550.
All reactions were performed using the same conditions, which included
0.25 µg of poly(dI-dC)/reaction.
To further investigate the DNA site specificity of NFATc3, the
binding of NFATc proteins to oligonucleotide probes corresponding to
putative NFAT sites within other cytokine promoters was examined in gel
shift assays under identical conditions, utilizing the same set of
nuclear extracts from COS cells transfected with NFATc expression
constructs. These extracts were examined under conditions of low
poly(dI-dC) to permit detection of weak binding (perhaps of no
physiologic significance). This approach was taken to reveal potential
differences in DNA binding specificity of the NFATc-transfected
extracts based on differences in the relative binding characteristics
of the same set of extracts with different DNA binding sites. In
addition to the distal murine IL-2 NFAT site, the sites used in binding
assays include the NFAT site within the murine IL-4
promoter(35) , the 3 site within the TNF
promoter(16) , and the GM550 site within the IL-3/GM-CSF
intergenic promoter(12) . In contrast to the IL-2 promoter NFAT
site, which bound all three NFATc proteins, the IL-4 promoter NFAT site
preferentially bound NFATc2 and NFATc3, whereas both the TNF
and
IL-3/GM-CSF sites preferentially bound NFATc2, but not NFATc1 or NFATc3 (Fig. 5C). Thus, the three NFATc family members exhibit
differences in binding specificity, since identical extracts and
binding conditions were used to assay binding to the various DNA sites.
Figure 6: NFATc3 activates NFAT DNA site-dependent transcription. Transiently transfected Jurkat cells were stimulated in triplicate with 1 µM ionomycin plus 20 ng/ml PMA 24 h after transfection. Culture supernatants were collected approximately 20 h after stimulation and assayed for secreted alkaline phosphatase activity. Results are expressed in arbitrary units of fluorescence intensity, reflecting relative alkaline phosphatase activity. All values are expressed relative to the alkaline phosphatase activity present in a control sample transfected with vector DNA alone, which was set to zero units of fluorescence intensity. Each point represents the mean of triplicate samples; error bars represent standard error of the mean. The data shown are representative of at least three independent experiments.
Figure 8: NFATc3 is modified in response to agents that increase intracellular calcium and activate protein kinase C. A, replicate aliquots of extracts from COS cells transfected with the indicated NFATc expression construct were Western blotted with either the monoclonal antibody 7A6 (anti-NFATc1), the monoclonal antibody 5H8 (anti-NFATc2), or a rabbit anti-NFATc3 polyclonal antisera. B, freshly isolated unfractionated thymocytes were stimulated for 15 min with ionomycin (1 µM), PMA (20 ng/ml), and/or FK506 (2 ng/ml) in the indicated combinations. Immunoprecipitation and subsequent Western blotting were performed with the rabbit NFATc3 antiserum. NFATc3` refers to the slower migrating form of the protein that could be phosphorylated, whereas NFATc3 is the more rapidly migrating form. Both forms give a characteristic blurry band that is not a loading or running artifact since Ig (immunoglobulin) runs normally on the same gel.
Figure 7: NFATc3 does not substantially contribute to the NFAT gel shift complex on the IL-2 ARRE. Nuclear extracts prepared from unfractionated thymus, spleen, and lymph node cell suspensions stimulated with ionomycin and PMA were used in gel shift assays. The contribution of NFATc1 and NFATc2 to the NFAT gel shift complex was determined by supershifts using the NFATc1 monoclonal antibody 7A6 and the NFATc2 monoclonal antibody 5H8.
The nature of the post-translational modification was investigated by using the NFATc3 antiserum to immunoprecipitate NFATc3 from whole cell thymic extracts, followed by Western blotting. The endogenous NFATc3 is a protein of approximately 190 kDa relative molecular mass, similar in size to the protein derived from the transfected cDNA (Fig. 8B). Thymus cells stimulated for 15 min with agents that increase intracellular calcium (ionomycin) or activate protein kinase C (PMA) demonstrate that NFATc3 undergoes regulated post-translational modification between a rapidly migrating form and a more slowly migrating form, NFATc3`, which could represent a phosphorylated form of the protein (Fig. 8B). Ionomycin treatment resulted in a slight reduction in the relative molecular mass of NFATc3 as compared with nonstimulated cells (lane 3 versus 1). The addition of the immunosuppressant FK506 reversed this change (lane 4 versus 3), causing in increase in the relative molecular mass. Phorbol ester also induced an apparent increase in mass relative to NFATc3 from nonstimulated cells (lane 5), which was partially reversed by the addition of ionomycin (lane 7). Finally, the addition of FK506 to cells stimulated with PMA plus ionomycin resulted in a reversal of the ionomycin-dependent changes in NFATc3 mobility (lane 7 versus 8). Thus, NFATc3 is subject to modification by agents that increase intracellular calcium or activate protein kinase C. Furthermore, the calcium-dependent modifications are sensitive to inhibition by the immunosuppressant FK506.
The NFATc family of transcription factors was originally defined by their binding to ARREs in the IL-2 gene(4) . This activity could be demonstrated in both T and B lymphocytes(38) , and the diffuse nature of the band on native gels suggested that the complex might be heterogeneous. Purification of the proteins that bound to the IL-2 ARRE from calf thymus led to the realization that the cytosolic component of NFAT consisted of two proteins, NFATc1 and NFATc2, that are 70% identical within the 280 amino acid DNA binding region, designated the RSD. Since genes activated as a result of antigen receptor signaling in the thymus are potentially different than those activated in mature T lymphocytes, we initiated a search for NFAT-related cDNAs in a cDNA library prepared from thymus induced to undergo negative selection in vivo (i.e. programmed cell death). A related cDNA clone was identified, which was designated NFATc3 in accord with the genome mapping nomenclature system. NFATc3 sequences were not identified in the proteins purified from calf thymus using the IL-2 ARRE as an affinity reagent(25) , implying that it does not interact with the IL-2 ARRE at as high an affinity as NFATc1 and NFATc2.
The rel
similarity domain of NFATc3 is 69% and 65% identical to that of NFATc1
and NFATc2, respectively. Comparison of the RSD of NFATc proteins (Fig. 2B) shows that the NH-terminal
portion is more highly conserved than the COOH-terminal portion,
exhibiting 79-89% amino acid identity within a 175-amino acid
region, which represents the minimal DNA binding
domain(36) .
Within this region there are three
stretches of 10-25 amino acids of near identity, separated by
areas in which amino acid differences between NFATc proteins appear to
be clustered. These subdomains of conserved and variable regions,
therefore, likely reflect those regions that are involved in
maintaining the structural integrity of the DNA binding domain and
those regions that might be involved in giving rise to the observed
differences in DNA binding specificity.
The identification of a third member of the NFATc family permits further definition of another unique region of protein sequence similarity, designated the SP repeat region, which spans approximately 110 amino acids and is composed of a consensus SP repeat motif repeated three times (Fig. 3C). The SP repeat motifs within this region are not only conserved in their spacing relative to each other, but also relative to the RSD, suggesting that it is the combination of repeated motifs rather than a single motif that may subserve a function. While the function of the SP repeat region remains unknown, the observations that each of the NFATc family proteins undergo a calcium-dependent reduction in relative molecular mass that is reversed by immunosuppressive agents that inhibit calcineurin suggest that this region may be the site of regulatory, proline-directed kinase/phosphatase activity. The presence of three SP repeat motifs within the SP repeat region suggests that a requirement for multiple phosphorylation/dephosphorylation events may be a hallmark of the calcium-regulated function of NFATc proteins. Such a requirement for multiple modifications could function to set a threshold for NFATc activation such that low basal levels of a regulatory activity, i.e. calcineurin, that might exist under conditions of variable basal intracellular calcium concentrations, would be insufficient to activate NFATc. Such a threshold mechanism for the activation of proteins that undergo multiple phosphorylations has been proposed for the ternary complex factor proteins Elk-1 and SAP-1, which interact with serum response factor to regulate transcription from serum response elements(39) . Alternatively, the multiplicity of SP repeat motifs within this region may be required for specific protein interactions.
Analysis of the DNA binding specificity of NFATc family members by gel mobility shift assay demonstrates distinct differences in the binding specificity of the three NFATc proteins for different ``NFAT'' sites or antigen receptor response elements in T cell activation genes. While the observed differences in DNA binding specificity may reflect differences in the affinity of binding of each of the NFATc family members with a specific DNA site, it remains possible that the observed differences in DNA binding specificity may reflect differences in the specificity of interaction with other DNA-binding proteins that form the NFAT gel shift complex, i.e. different AP1-related proteins that may constitute the nuclear NFAT component. Our present data indicate that NFATc3 may not make a substantial contribution to the NFAT DNA binding complex at the IL-2 ARRE in extracts of cells from the spleen, thymus, and lymph node. Hence, NFATc3 may bind to the regulatory regions of as yet unidentified genes in response to T cell antigen receptor signaling. Given the prominent affects of the immunosuppresive agents FK506 and cyclosporin A in T cell development, such genes may play a central role in determining developmental pathways in the thymus.
The changes in the relative molecular mass of NFATc3 upon stimulation with agents that either increase intracellular calcium concentration or activate protein kinase C indicates that NFATc3, like NFATc1(25) , is the target of two distinct signaling pathways. The reversal by FK506 of changes induced by increasing intracellular calcium suggests that NFATc3 may be directly or indirectly regulated the calcium/calmodulin-dependent phosphatase calcineurin, as it is calcineurin that is the target of the complex between FK506 and FKBP12(40) . The calcium-dependent decrease in the apparent relative mass of NFATc3 is consistent with a dephosphorylation event, possibly mediated by calcineurin (11) or by a calcineurin-regulated phosphatase such as phosphatase 1(41) . The site of this phosphorylation/dephosphorylation is likely the SP repeat region, as this is the only region of similarity among NFATc proteins other than the DNA binding domain.
The Nfatc3 gene has been mapped by somatic hybrid cell lines and fluorescence in situ hybridization to mouse chromosome 8 band D within a region of conserved synteny with the long arm of human chromosome 16. We have previously mapped Nfatc1 to mouse chromosome 18 and Nfatc2 to mouse chromosome 2 into regions of known homology with human chromosomes 18 and 20, respectively(29) . These results indicate that the genes encoding the NFATc family are not clustered in the human genome. Nfatc3 maps in the vicinity of a mutant locus called Nan (neonatal anemia), which is characterized by lethality at day 10-11 gestation in homozygous embryos due to lack of hematopoiesis. As described above, although NFATc3 appears to be expressed predominantly in lymphoid tissues, a more detailed analysis of NFATc3 expression in hematopoietic tissues has not been performed. Thus, a role for NFATc3 in hematopoiesis and in the development of the Nan phenotype remains a tenable hypothesis.
The finding that NFATc3 is expressed at high levels in the thymus and is capable of activating transcription, yet does not appear to bind significantly to NFAT-dependent ARREs, suggests that it may play a role in regulating the transcription of genes with ARREs distinct from those that have been identified. The hypothesis that such genes regulated by NFATc3 are involved in T cell development in the thymus is supported by the finding that endogenous thymic NFATc3 undergoes post-translational modifications in response to the same intracellular signals that regulate developmentally important events in thymocyte maturation. Definition of the physiologically relevant binding sequence for NFATc3 and thymus-specific nuclear partners of NFATc3 will be essential to understand its role in the complex pathways directing lymphocyte development.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank].