Nuclear Factor of Activated T Cells 2 Transactivation in Mast Cells

A NOVEL ISOFORM-SPECIFIC TRANSACTIVATION DOMAIN CONFERS UNIQUE Fc{epsilon}RI RESPONSIVENESS*

M. Benjamin Hock {ddagger} and Melissa A. Brown {ddagger} § 

From the §Department of Pathology and {ddagger}Graduate Program in Genetics and Molecular Biology, Emory University School of Medicine, Atlanta, Georgia 30322

Received for publication, January 30, 2003 , and in revised form, May 7, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Murine nuclear factor of activated T cells (NFAT)2.{alpha}/{beta} differ by 42 and 28 unique amino-terminal amino acids and are differentially expressed. Both isoforms share conserved domains that regulate DNA-binding and subcellular localization. A genetic "one-hybrid" assay was used to define two distinct transactivation (TA) domains: in addition to a conserved TAD present in both isoforms, a second, novel TAD exists within the {beta}-specific amino terminus. Pharmacologic inhibitors Gö6976 and rottlerin demonstrate that both conventional and novel protein kinase C (PKC) family members regulate endogenous mast cell NFAT activity, and NFAT2 TA. Overexpression of dominant active PKC{theta} (which has been implicated in immune receptor signaling) induces NFAT2.{alpha}/{beta} TA. Mutations within the smallest PKC{theta}-responsive transactivation domain demonstrate that the PKC{theta} effect is at least partially indirect. Significantly, the {beta}-specific domain confers greater ability to TA in response to treatment with phorbol 12-myristate 13-acetate/ionomycin or lipopolysaccharide, and unique sensitivity to Fc{epsilon}RI signaling. Accordingly, overexpression of NFAT2.{beta} results in significantly greater NFAT- and interleukin-4 reporter activity than NFAT2.{alpha}. These results suggest that whereas NFAT2 isoforms may share redundant DNA-binding preferences, there are specialized functional consequences of their isoform-specific domains.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear factor of activated T cells (NFAT)1 transcription factors regulate critical events in vertebrate cell development and differentiation (for review, see Ref. 1). NFAT was first characterized as an essential component of inducible IL-2 transcription in T cells (2), but is now known to denote a family of transcription factors that include four Ca2+-responsive NFAT family members (NFAT1–4) (1, 3). NFAT family members are often expressed as multiple isoforms, and exhibit cell type-specific expression patterns (4, 5). Although they function in a wide variety of cell types, their role in regulating the activation-dependent expression of many immunologic effector molecules (including: IL-2, IL-4, granulocyte-macrophage colony-stimulating factor, interferon-{gamma}, and CD95L) is best characterized (3). Based largely on studies of activated T cells, it has been established that in addition to restricted patterns of expression, NFAT activity is controlled at several levels: subcellular localization, DNA binding, and transcriptional transactivation.

The motifs that regulate the subcellular localization of NFAT, a latent cytoplasmic factor, are located within a conserved amino-terminal regulatory region, the NFAT-homology region. Upon cellular activation, a sustained Ca2+ flux activates the Ca2+/calmodulin-dependent phosphatase, calcineurin (Cn). Cn directly associates with NFAT molecules through recognition/docking sites, including the SPRIEIT sequence (NFAT1–4) and CnBP-B (NFAT2–4) (6, 7). Activated Cn dephosphorylates a conserved set of serine-proline repeats (SP boxes), which induces NFAT translocation to the nucleus (8). Kinases, including glycogen synthase kinase re-phosphorylate nuclear NFAT and promote its export back to the cytoplasm, thus regulating its access to DNA (9). NFAT molecules associate with DNA as monomers via a highly conserved domain, the Rel-homology domain (related to Rel-family transcription factors NF{kappa}B and Dorsal) (10). The Rel-homology domain consists of ~300 amino acids and binds the consensus sequence: 5'-(A/T)GGAAAA-3'. NFAT activity often depends on association with AP-1 molecules. In these cases, AP-1 and NFAT bind co-operatively at composite DNA elements and activate transcription (11, 12). The requirement for co-operative binding of NFAT and AP-1 family members has been one way to account for the dependence on both PMA (through the activation of a kinase cascade that includes MAP kinases and PKC family members) and ionomycin signals (that activate Cn) for expression of some NFAT-regulated genes (3).

Regions of the NFAT molecule that contribute to transcriptional transactivation have been identified within NFAT1, -2, and -4. NFAT1 transactivation activity is localized to amino acids 1–415 (13), a region that includes the SPRIEIT sequence. Two transactivation domains (TADs) have been identified within the human NFAT2 molecule: TAD-A (amino acids 113–205) and TAD-B (amino acids 690–930 within the carboxyl terminus of NFAT2.C) (4). In NFAT4, a carboxyl-terminal, isoform-specific region (amino acids 1030–1044) also contributes to transcriptional activation (14).

Despite the conservation of DNA-binding and regulatory domains, the phenotypes of NFAT-null mice illustrate that some NFAT family members have specialized functions. For example, deletion of the NFAT1 gene results in dysregulated lymphoproliferation and a Th2-dominant cytokine response (15). In contrast, NFAT2 –/– lymphocytes are defective in proliferation and production of Th2 cytokines (16, 17). Such phenotypes suggest that NFAT2 activity drives Th2 cytokine production and proliferation, and is modulated by the repressive action of NFAT1. NFAT2 also has an essential role in cardiac valve formation (18, 19). These studies illustrate the conserved and essential role of NFAT gene products in development and expression of immunologic effector molecules, but also suggest functional specialization among family members. The mechanisms that underlie the ability of NFAT family members to regulate unique programs of gene expression remain undefined.

The existence of multiple isoforms of NFAT1, -2, and -4 suggests that there is even more complexity in the regulation and function of NFAT family members. Strategies used to target NFAT genes for deletion result in the loss of expression of all isoforms derived from the targeted locus. Thus, the contribution of individual NFAT isoforms to unique patterns of gene expression has not yet been evaluated in vivo. We previously cloned two isoforms of NFAT2 from a murine mast cell cDNA library that differ only at their amino termini (5). NFAT2.{alpha} and NFAT2.{beta} contain 42 and 28 unique amino acids, respectively. These two murine isoforms do not contain the alternative carboxyl termini that are described for human isoforms: NFAT2.A/B/C (20). However, the amino termini are conserved: hNFAT2.A/B corresponds to mNFAT2.{alpha}, whereas hNFAT2.C has the mNFAT2.{beta} amino terminus. Murine NFAT2.{alpha} is expressed predominantly in the spleen, whereas NFAT2.{beta} is also expressed in the liver and kidney. In both T and mast cell lines, the expression of NFAT2.{alpha} is inducible, whereas NFAT2.{beta} is constitutively expressed at low levels in T cells, and is up-regulated only in mast cells upon cell activation.

Because the sequences that regulate DNA-binding and nuclear transport are identical in murine NFAT2.{alpha} and NFAT2.{beta}, we hypothesized that the isoform-specific regions confer distinct abilities to transactivate transcription, perhaps allowing for cell- and signal-specific NFAT activities. In this study we used a genetic one-hybrid assay to identify transactivation domains (TADs) within NFAT2.{alpha} and NFAT2.{beta}. Two regions act independently to confer transcriptional activation in response to PMA and ionomycin. Both conventional and novel PKCs regulate this activity. In addition, a novel acidic activation domain (Glu5-Asp19) within the {beta}-specific amino terminus imparts NFAT2.{beta} with greater ability to transactivate in response to a broad range of PMA and ionomycin concentrations. Significantly, it also provides NFAT2.{beta} with the unique ability to respond to Fc{epsilon}RI cross-linking. These data demonstrate that there is functional specialization among NFAT2 isoforms that is regulated at the level of transactivation in response to specific activation signals.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Cultures and Reagents—CFTL-15 mast cells were cultured as previously described (5) except 3.3 ng/ml recombinant murine IL-3 (BIOSOURCE Intl., Camarillo, CA) was substituted for WEHI3B supernatant. MC9 mast cells (21) (obtained from American Type Culture Collection) were cultured in RPMI 1640 supplemented with 15% fetal bovine serum and 30% WEHI3B supernatant. COS-1 cells (American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium (4.5 g/liter glucose and 1.5 g/liter sodium bicarbonate) supplemented with 10% fetal bovine serum (Invitrogen). Cells were stimulated with PMA (Sigma), ionomycin (Calbiochem), or LPS (Sigma). PMA and ionomycin concentrations (20 ng/ml and 1 µg/ml, respectively) were uniform with the exception of the PMA/ionomycin dose-response shown in Fig. 3B, which utilized five 10-fold dilutions from 0.2 µg/ml PMA and 10 µg/ml ionomycin. Fc{epsilon}RI cross-linkage was achieved through IgE/{alpha}-IgE treatment, briefly: CFTL-15 mast cells were preincubated for 2 days in 0.5 µg/ml purified mouse IgE (BD Biosciences); 24 h after transfection, cells were resuspended in 200 µl of complete media with 5.0 µg/ml IgE at 4 °C for 30 min; cells were washed twice in serum-free RPMI to remove unbound IgE, and resuspended in 200 µl of complete media with 5.0 µg/ml rat {alpha}-mouse IgE (Southern Biotech, Birmingham, AL) at 37 °C for 45 min. Cells were transferred to 12-well plates in complete media until harvest (48 h post-transfection). PKC inhibitors Gö6976 and rottlerin (Calbiochem) were dissolved in Me2SO, and added to cells 30 min prior to PMA/ionomycin treatment.



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FIG. 3.
NFAT2.{alpha}/{beta} require both PMA and ionomycin signals for maximal TA and have similar sensitivities to PMA/ionomycin signal strength. A, {alpha}1–429 or {beta}1–415 stimulated as indicated ± 30 min pretreatment with 0.5 µg/ml cyclosporin A. B, {alpha}1–429 and {beta}1–415 transactivation in response to increasing doses of PMA/ionomycin. One-hybrid assay using 0.02 µg of effector construct. Data are representative of three independent experiments.

 

Transfections—5.0 x 106 CFTL-15 cells were electroporated in 0.5 ml of serum-free RPMI at 400 V and 425 µF in 0.4-cm gap cuvettes with a Gene-Pulser II (Bio-Rad). All CFTL-15 shocks contained 50 µg of salmon sperm DNA (Invitrogen). Samples were incubated for 10 min with DNA at room temperature prior to electroporation, and were allowed to recover on ice for 10 min post-electroporation.

One-hybrid Constructs and CAT Assays—PCR-generated fragments of murine NFAT2.{alpha} and NFAT2.{beta} were cloned in-frame into the Gal4 DBD expression construct, pM (Clontech). The isoform-specific regions are represented by {alpha}1–44 and {beta}1–30. Single amino acid mutations were constructed with the QuikChangeTM mutagenesis kit (Stratagene, La Jolla, CA). CFTL-15 assays were performed with 0.001–10 µg of effector construct and 5 µg of G5CAT reporter construct (Clontech). Within an experiment, equal molar quantities of effector constructs were used. PKC{theta} expression constructs were the kind gift of A. Altmon (La Jolla Institute for Allergy and Immunology).

For overexpression analysis, NFAT2.{alpha} and NFAT2.{beta} were cloned into the expression vector pcDNA3 (Invitrogen); 20 µg of the expression constructs were transiently transfected into CFTL-15 cells with 20 µgof either NFAT- or IL-4-CAT reporter constructs. Twenty-four hours post-electroporation, CFTL-15 cells were split into two to three samples and treated with PMA/ionomycin, or IgE/{alpha}-IgE. Forty-eight hours post-electroporation, CAT extracts were harvested by the Tris/EDTA/NaCl (TEN)/Triton X-100 method (22). CAT activity was measured in a scintillation:diffusion assay as previously described (23). All CAT results are representative of at least three independent experiments. The NFAT reporter contains 3 tandem repeats of the IL-2 NFAT site upstream of the IL-2 promoter (–72 to +25) (24). The NFAT sites each contain the composite NFAT:AP-1 "NFAT-responsive element" (Fig. 4A). The NFAT reporter was the kind gift of T. J. Murphy (Emory University). The IL-4 reporter construct contains sequences corresponding to –797 to +5 base pairs of the murine IL-4 promoter and has been previously described (23).



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FIG. 4.
NFAT activity in CFTL-15 mast cells is dependent on both conventional and novel PKCs. A, cartoon depicting NFAT-CAT reporter that contains three tandem NFAT-response elements derived from the IL-2 proximal promoter. B, NFAT reporter activity in mast cells preincubated with carrier (Me2SO), 5 µM Gö6976, or 15 µM rottlerin for 30 min prior to stimulation with either PMA/ionomycin or IgE/{alpha}-IgE (±S.D.). Data are representative of three independent experiments. B, PKC{theta} transcript and protein are detectable in untreated and PMA/ionomycin stimulated CFTL-15 mast cells. C, upper, reverse transcriptase-PCR and B, lower, immunodetection of whole cell lysate with {alpha}-PKC{theta} clone E-7. D, NFAT reporter activity increases in response to dominant active PKC{theta} overexpression. NFAT reporter activity in mast cells ± dominant active PKC{theta} (A148E) expression construct (20 µg) is shown. Data are representative of three independent experiments.

 

Cell Extracts and Immunodetection—Extracts from COS-1 and CFTL-15 cells were prepared by collecting and washing approximately 1.0 x 106 cells in cold phosphate-buffered saline. Cells were resuspended in 200 µl of 1.2x Laemmli sample buffer (25) on ice. Extracts were heated to 95 °C for 5 min, then either sonicated or passaged through a 22-gauge needle to disrupt chromatin. Extracts were then centrifuged at 10,000 x g for 10 min. After gel electrophoresis, proteins were electroblotted to nitrocellulose for 20 min at 15 V in transfer buffer (48 mM Tris, 39 mM glycine, 20% methanol). Blots were blocked in 5% dry milk/TBST (0.05% Tween 20) for 1 h at room temperature. Primary antibodies specific for either Gal4 DBD (clone RK5C1, Santa Cruz Biotechnology, Santa Cruz, CA) or PKC{theta} (clone E-7, Santa Cruz Biotechnology) were added to the same solution for an additional hour. After 3 washes in TBST (5 min each), blots were treated with 1:10,000 dilutions of secondary antibodies conjugated to horseradish peroxidase in 5% dry milk/TBST (0.05% Tween 20) for 1 h at room temperature.

Reverse Transcriptase-PCR Analysis—RNA was harvested from 1.0 x 107 cells with RNA STAT-60 reagent (Tel-Test Inc., Friendswood, TX). 1.0 µg of DNase I-treated RNA was used as a template in a Superscript IITM (Invitrogen) first-strand synthesis reaction. PCR primers to detect PKC{theta} transcripts were: PKC{theta} forward, 5'-CTCGTCAAAGAGTATGTCGAATCA-3' and PKC{theta} reverse, 5'-AATTCATTCAGTCCTTTGTGTCACTCA-3'.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Two Distinct NFAT2 Transactivation Domains Function in Mast Cells—To examine the ability of murine NFAT2 isoforms to transactivate transcription, one-hybrid effector constructs (Fig. 1A) were used. These constructs express the Gal4 DBD fused to discrete regions of NFAT2, but exclude the NFAT Rel-homology domain. The Gal4 DBD (amino acids 1–147) contains a nuclear localization signal that targets one-hybrid proteins to the nucleus. As a result, these molecules localize exclusively to reporter cis-elements. Furthermore, transactivation measured in the assay is not a result of association with AP-1 family members (which make contacts within the NFAT Rel-homology domain). Effector constructs were co-transfected into mast cells with a reporter whose expression is regulated by five Gal4 cis-elements and a minimal E1b promoter. These experiments were performed in the non-transformed, IL-3-dependent mast cell line, CFTL-15. These cells express functional Fc{epsilon}RI, and release a number of mast cell mediators when activated through this receptor including histamine, IL-4, IL-13, tumor necrosis factor {alpha}, and IL-6 (26).2 They also express MMCP-4, a serine protease expressed by mature connective tissue mast cells (27).



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FIG. 1.
Analysis of NFAT2 transactivation in mast cells. A, upper, schematic representation of structural differences between NFAT2.{alpha}/{beta} isoforms and location of common regulatory elements including: SPRIEIT sequence (black box), SP boxes (gray boxes), and the Rel-homology domain. A, lower, structure of NFAT2-Gal4 fusion one-hybrid effector constructs. B, CFTL-15 mast cells co-transfected with 2.5 µg of the indicated effector construct and a Gal4 reporter gene were stimulated with PMA and ionomycin 24 h post-transfection. Data are normalized to transactivation activity of the Gal4 DBD alone (±S.D.) and is representative of three independent experiments. C, {beta}1–30 dose responsive transactivation. The one-hybrid assay was performed with the indicated quantities of isoform-specific effector constructs. Data are representative of three independent experiments. D, demonstration of one-hybrid effector molecule expression. Whole cell lysates from COS-1 cells transfected with the indicated one-hybrid effector constructs were subjected to Western blot analysis. Immunodetection of Gal4 fusion proteins was performed with {alpha}-Gal41–147 clone RK5C1.

 

As shown in Fig. 1B, the "full-length" amino-terminal constructs, {alpha}1–429 and {beta}1–415, as well as 31–415 (which excludes the isoform-specific domains) were active in this assay. {beta}1–30 and 91–415 represent the smallest independent constructs that can transactivate. All active constructs possess both basal and stimulation responsive (20 ng/ml PMA and 1 µg/ml ionomycin) activity. The common region encompassing amino acids 91–415 includes the previously defined human NFAT2 transactivation domain (TAD-A, amino acids 113–205), whereas {beta}1–30 represents a novel TA domain. Amino acids 30–90 and the carboxyl terminus (amino acids 550–704), regions shared by both NFAT2.{alpha} and NFAT2.{beta}, as well as the isoform-specific region of NFAT2.{alpha} (amino acids {alpha}1–44), have no independent transactivation ability. {alpha}1–44 is inactive over a broad range of effector concentrations, conditions under which {beta}1–30 demonstrates a concentration-dependent ability to transactivate (Fig. 1C).

Regions that have no independent ability to transactivate can contribute to the activity of adjacent domains. For example, the basal and inducible activity of {beta}1–90 is greater than that of {beta}1–30, yet constructs containing amino acids 30–90 alone are inactive. Deletion of 30–90 from the 31–415 construct (represented by 91–415) has only a minimal affect on activity, indicating that this modulating influence is exerted primarily on the {beta}1–30 transactivation domain.

These differences in transactivation are not the result of significant variations in the inherent stability of the hybrid effector molecules or in their ability to be expressed. Whole cell lysates were isolated from COS-1 cells transiently transfected with one-hybrid effector constructs and subjected to Western blot analysis using Gal4-specific antibodies. A representative experiment is shown in Fig. 1D. Chimeric Gal4 proteins of the expected molecular weight are expressed in all transfectants. In addition, the expression levels appear uniform with the exception of {beta}1–30 and {beta}1–90. Whereas differences in NFAT2 isoform stability have not been reported, the acidic {beta}-specific amino terminus may confer a shorter protein half-life on these constructs. This result also indicates that the levels of transactivation for these two constructs (Fig. 1B) may be underestimated by this assay. Therefore, we are unable to determine whether the overall ability of NFAT2.{beta} to transactivate represents additive or synergistic contributions from the two distinct transactivation domains (91–415 and the {beta}-specific domain).

{beta}1–30 Contains a Novel Acidic Activation Domain: Glu5-Asp19The {beta}-specific amino terminus is highly charged (pI = 2.9) and contains 7 acidic residues (Asp/Glu) interspersed by a number of hydrophobic residues including phenylalanine, a pattern conserved in many acidic activation domains (AAD) (28). An alignment of the amino termini of human NFAT2.C and murine NFAT2.{beta} (Fig. 2B) reveals a conserved unit of acidic/hydrophobic residues. We tested whether the acidic "core" of this domain, Glu5-Asp19 is able to transactivate transcription in mast cells. The results shown in Fig. 2A demonstrate that the activity of Glu5-Asp19 fully complements the ability of {beta}1–30 to drive transcription, and respond to PMA/ionomycin. Thus, a novel 15-amino acid acidic activation domain exists within the {beta}-specific amino terminus of NFAT2.



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FIG. 2.
NFAT2.{beta}-specific amino terminus contains a conserved, 15-amino acid acidic activation domain: Glu5-Asp19. A, Glu5-Asp19 is able to complement the full TA ability of {beta}1–30. One-hybrid assay was performed with the indicated quantities of effector constructs. Data are representative of three independent experiments. B, alignment of murine and human isoform-specific amino termini. The Glu5-Asp19 activation domain is boxed.

 

NFAT2.{alpha}/{beta} Exhibit Quantitative Differences in Their Basal and Inducible Transactivation in Response to PMA and Ionomycin—Although it is well established that Ca2+-dependent activation of Cn regulates NFAT nuclear translocation, and PMA-induced signals regulate the activity of AP-1 (3), the influence of these signals on transactivation has not been well characterized. We examined the ability of either PMA or ionomycin treatment alone to induce transactivation of NFAT2.{alpha} and NFAT2.{beta}. As shown in Fig. 3A, PMA treatment alone results in a modest increase in transactivation, suggesting that PMA-induced signals do contribute to transactivation. However, signals induced by ionomycin or PMA/ionomycin have the most significant affects on inducible activity. The addition of cyclosporin A inhibits PMA/ionomycin-induced transactivation (Fig. 3A). Because one-hybrid effector proteins are targeted to the nucleus via the Gal4 nuclear localization signal, this finding suggests that calcineurin participates in the regulation of both NFAT2 subcellular localization and transactivation.

At the fixed doses of PMA and ionomycin used in these assays, {beta}1–415 consistently transactivates better than {alpha}1–429 (Figs. 1B and 3A). It is possible that the isoform-specific regions confer differential ability to activate transcription in response to varying activation signal strength. For example, NFAT2.{alpha} may respond equivalently to NFAT2.{beta} only at high signal strengths. To test this possibility, transfected cells were treated with increasing concentrations of PMA and ionomycin. As shown in Fig. 3B, the absolute level of transactivation differs between the two isoforms over the entire activator concentration range. However, both isoforms demonstrate inducible transactivation at the same PMA/ionomycin concentrations, indicating that there is no qualitative transactivation difference in response to signal strength.

Endogenous Mast Cell NFAT Activity Is Influenced by Both Novel and Conventional PKCs—Several Ser/Thr kinases have been demonstrated to regulate NFAT transactivation, including: CaMK IV, Cot-1, PIM-1, and PKC{zeta} (2932). The role of PKC family members in the transduction of immunoreceptor signals is clearly established (for review see Ref. 33). In T cells, T cell receptor-proximal kinases activate PLC{gamma}, leading to the generation of diacylglycerol and inositol triphosphate. Both of these signaling intermediates are necessary to activate conventional PKCs (including {alpha}, {beta}I, and {beta}II isoforms). Only diacylglycerol-responsive signals (mimicked by PMA treatment) are required for the activation of novel PKCs ({delta}, {epsilon}, µ, and {theta}). To further characterize the molecular mechanisms that regulate NFAT activity, we used pharmacologic inhibitors Gö6976 (specific for conventional PKCs) (34) and rottlerin (specific for novel PKCs {delta} and {theta}) (35). We first examined the effect of these inhibitors on endogenous NFAT activity using an NFAT reporter construct (Fig. 4A). Both Gö6976 and rottlerin inhibit NFAT reporter activity induced in response to PMA/ionomycin treatment and IgE receptor cross-linking, indicating that both conventional and novel PKCs regulate physiologic mast cell NFAT activity (Fig. 4B).

The novel PKC, PKC{theta}, has been implicated in T cell receptor signaling pathways. In activated T cells, PKC{theta} is rapidly recruited to the T cell receptor (36). PKC{theta} also associates with the actin cytoskeleton (37). Both PKC{theta} mRNA and protein are detectable in CFTL-15 mast cells (Fig. 4C). In addition, overexpression of dominant active PKC{theta} (A148E) increases basal and ionomycin-induced NFAT reporter activity (Fig. 4D).

PKC{theta} Induces NFAT2 Transactivation—Because endogenous NFAT activity depends on Fc{epsilon}RI- or ionomycin-induced Ca2+ flux to drive nuclear localization, our PKC experiments using NFAT reporter constructs do not distinguish between effects on translocation versus transcriptional activation. Furthermore, the NFAT reporter contains composite NFAT:AP-1 elements, and does not discriminate between NFAT- and AP-1-dependent activity. Thus, one-hybrid constructs were used in transfection experiments to examine affects of PKC inhibitors on NFAT2 transactivation in isolation. As shown in Fig. 5, PMA/ionomycin-induced transactivation of both {alpha}1–429 and {beta}1–415 is inhibited by treatment with either Gö6976 or rottlerin. These results demonstrate that both novel and conventional PKCs participate in NFAT2 transactivation.



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FIG. 5.
Transactivation of NFAT2 isoforms depends on both conventional and novel PKCs. A PMA/ionomycin-induced transactivation of both {alpha}1–429 and {beta}1–415 is reduced by inhibitors of conventional and novel PKCs. Transactivation of {alpha}1–429/{beta}1–415 (0.02 µg) after 30 min preincubation with carrier, 5 µM Gö6976, or 15 µM rottlerin.

 

We next asked whether PKC{theta} regulates NFAT2 transactivation. As shown in Fig. 6A, transactivation of both NFAT2.{alpha} and NFAT2.{beta} is induced by co-expression of PKC{theta} (A148E). This response is mediated by both the {beta}-specific transactivation domain, as well as the amino-terminal common region (31–415) (Fig. 6B). Transactivation of p53 and VP-16 one-hybrid constructs is not induced by PKC{theta} A/E overexpression (nor by PMA/ionomycin treatment) (Fig. 6C), indicating that the PKC{theta} effect is specific for NFAT2.



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FIG. 6.
NFAT2 transactivation is induced by dominant active PKC{theta} expression. A, both NFAT2 isoforms transactivate in response to PKC{theta} overexpression. Basal transactivation of {alpha}1–429 and {beta}1–415 (0.02 µg) ± dominant active PKC{theta} (A148E) expression construct (20 µg). B, both distinct TADs respond to PKC{theta}. Transactivation of the indicated one-hybrid effectors ± dominant active PKC{theta} (A148E) expression construct (20 µg). C, p53 and VP16 TADs are not induced by either PMA/ionomycin treatment or dominant active PKC{theta} expression. Transactivation of p53 and VP16 (20 µg) ± dominant active PKC{theta} (A148E) expression construct (20 µg) is shown. D, transactivation of NFAT2.{alpha}/{beta} is equally sensitive to PKC{theta} overexpression. Cells were transfected with 0.02 µg of effector construct and 0–40 µg of PKC{theta} (A148E). E, {beta}1–30 WT and MUT (T2A/S21A) transactivation are equally responsive to PKC{theta} A148E overexpression. Cells were transfected with 0.02 µg of effector construct and 0–20 µg of PKC{theta} (A148E). Data are representative of three independent experiments.

 

To determine whether NFAT2.{alpha} and NFAT2.{beta} are equally sensitive to PKC{theta} signals, we performed a dose-response experiment. Like PMA/ionomycin treatment, overexpression of dominant active PKC{theta} equally affects both NFAT2 isoforms (Fig. 6D). PKC enzymes phosphorylate the motif: S/T-X-K/R. Several of these consensus motifs are present throughout 91–415, but are absent in the {beta}1–30 sequence. However, two residues, Thr2 and Ser21 exist within {beta}1–30 that could be non-consensus targets of phosphorylation. To evaluate this possibility, alanine substitutions of Thr2 and Ser21 were introduced into {beta}1–30 and {beta}1–415, and the consequences on transactivation ability were assessed. Transactivation of the non-phosphorylatable, doubly mutated effectors, {beta}1–30(T2A/S21A) and {beta}1–415(T2A/S21A) (data not shown) is unaffected. The activity of {beta}1–30(T2A/S21A) remains similar to the wild type construct over a range of PKC{theta} A/E input concentrations (Fig. 6E). Thus PKC{theta} appears to influence {beta}1–30 transactivation through an indirect mechanism.

The {beta}-Specific Transactivation Domain Confers Unique Responsiveness to IgE Receptor Cross-linking—Mast cells are activated by a variety of agonists including neuropeptides, proteases, bacterial products, and specific antigen (38, 39). Antigen cross-linkage of the high affinity IgE receptor (Fc{epsilon}RI) expressed on mast cells results in the activation of signaling cascades mediated by Ca2+ and diacylglycerol and is perhaps the best characterized mode of mast cell activation (40). The use of PMA and ionomycin to activate mast cells can also provide these intracellular signals and bypasses the need for cell surface receptor engagement. However, these agents cannot faithfully mimic early receptor-proximal events that may affect signal strength and outcome of the response. Thus, we considered the possibility that physiological activators (that deliver quantitatively or qualitatively different signals) would lead to differences in the ability of NFAT2 isoforms to transactivate.

Cross-linking of Fc{epsilon}RI by treatment of transfected cells with IgE/{alpha}-IgE results in inducible transactivation of {beta}1–30 and {beta}1–415 but not {alpha}1–44, {alpha}1–429, or 31–415 (Fig. 7A). Even under conditions where cells were treated with a range of IgE/{alpha}-IgE concentrations, {alpha}1–429 and 31–415 are unable to respond (Fig. 7B). We repeated this experiment in a second IgE-responsive, non-transformed mast cell line, MC9. Fig. 7C demonstrates that NFAT2.{beta} is selectively responsive to IgE-mediated signals, and that the {beta}1–30 TAD is required for significant inducible transactivation.



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FIG. 7.
Differential sensitivity of NFAT2.{alpha}/{beta} to physiologic stimuli. A, transactivation in response to high affinity IgE receptor stimulation is mediated by {beta}1–30 TAD. CFTL-15 cells transfected with the indicated one-hybrid effector constructs (0.02 µg) were stimulated with either PMA/ionomycin, or via IgE/{alpha}-IgE treatment to cross-link Fc{epsilon}RI. B, deletion of {beta}1–30 ablates Fc{epsilon}RI-responsive transactivation. CFTL-15 cells transfected with the indicated one-hybrid effector constructs (0.02 µg) were stimulated with increasing doses of IgE/{alpha}-IgE as described under "Experimental Procedures." C, {beta}1–30 confers Fc{epsilon}RI-responsive transactivation in MC9 mast cells. MC9 cells transfected by DEAE-dextran (58) with 8 µg of G5CAT reporter and 2 µg of the indicated one-hybrid effector constructs were stimulated with increasing doses of IgE/{alpha}-IgE as described under "Experimental Procedures." D, LPS induces NFAT2.{alpha}/{beta} transactivation. Transactivation of {alpha}1–429 and {beta}1–415 (0.02 µg) in CFTL-15 cells were treated with 0.001–100 µg/ml LPS. Data are representative of three independent experiments.

 

This selectivity appears to be specific for IgE-mediated signals. For example, LPS induces signals through toll-like receptor 4, a receptor expressed on mast cells (data not shown and Ref. 41). Although LPS is known to transduce signals through the activation of NF{kappa}B (42), we show in Fig. 7D that both {alpha}1–429 and {beta}1–415 transactivate in response to a range of concentrations. However, as we observed with PMA/ionomycin treatment, there were no qualitative differences in ability of either NFAT2 isoform to transactivate in response to this physiologic stimuli.

Overexpression of NFAT2.{beta} Induces More NFAT- and IL-4 Reporter Activity Than NFAT2.{alpha}Demonstration of functional differences among NFAT2 isoforms in vivo will require the use of technologies that eliminate expression of specific isoforms. In the absence of such conclusive experiments, we tested whether overexpression of either NFAT2 isoform would result in differential activation of an exogenous reporter construct in response to Fc{epsilon}RI signaling. CFTL-15 mast cells were co-transfected with an NFAT2.{alpha} or NFAT2.{beta} expression construct and an NFAT-dependent promoter/reporter construct. CAT reporter activity was measured 24 h after activation by Fc{epsilon}RI cross-linking. Fig. 8A demonstrates that overexpression of NFAT2.{beta} results in greater NFAT reporter activity in response to Fc{epsilon}RI signal than either NFAT2.{alpha} or the vector control, pcDNA3. A reporter construct containing 797 bp of the proximal IL-4 promoter is also more active in cells overexpressing NFAT2.{beta} (Fig. 8B). The modest increase in Fc{epsilon}RI-induced reporter activity observed in NFAT2.{alpha} transfectants appears to conflict with our data demonstrating that NFAT2.{alpha} is unable to mediate significant transactivation in response to Fc{epsilon}RI signals (Fig. 7, AC). At least two possible mechanisms may reconcile these findings: 1) NFAT2.{alpha} can still associate with AP-1 family members, and thus recruit the AP-1-dependent transactivation ability to the promoter, and/or 2) overexpression of NFAT2.{alpha} may titrate out molecules that regulate NFAT subcellular localization (calcineurin and glycogen synthase kinase for example), resulting in an increased ability of endogenous NFAT2.{beta} to enter the nucleus and activate transcription.



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FIG. 8.
NFAT2.{beta} overexpression induces greater NFAT and IL-4 reporter activity. A, NFAT reporter activity in CFTL-15 cells transfected with 20 µg of the indicated NFAT2 expression construct. Cells were stimulated via IgE/{alpha}-IgE treatment to cross-link Fc{epsilon}RI. B, IL-4 promoter (–797 bp) activity in CFTL-15 cells transfected with 20 µg of the indicated construct. Cells were stimulated via IgE/{alpha}-IgE treatment to cross-link Fc{epsilon}RI. Data are representative of three independent experiments (±S.D.).

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
NFAT2.{alpha} and NFAT2.{beta} differ by 42 and 28 unique amino-terminal amino acids but share common sequences that regulate nuclear import and export as well as DNA binding activity. In this study we provide evidence that there are differences in the regulation of these two isoforms at the level of transcriptional activation ability. Two distinct domains contribute to NFAT2 transactivation. One is located within the conserved NFAT homology region present in both isoforms. A second domain is contained within the {beta} isoform-specific region. The activity of both domains appears to be regulated by novel and conventional PKCs, including PKC{theta}. The presence of the {beta}-specific sequence is associated with both quantitative and qualitative differences in transactivation ability: NFAT2.{beta} demonstrates a greater ability to transactivate in response to both PMA/ionomycin and LPS over a range of signal strengths when compared with NFAT2.{alpha}. In addition, NFAT2.{beta} but not NFAT2.{alpha} can mediate high levels of transcriptional activation in response to Fc{epsilon}RI cross-linking.

We demonstrate that the {beta}-specific domain is necessary for optimal responsiveness to all these signals. Deletion of amino acids 1–30 from {beta}1–415 (represented by the 31–415 construct) ablates Fc{epsilon}RI responsiveness (Fig. 7, B and C) and significantly reduces PMA/ionomycin inducible activity (Fig. 1B). A 15-amino acid sequence, Glu5-Asp19, located within {beta}1–30 is responsible for this activity and has the hallmarks of an AAD. AADs are comprised of acidic amino acids interspersed with hydrophobic residues (Phe in particular) and are among the most potent transcriptional activators (28). It has been shown that AADs of p53 and VP16 make contacts with the RNA polymerase II holoenzyme through the co-activator, hTAFII31 (43, 44). These protein-protein contacts depend on a motif: F-X-X-{Phi}-{Phi}, that occurs in two orientations within Glu5-Asp19 (forward, amino acids 12–16, and reverse, amino acids 14–18). Studies to determine whether the {beta}-specific TAD also mediates its transactivation function through this co-activator are underway.

Proteins containing acidic activation domains such as VP16 and p53 are also characterized by a short half-life (45, 46). Molinari et al. (47) have used multimers of acidic activation domains to show that protein half-life is inversely correlated with transactivation activity; they and others speculate that this is one mechanism used to regulate the activity of strong transactivators. The observation that {beta}1–30 has the lowest steady-state protein level among our effector constructs may be a reflection of this phenomenon.

The varied abilities of NFAT2 isoforms to transactivate transcription in response to cell type-specific signals likely contributes to unique patterns of gene expression. How might this be accomplished? At the level of signal transduction, distinct signals can elicit differential co-activator activation and recruitment and lead to varying levels of transcriptional activation. We speculate that IgE receptor cross-linking results in the activation of one or more co-activators recruited by the {beta}-specific, but not the common (91–415) transactivation domain, leading to selective transactivation. Signals downstream of the IgE receptor on mast cells may also facilitate increased association of co-factors such as CBP/p300 (which can contribute enzymatic activities (histone acetyltransferase activity in this case) (48)) with NFAT2.{beta} (via the {beta}1–30 and 91–415 domains). These co-activators could act to increase local acetylation and enhance transcriptional activation relative to NFAT2.{alpha} by reducing the "chromosomal barrier" to transcription.

In contrast to our findings, Monticelli and Rao (49) recently reported that overexpression of "constitutively active" NFAT1 and NFAT2 (including isoforms corresponding to {alpha} and {beta}) did not result in differential IL-4 expression in response to PMA/ionomycin treatment in primary T cells. These apparently contradictory observations may reflect either signal- or cell-specific differences in the ability of NFAT2 isoforms to induce transcription. Alternatively, the IL-4 gene may be representative of a subset of NFAT-target genes that have low energetic barriers to transcription (because of their chromosomal context), and thus may not be differentially regulated by NFAT2 isoforms of distinct transactivation capabilities.

The presence of consensus PKC sites (S/T-X-K/R) throughout the 31–415 "common region," and within {alpha}1–44 led us to examine the role of PKC family members in NFAT2 transactivation. The novel PKC, PKC{theta}, has received particular attention for its role in transduction of T cell receptor signals upstream of c-Jun NH2-terminal kinase and NF{kappa}B (50). Our demonstration that PKC{theta} message and protein are detectable in CFTL-15 mast cells (Fig. 4C), taken together with its well characterized role in immunoreceptor signaling, suggests that PKC{theta} may be implicated in Fc{epsilon}RI-dependent NFAT activation. These data are consistent with recent evidence showing that 1) Nef-dependent NFAT activation in T cells requires PKC{theta} (51), 2) PKC{theta} (but not {alpha} or {delta}) specifically synergizes with calcineurin to induce endogenous NFAT activity in Jurkat T cells (52), and finally 3) FasL promoter activity in Jurkat cells requires an NFAT cis-regulatory element to mediate responsiveness to dominant active PKC{theta} and calcineurin (35). Of note, studies of PKC{theta} –/– thymocytes did not indicate a defect in NFAT activity (53). Our observation may represent a cell-specific phenomenon. Alternatively, PKC family members may be redundant with respect to this function. The inhibitor studies presented here demonstrate that both novel and conventional PKCs regulate NFAT activity, thus PKC{theta} is not likely to be the exclusive mediator of Fc{epsilon}RI signals to NFAT. Both NFAT2.{alpha} and NFAT2.{beta} are responsive to PKC{theta} overexpression, yet only the {beta} isoform responds to IgE receptor cross-linking. Therefore it is likely that additional Fc{epsilon}RI-dependent signals are required to induce transactivation of the {beta}-specific domain under physiologic conditions.

PKC{theta} responsiveness of the non-phosphorylatable construct {beta}1–30(T2A/S21A) demonstrates that at least part of the PKC{theta} effect on NFAT2 TA is indirect. PKC{theta} may act in concert with calcineurin to induce a co-activator required for NFAT2 transactivation. We and others have shown that NFAT transactivation is cyclosporin A-sensitive, and the requirement for active calcineurin in other models of PKC{theta}-responsive NFAT activity has been demonstrated (54). Cyclosporin A treatment also inhibits transactivation from the NFAT1 amino-terminal region in Jurkat T cells (32).

We also made the unexpected observation that LPS treatment of mast cells induced transactivation from both NFAT2.{alpha} and NFAT2.{beta}. Toll-like receptor signaling is known to induce degradation of I{kappa}Bs and NF{kappa}B activation (42), but has not previously been shown to influence NFAT activity. Endogenous NFAT activity (as measured by an NFAT reporter assay, data not shown) was not detectable, indicating that LPS signals regulate NFAT activity after nuclear localization (toll-like receptor 4 has not been reported to induce the sustained Ca2+ flux necessary to drive NFAT nuclear localization). These data suggest that a convergence of signals is required for NFAT function in response to LPS. Our finding may represent a clinically important example of signaling cross-talk. The inflammation associated with asthma, which depends, in part, on IgE-dependent activation of mast cells, is regulated by NFAT-dependent gene expression (55) and can be altered by LPS exposure (56). A likely candidate to mediate cross-talk is the atypical PKC, PKC{zeta}. PKC{zeta} induces NFAT1 transactivation, can induce NFAT1/2-dependent NFAT reporter activity, and physically associates with NFAT1/2 (32). PKC{zeta} also plays a critical role in LPS-induced activation of c-Jun NH2-terminal kinase, mitogen-activated protein kinase, and extracellular signal-regulated kinase in macrophages (57).

As our demonstration that LPS induces NFAT2 transactivation suggests, the range of signals that converge on NFAT is broad. The confluence of other signaling cascades is especially relevant in mast cells, which are able to respond to a variety of signals, and are in physiologic locations where combinatorial activation is likely. We propose that the expression of selected NFAT isoforms, of varying ability to transactivate transcription, allows a cell to "tune" its responsiveness to an array of cellular signals. Signal-specific transactivation is one mechanism through which functionally distinct NFAT isoforms can differentially regulate gene expression.


    FOOTNOTES
 
* This work was supported by National Institutes of Health Grant CA047992 [GenBank] (to M. A. B.). 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. Back

To whom correspondence should be addressed: Northwestern University School of Medicine, Dept. of Microbiology and Immunology, Tarry Medical Research Bldg., Rm. 7-711, mail code S213, 320 East Superior St., Chicago, IL 60611-3010. Tel.: 312-503-0108; Fax: 312-503-1339; E-mail: m-brown12{at}northwestern.edu.

1 The abbreviations used are: NFAT, nuclear factor of activated T cells; PKC, protein kinase C; TA, transactivation; DBD, DNA-binding domain; PMA, phorbol 12-myristate 13-acetate; Cn, calcineurin; IL, interleukin; LPS, lipopolysaccharide; CAT, chloramphenicol acetyltransferase; TA, transactivate; AAD, acidic activation domains. Back

2 M. Brown, unpublished data. Back


    ACKNOWLEDGMENTS
 
We thank A. Altman and T. J. Murphy for generous gifts of plasmids. We also greatly appreciate critical readings of the manuscript by Drs. J. Boss, P. Wade, and M. Sherman.



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