©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
FcRI-mediated Induction of Nuclear Factor of Activated T-cells (*)

Lori E. Hutchinson , Michael A. McCloskey

From the (1)Department of Zoology and Genetics and the Signal Transduction Training Group, Iowa State University, Ames, Iowa 50011

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Nuclear factor of activated T-cells (NFAT) is a transcriptional activator that binds to the interleukin-2 promoter and is believed to be responsible for T-cell-specific interleukin-2 gene expression. Here we demonstrate using electrophoretic mobility shift assays that nuclear NFAT can be induced in the rat basophilic leukemia (RBL-2H3) mast cell line and rat bone marrow-derived mast cells upon cross-linkage of the high affinity receptor (FcRI) for immunoglobulin E (IgE). Receptor-dependent activation of NFAT was mimicked by the combination of the protein kinase C activator phorbol myristate acetate and the calcium ionophore ionomycin. The induced binding activity was specific for the NFAT recognition motif because competition with nonradioactive NFAT oligonucleotide abolished the DNA binding activity, whereas nonradioactive oligonucleotides recognized by the transcription factors NFB, glucocorticoid receptors, and TFIID did not. An oligonucleotide representing the AP-1 recognition sequence also blocked the NFAT DNA binding activity, as did a combination of anti-Fos and anti-Jun antibodies. Using electrophoretic mobility shift assays, AP-1-binding proteins were found to be induced in RBL-2H3 cells under the same conditions as was the NFAT binding activity. Together these data suggest that the NFAT complex in mast cells contains Fos and Jun proteins as does NFAT in T-cells. The appearance of nuclear NFAT binding activity was dependent in part upon calcium mobilization, as buffering the antigen-induced calcium rise with intracellular BAPTA strongly inhibited NFAT activation. Prevention of calcium influx with external EGTA also inhibited NFAT activation, indicating that release of calcium from internal stores was insufficient for sustained activation of mast cell NFAT. Cyclosporin A, a potent inhibitor of the calmodulin-dependent phosphatase calcineurin, blocked the induction of NFAT-DNA binding activity, implicating calcineurin as a key signaling enzyme in this pathway. These results suggest that NFAT is present in the mast cell line RBL-2H3 and in primary bone marrow-derived mast cells, is similar in subunit composition to the T-cell NFAT, and may play a role in calcium-dependent signal transduction in mast cells.


INTRODUCTION

Nuclear factor of activated T-cells (NFAT)()is a transcription complex believed to mediate the final step in the signal transduction pathway linking T-cell receptor engagement with the expression of the interleukin-2 (IL-2) gene (for reviews, see Ullman et al.(1990) and Rao (1994)). Although selective expression of NFAT is thought to account for the T-cell specificity of IL-2 gene expression, recent evidence suggests the possibility that NFAT may be one in a family of related transcription factors that regulate the transcription of cytokine genes in other leukocytes. The present work was undertaken to determine whether NFAT-related activity can be induced in mast cells via cross-linkage of the receptor with high affinity for IgE (FcRI).

Although the exact molecular make-up of the NFAT complex is unknown, there is evidence that it is composed of a lymphocyte-specific cytoplasmic component (or components) present in unactivated cells, designated NFATp/c, and a ubiquitous, inducible nuclear component containing Fos and Jun proteins (Jain et al., 1992; Northrop et al., 1993). Cross-linkage of the T-cell receptor results in two intracellular signals required for the assembly of active NFAT in the nucleus: activation of protein kinase C and mobilization of cytosolic calcium. The activation of PKC is necessary for the transcription of Fos and Jun genes (Jain et al., 1992; Northrop et al., 1993), and an increase in intracellular calcium is required for the translocation of NFATp into the nucleus (Flanagan et al., 1991). The translocation of NFATp from the cytoplasm to the nucleus is thought to be controlled either directly or indirectly by the calcium-dependent protein phosphatase calcineurin (Clipstone and Crabtree, 1992). The role of calcineurin in this signaling pathway has been further substantiated by the fact that a constitutively active form of calcineurin can bypass the calcium requirement for IL-2 transcriptional activation (O'Keefe et al., 1992). The immunosuppressive drugs cyclosporin A (CsA) and FK506 block transcription of the IL-2 gene by inhibiting the phosphatase activity of calcineurin and preventing the intracellular translocation of NFATp (McCaffrey et al., 1993a; Clipstone and Crabtree 1992; Jain et al., 1993; Mattila et al., 1990). Once assembled in the nucleus, the NFAT complex initiates transcription by binding to two sequence-specific binding sites located in the enhancer region of the IL-2 promoter (Shaw et al., 1988).

The signal transduction pathway emanating from the FcRI in mast cells shares many relevant features with the T cell receptor-initiated pathway, including an increase in intracellular calcium (Beaven et al., 1984) and activation of PKC, which in turn modulates the expression of AP-1 binding proteins such as Jun and Fos (Lewin et al., 1993; Baranes and Razin, 1991). Antigen-activated mast cells produce cytokines such as IL-3, GM-CSF, TNF-, IL-4, IL-5, IL-6, and IL-1 (Kaye et al., 1992; Wodnar-Filipowicz et al., 1989; Plaut et al., 1989), and NFAT binding sites have been identified in the regulatory regions of several of these genes (reviewed in Rao, 1994). The NFAT binding site located in the intergenic enhancer region of the IL-3/GM-CSF genes has been shown to bind NFAT from activated T-cells (Cockerill et al., 1993), and CsA inhibits the induction of DNA binding activity directed to this site in Jurkat cells treated with PMA and ionomycin. CsA blocks the FcRI-dependent induction of IL-1, TNF-, and IL-6 mRNA in mouse bone marrow-derived mast cells, apparently through the mediation of some of the same immunophilin proteins present in T-cells (Hultsch et al., 1991; Kaye et al., 1992). This CsA sensitivity suggests a possible role for calcineurin in the FcRI-mediated expression of cytokine genes in mast cells. Given the presence of NFAT binding sites in the regulatory regions of cytokines induced by FcRI cross-linkage, a key question is, does NFAT mediate transduction of signals from the FcRI to the nucleus?

Here we show that antigenic stimulation through the Fc receptor induces NFAT DNA binding activity in two types of rat mast cells. The presumed NFAT complex in mast cells, like that in T-cells, contains AP-1 proteins in association with (an)other species conferring specificity for the NFAT recognition motif. Induction of NFAT binding activity by the FcRI was mimicked by simultaneous treatment with the PKC activator PMA and the calcium ionophore ionomycin, required calcium mobilization, and was blocked by the calcineurin inhibitor, CsA. The presence of NFAT in mast cells and its activation via the FcRI suggest that it may mediate the terminal steps of signal transduction via the FcRI, namely the transcription of one or more cytokine genes.


EXPERIMENTAL PROCEDURES

Cell Culture and Stimulation

The RBL-2H3 (RBL) cell line was maintained in 75-cm flasks as monolayers in Eagle's minimum essential medium (Earle's salts) supplemented with 20% fetal bovine serum (Life Technologies, Inc.), 100 units/ml penicillin G, and 100 µg/ml streptomycin as described previously (Barsumian et al., 1981). The cells were cultured at 37 °C in a 5% CO atmosphere in a humidified incubator. Every 4 days the cells were removed by trypsinization and passed to new flasks at a cell density of 2 10 cells/20 ml of medium. Rat bone marrow-derived mast cells (RBMMC) were cultured from bone marrow of 3-5-week-old Fisher 344 rats in the presence of recombinant murine stem cell factor and rat interleukin-3. Details of the culture methods and functional and molecular properties of this cell population are described elsewhere.()

For activation experiments, 4-day-old confluent monolayer cultures were used. When IgE-dependent activation was required, the cells were sensitized 12 h before the experiment by replacing the culture medium with medium containing approximately 3 µg/ml anti-2,4,6-trinitrophenyl (TNP) IgE produced from the IGEL a2 hybridoma (ATCC TIB 142) (Rudolph et al., 1981). On the day of the experiment, cells were removed from the culture flasks by rinsing the monolayers twice with phosphate-buffered saline containing 0.66 mM EDTA and then incubating the cells in the same solution for 4 min at 37 °C. The cells were dislodged from the flasks, transferred to a centrifuge tube, and diluted 2:1 with Eagle's minimum essential medium supplemented with 10 mM HEPES, 0.5 mg/ml BSA, 100 units/ml penicillin G, and 100 µg/ml streptomycin. The cells were pelleted, resuspended in the same medium, and incubated for 90 min before stimulation. This incubation permitted decay of the initial NFAT activity induced by the cell isolation protocol itself (see ``Discussion'').

Cells were activated by adding concentrated stock solutions of the triggering agents directly to the cell suspensions (1 10 cells/ml). Trinitrophenylated bovine serum albumin (TNP-BSA) was used at a final concentration of 50 ng/ml, and PMA and ionomycin were used at 32 nM and 2 µM, respectively. CsA (Sandoz Research Institute, Hanover, NJ) stock solutions were prepared by first dissolving 1 mg of CsA in 100 µl of 100% ethanol containing 20 µl of Tween 20. The volume was adjusted to 1 ml with culture medium, and appropriate dilutions of this stock solution were made directly into the cell suspensions immediately following the 90-min incubation. The cells were pretreated with CsA 15 min before activation. In treatments where cells were triggered in the absence of calcium, the cells were incubated for 30 min in medium containing the calcium chelators before cell activation. Solid EGTA was added at a concentration of 3.2 mM to basal medium (1.8 mM Ca, 0.8 mM Mg) and the pH adjusted to 7.40 with NaOH. This yields an estimated free [Ca] of 80 nM. For chelation of extracellular calcium, cells were pelleted and resuspended in fresh medium containing EGTA. BAPTA-AM was added directly to the cell suspension as a concentrated stock solution in methyl sulfoxide to attain a final concentration of 20 µM.

Nuclear Extraction

Nuclear extracts were prepared essentially as described (Fiering et al., 1990) with the following modifications. Between 1-5 10 cells were used per time point. To begin the extractions the cells were pelleted and washed once in ice-cold phosphate-buffered saline. All subsequent steps were performed at 4 °C in a 1.5-ml microcentrifuge tube with the following protease inhibitors added to all buffers immediately before use: 1 mM phenylmethylsulfonyl fluoride and 2 µg/ml antipain, leupeptin, pepstatin A, and aprotinin. After washing, the cells were pelleted by centrifugation and resuspended in 1 ml of buffer A (10 mM HEPES, pH 7.8, 15 mM KCl, 2 mM MgCl, 1 mM dithiothreitol, 0.1 mM EDTA). The cells were pelleted again, resuspended in 1 ml of buffer B (buffer A plus 0.05% Nonidet P-40), and allowed to incubate on ice for 1 min. The cell suspension was then pipetted up and down once. At this point >90% of the cells were lysed, yielding intact nuclei as determined by trypan blue staining and light microscopy. Intact nuclei were pelleted and resuspended in 315 µl of buffer C (50 mM HEPES, pH 7.8, 50 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 10% glycerol) and 35 µl of 3 M (NH)SO, pH 7.8. The resultant solution was mixed at 4 °C for 30 min to extract nuclear proteins and then centrifuged at 150,000 g for 15 min. An equal volume of 3 M (NH)SO, pH 7.8, was added to the supernatant. Precipitated proteins were pelleted by centrifugation at 100,000 g for 10 min and resuspended in 50-100 µl of buffer C. The protein samples were desalted on P6DG columns (Bio-Rad), and protein concentrations were determined using a dye binding assay (Bradford, 1976). The samples were stored in aliquots at -80 °C.

Electrophoretic Mobility Shift Assays

Electrophoretic mobility shift assays were done as described previously (Fiering et al., 1990). Binding reactions for NFAT were carried out in a 20-µl volume containing 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 5% glycerol, 1.7 µg of poly(dI-dC), and 10-30 µg of nuclear protein. Binding reactions for AP-1 were carried out in a 20-µl volume consisting of 50 mM Tris-HCl, pH 7.5, 13 mM MgCl, 1 mM EDTA, 5% glycerol, 1.7 µg of poly(dI-dC), and 10 µg of nuclear proteins (Mattila et al., 1990). Competitor oligonucleotides and anti-Fos and anti-Jun antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, catalog nos. sc-253X (Fos) and sc-44X (Jun)) were added at concentrations of 25 ng/20 µl and 1 µg/20 µl, respectively. Reaction mixtures containing competitor oligonucleotides or antibodies were incubated on ice for 10 min before the addition of 0.1-0.5 ng of double-stranded oligonucleotides end-labeled with [-P]dATP. The probe for NFAT was based upon the distal NFAT recognition site in the promoter for human IL-2 (Emmel et al., 1989). It was prepared for the binding assay by annealing the single-stranded oligonucleotides 5`-GGAGGAAAAACTGTTTCATACAGAAGGCGT-3` and 5`-ACGCCT-TCTGTATGAAACAGTTTTTCCTCC-3` (Midland Certified Reagent Company, Midland, TX) in the presence of 50 mM NaCl. The probes for AP-1, NFB, TFIID, and GRE were purchased as double-stranded oligonucleotides with the sequences 5`-CGCTTGATGAGTCAGCCGGAA-3`, 5`-AGTTGAGGGGACTTTCCCAGGC-3`, 5`-GCAGAGCATATAAGGTGAGGTAGGA-3`, and 5`-TCGACTGTACAGGATGTTCTAGCTACT-3`, respectively. These oligonucleotides were end-labeled with [-P]dATP as needed (Promega). After a 40-min incubation on ice, the binding reactions were electrophoresed on prerun 5% polyacrylamide gels in 0.5 TBE (0.5 times TBE = 45 mM Tris borate, 1 mM EDTA) for 2.5 h at 150 V. The gels were dried on Whatman No. 3MM filter paper and autoradiographed.


RESULTS

Induction of NFAT Binding Activity in RBL-2H3 Cells and Rat Bone Marrow-derived Mast Cells

Because NFAT activity in the Jurkat human T-cell line has been extensively characterized (Durand et al., 1988; Shaw et al., 1988; Emmel et al., 1989, Clipstone and Crabtree, 1992), we used this cell line as a positive control in the present experiments. To test whether NFAT binding activity is present in activated mast cells, we stimulated RBL-2H3 cells and RBMMC either through the FcRI or with PMA and ionomycin. Fig. 1and Fig. 2show that in fact, NFAT binding activity was induced in RBL cells and RBMMC after a 2-h incubation with PMA and ionomycin (lanes 5 and 3, respectively), and also after a 2-h stimulation via the Fc receptor with the multivalent antigen TNP-BSA (lanes6 and 2, respectively). Both stimulation protocols caused the appearance of a band that migrated to a position similar to that of the band induced in Jurkat cells treated with PMA and ionomycin. Triggering RBMMC through the Fc receptor induced only modest NFAT activity, compared with that induced by activating the cells with PMA and ionomycin (Fig. 2).


Figure 1: EMSA showing inducibility of NFAT in RBL cells by either PMA and ionomycin or by cross-linkage of the Fc receptor. Nuclear extracts were prepared from RBL and Jurkat cells that were unstimulated or stimulated with PMA (32 nM) and ionomycin (2 µM) (P/I) for 2 h and RBL cells stimulated with 50 ng/ml TNP-BSA (Ag) for 2 h as indicated. Equivalent amounts (20 µg) of nuclear proteins from each treatment were incubated with double-stranded P-labeled oligonucleotide containing the NFAT binding site. Retarded bands representing NFAT complexes are indicated by an arrow. The first lane (fp) shows free probe without nuclear extract. Data are representative of five independent experiments.




Figure 2: EMSA showing inducibility of NFAT in RBMMC by either PMA and ionomycin or by cross-linkage of the Fc receptor. Nuclear extracts were prepared from RBMMC that were unstimulated, stimulated with PMA (32 nM) and ionomycin (1 µM) (P/I), or stimulated with 50 ng/ml TNP-BSA (Ag) for 2 h as indicated. Equivalent amounts (20 µg) of nuclear proteins from each treatment were incubated with double-stranded P-labeled oligonucleotide containing the NFAT binding site. Retarded bands representing NFAT complexes are indicated by an arrow. Data are representative of three independent experiments.



Specificity and Subunit Composition of NFAT Binding Activity in RBL Cells

To determine the specificity of the NFAT DNA binding activity induced in RBL cells, unlabeled oligonucleotides containing various recognition sequences were added in excess to the EMSA reaction. As expected, excess unlabeled NFAT oligonucleotide completely reversed the NFAT DNA binding activity present in the nuclear extracts from RBL and Jurkat cells (Fig. 3A). In contrast, the presence of excess unlabeled NFB, TFIID, and GRE oligonucleotides had no effect on the NFAT binding activity in either RBL or Jurkat nuclear extracts. Hence, the NFAT DNA binding activity induced in RBL cells by stimulation via the FcRI was highly specific for the NFAT recognition motif. As stimulation of RBL cells via the FcRI induces expression of the AP-1 transcription complex (Baranes and Razin, 1991; Lewin et al., 1984), which in T-cells assembles with NFATc/p to form the NFAT transcription complex, we tested the hypothesis that AP-1 forms part of this complex in mast cells. As shown in Fig. 3A, unlabeled AP-1 oligonucleotides indeed removed the NFAT band both in RBL and in Jurkat cells at least as completely as the unlabeled NFAT oligonucleotides did when present at the same concentration. This is consistent with the idea that, as with T-cells, AP-1 forms part of the NFAT complex in RBL cells. To further explore this possibility, we performed EMSA in the presence of antibodies to the AP-1 proteins Fos and Jun. Using nuclear proteins from RBL cells stimulated through the FcRI receptor, Fig. 3B shows that either the Fos or the Jun antibody alone slightly reduced the NFAT binding activity and caused the appearance of a faint band of decreased mobility (lanes2 and 3). When used in combination, the Fos and Jun antibodies completely abrogated the NFAT DNA binding activity induced by FcRI cross-linkage (lane4).


Figure 3: The NFAT complex in RBL and Jurkat cells contains AP-1 proteins. A, EMSA was performed using a P-labeled NFAT probe and nuclear extracts (20 µg) from Jurkat cells stimulated with PMA and ionomycin for 2 h, or RBL cells triggered with antigen for 60 min. As indicated, either no competitor DNA or a 200-fold excess of unlabeled NFAT, AP-1, NFB, TFIID, or GRE oligonucleotide was added. B, EMSA was performed using a P-labeled NFAT probe and nuclear extracts (20 µg) from RBL cells triggered with antigen for 2 h. As indicated, either no antibodies or 1 µg of anti-Fos antibody (F), 1 µg of anti-Jun antibody (J), or 1 µg each of anti-Fos and anti-Jun antibodies (F/J) were added to the binding reaction. Data are representative of three independent experiments.



Kinetics of Appearance of Nuclear NFAT and AP-1 Activity

When triggered through the FcRI, NFAT binding activity was detectable within 10 min after exposure to antigen, and the activity steadily increased for up to 120 min (Fig. 4A). A low background level of NFAT binding activity could be detected in unstimulated cells. This background activity varied between experiments but did not mask the increase in NFAT induced by cell activation.


Figure 4: Time course of appearance of NFAT and AP-1 activity in RBL cells in response to cross-linkage of the Fc receptor. Nuclear extracts were prepared from RBL cells at the indicated times (in min) after the addition of 50 ng/ml TNP-BSA. Equivalent amounts of nuclear proteins (20 µg (A) or 10 µg (B)) from each time point were incubated with double-stranded P-labeled oligonucleotide containing the NFAT or AP-1 binding sites as indicated. Retarded bands representing NFAT (A) and AP-1 (B) complexes are indicated by arrows. Data are representative of three independent experiments.



Having evidence that AP-1 proteins contribute to the NFAT transcriptional complex present in RBL cells, we examined the kinetics of appearance of AP-1 activity following cell activation through the FcRI. Fig. 4B shows that AP-1 activity appeared in RBL nuclear extracts in a similar kinetic pattern as did NFAT, although virtually no AP-1 binding activity was present prior to activation. A slight induction was detected at 10 min, followed by a gradual increase in AP-1 activity up to 120 min. The absolute amount of binding-competent AP-1 appears greater at all time points than that of the NFAT complex, given the smaller amount of extract used in the AP-1 assays; this suggests that the rate of appearance of AP-1 does not limit the rate of appearance of NFAT DNA binding activity.

Calcium Dependence of NFAT Induction by FcRI Cross-linkage

To determine whether the appearance of NFAT in the nucleus of RBL cells is calcium-dependent, cells were activated in the presence of extracellular EGTA sufficient to buffer extracellular calcium to 80 nM. Alternatively, cells were incubated with the cell permeant BAPTA-AM (20 µM) for 30 min at 37 °C, conditions previously determined to block the calcium rise produced by FcRI cross-linkage.() The 30-min pretreatment with EGTA did not by itself induce NFAT binding activity above the level present in the cells incubated in medium alone (Fig. 5, lanes1 and 6). Similarly, incubating RBL cells in the presence of BAPTA-AM for 30 min prior to beginning the experiment and for 60 min after beginning the experiment did not induce the appearance of NFAT above the level of the control (Fig. 5, lanes1, 3, and 4). However, preincubation of cells with BAPTA-AM did inhibit completely the induction of NFAT binding activity by antigen (Fig. 5, lanes2 and 5). Triggering RBL cells with antigen in the presence of extracellular EGTA induced very modest NFAT binding activity, considerably less than that induced in the presence of external millimolar free [Ca] (Fig. 5, lanes2 and 7). Nevertheless, prevention of calcium influx with EGTA did not inhibit NFAT induction as completely as did intracellular BAPTA, which quenches the rise in intracellular calcium due to both release from internal stores and influx across the plasma membrane. Hence, intracellular release apparently can support a modest NFAT induction, but a large, sustained induction requires influx of extracellular calcium.


Figure 5: The induction of NFAT in RBL cells is dependent on the availability of calcium. EMSA was performed using a P-labeled NFAT probe and nuclear proteins (20 µg) from RBL cells unstimulated or stimulated with antigen for 60 min as shown: basal medium, after incubation with 20 µM BAPTA-AM or in the presence of excess EGTA (extracellular free [Ca] 80 nM). Data are representative of three independent experiments.



CsA Sensitivity of NFAT Induction by FcRI Cross-linkage

To determine whether the calcium-dependent protein phophatase calcineurin is involved in the induction of NFAT binding activity in mast cells, we activated RBL cells with antigen in the presence of the calcineurin inhibitor CsA at concentrations from 1 to 1000 ng/ml. CsA at 1 and 10 ng/ml had no effect on antigen-induced NFAT binding activity, whereas 100 ng/ml CsA reduced the activity markedly and 1000 ng/ml completely blocked nuclear NFAT induction (Fig. 6).


Figure 6: The induction of NFAT in RBL cells is inhibited by CsA. EMSA was performed using a P-labeled NFAT probe and nuclear proteins (20 µg) from RBL cells unstimulated or stimulated with antigen for 2 h in the presence of 1, 10, 100, and 1000 ng/ml CsA. Data are representative of three independent experiments.




DISCUSSION

Using specific DNA binding as a criterion, activation of RBL cells by cross-linkage of the Fc receptor induced NFAT activity quantitatively similar to that seen in Jurkat cells stimulated with PMA and ionomycin (Fig. 1). NFAT was detected in RBL nuclear extracts within 10 min following antigenic stimulation, with the activity steadily increasing for at least 2 h (Fig. 4A). This is similar to the kinetics of appearance of nuclear NFAT activity in Jurkat human T-cells stimulated with PMA and ionomycin (Shaw et al., 1988), although somewhat faster than the appearance of AP-1-dependent NFAT activity in murine Ar-5 T-cells stimulated with immobilized anti-CD3 antibodies (Jain et al., 1993b). Because RBL cells are a transformed analog of mast cells, the possibility exists that the presence of NFAT activity in these cells is an artifact of the transformation process. To address this possibility, we also assayed a non-transformed mast cell population derived from rat bone marrow. These cells showed nuclear NFAT activity inducible under the same activation regimens used with RBL cells (Fig. 2).

As in previous studies with lymphocytes (see, e.g., Shaw et al.(1988)), modest NFAT activity sometimes was detected in nuclear extracts from unstimulated RBL cells. This activity appeared within 15-30 min after cells were removed from the culture flasks and was dependent upon the presence of extracellular calcium. Nuclear extracts prepared from cells eluted with EDTA and not restored to medium that contained mM Ca did not possess NFAT DNA binding activity. To reduce basal NFAT activity to a low, albeit detectable level, cells were incubated in calcium-replete medium for 90 min prior to antigenic stimulation. We have observed large, transient rises in intracellular [Ca] in RBL cells exposed to an air-water interface, suggesting that adventitious calcium influx during cell preparation may be responsible for basal NFAT activity (see below).

Two findings indicate directly that the induction of NFAT activity by cross-linkage of the FcRI is likely to require an elevation of cytosolic [Ca]. First, removal of the steep [Ca] gradient across the plasma membrane by buffering external free [Ca] at 80 nM with extracellular EGTA markedly, although not completely, reduced the NFAT activity induced by 60 min (Fig. 5). Second, pretreatment of cells with BAPTA-AM under conditions found to block the antigen-induced calcium signal completely inhibited induction of NFAT activity by antigen (Fig. 5). Thus, the appearance of NFAT DNA binding activity in the nucleus of RBL cells could be part of a Ca-dependent signaling pathway like that postulated to mediate the translocation of NFATp/c in T-cells. That some nuclear NFAT activity appeared when influx of extracellular Ca alone was prevented suggests that sufficient Ca may be mobilized from intracellular stores to initiate formation of the active NFAT complex but that sustained activity requires Ca influx.

What role does calcium play in the induction of mast cell NFAT activity? The ability of CsA, a potent calcineurin inhibitor, to block NFAT induction suggests that as with T-cells, calcium-dependent activation of this serine/threonine phosphatase is a key step in activation of NFAT via the FcRI. Antigen-induced NFAT activity in RBL cells was not affected by CsA at a concentration of 1 and 10 ng/ml but was significantly reduced by CsA at 100 ng/ml and was completely blocked at 1000 ng/ml (Fig. 6). This is similar, although not identical, to the dose-response curve for CsA inhibition of NFAT activity obtained with Jurkat cells stimulated with PMA and ionomycin. Emmel et al.(1989) found that CsA at concentrations of 1 and 10 ng/ml inhibited only slightly the induction of NFAT activity in Jurkat cells, and detectable activity was still present even at 1 µg/ml CsA. Also in Jurkat cells, Mattila et al.(1990) found no inhibition of NFAT binding activity with 1 ng/ml CsA, but a significant reduction in NFAT binding with 10 and 100 ng/ml CsA. Thus, the effective concentration of CsA for inhibiting NFAT induction in RBL cells is similar to that found in Jurkat cells. In mouse bone marrow-derived mast cells cultured in IL-3, CsA has been shown to inhibit antigen-induced production of IL1-, tumor necrosis factor-, and IL-6 mRNA at IC values of 4.4, 72, and 143 ng/ml, respectively (Kaye, et al., 1992). Our results showing that CsA inhibits substantially the induction of NFAT activity at a similar effective concentration (100 ng/ml) suggests that NFAT may play a role in the transcriptional activation of these genes in mast cells.

AP-1 and NFAT activity appeared in the nucleus with a similar time course following antigenic stimulation of RBL cells (Fig. 4, A and B). An NFAT nuclear complex in T-cells is thought to contain AP-1, based in part upon the finding that AP-1 oligonucleotides reverse binding of this putative complex to the NFAT recognition sequence, whereas the latter has no effect on binding of AP-1 to the AP-1 recognition motif (Jain et al., 1993a; Northrop, et al., 1993). The identical observations with RBL cells (Fig. 3A) suggest that the NFAT complex therein also contains AP-1, although AP-1 in isolation cannot bind tightly to the NFAT recognition motif (data not shown) (Northrop, et al., 1993; Jain et al., 1992). Here, we show that antibodies to the AP-1 proteins c-Fos and c-Jun, in combination, completely block NFAT DNA binding, thus providing independent evidence that, as in T-cells, these proteins contribute to the mast cell NFAT complex (Fig. 3B).

Of a variety of tissues tested so far (Verweij et al., 1990), transcription mediated by the distal NFAT site in the IL-2 promoter appears restricted to T- and B-lymphocytes, and to a subpopulation of cells in the dermis, where skin mast cells are concentrated. DNA binding activity specific for this site in B-cells also was confirmed in EMSA studies of Verweij et al.(1990) and Yaseen et al.(1993), although in the latter study transcriptional activity was not demonstrated. Given that unstimulated lymphocytes generally do not exhibit substantial NFAT activity, and that other tissues tested were not stimulated as were the T-cells used for comparison, the conclusion on tissue specificity remains tentative. Future examination of other cell types may reveal, as found here, that stimulation via relevant receptors or with PMA and ionomycin, induces NFAT activity.

Two recent studies indicate that assembly of the NFAT complex(es) is likely to involve more components and more intricate mechanisms than originally envisaged. McCaffrey et al. (1993b) cloned a cDNA corresponding to the preexisting cytoplasmic component NFATp from murine T-cells. A truncated recombinant protein expressed from this cDNA was shown to bind directly the murine distal NFAT recognition motif from the IL-2 promoter, to combine with Fos and Jun proteins to form another DNA-binding complex, and to be constitutively expressed in T-cells but not L-cells (McCaffrey et al., 1993b). There appear to be three alternatively spliced products of this gene. A similar study with human T-cells adds a new twist to the shuttle model for NFAT assembly. Northrop et al.(1994) cloned a human gene encoding another cytosolic protein, NFATc, which was transcribed differentially from NFATp in different tissues. Unexpectedly, NFATc transcripts were absent or barely discernible in unstimulated T-cells and lymphoid tissue, although NFATp transcripts were present constitutively. PMA and ionomycin induced transcription of NFATc but had no effect on NFATp (Northrop et al. 1994). It will be interesting to determine which if any of the constitutive and inducible cytosolic components participate in nuclear NFAT assembly in mast cells.

The present results indicate that at least two types of mast cells, RBL-2H3 cells and rat bone marrow cultured mast cells, in addition to lymphocytes, express latent NFAT DNA binding activity. As with lymphocytes, the role of AP-1 in, and the precise subunit composition of the mast cell NFAT complex are key questions for ongoing research. Mast cells provide a unique opportunity for studies of NFAT assembly, in that they can be stimulated much more readily through a physiologically relevant pathway, antigen binding, than can T-cells. Future studies may soon reveal whether mast cell NFAT mediates the transcription of one or more cytokine genes elaborated in response to FcRI cross-linkage.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM48144. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: NFAT, nuclear factor of activated T-cells; NFATp, nuclear factor of activated T-cells, preexisting; NFATc, nuclear factor of activated T-cells, cytosolic; FcRI, receptor with high affinity for IgE; IL, interleukin; EMSA, electrophorectic mobility shift assay; RBL, rat basophilic leukemia; RBMMC, rat bone marrow-derived mast cells; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; BSA, bovine serum albumin; TNP-BSA, trinitrophenylated bovine serum albumin; BAPTA-AM, 1,2-bis(2-aminophenoxy)eth-ane-N,N,N`,N`-tetraacetic acid; TBE, Tris-borate-EDTA buffer; CsA, cyclosporin A.

M. A. McCloskey, M. Haggerty, and Y.-x. Qian, submitted for publication.

M. A. McCloskey, unpublished results.


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