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The Journal of Immunology, 2004, 173: 5721-5729.
Copyright © 2004 by The American Association of Immunologists

A New Pharmacological Effect of Salicylates: Inhibition of NFAT-Dependent Transcription1

Mónica Aceves*, Ana Dueñas{dagger}, Cristina Gómez*, Edurne San Vicente{dagger}, Mariano Sánchez Crespo* and Carmen García-Rodríguez2,*

* Instituto de Biología y Genética Molecular, Universidad de Valladolid-Consejo Superior de Investigaciones Cientificas, and {dagger} Unidad de Investigación, Hospital Clínico Universitario, Valladolid, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The anti-inflammatory effects of salicylates, originally attributed to inhibition of cyclooxygenase activity, are currently known to involve additional mechanisms. In this study we investigated the possible modulation by salicylates of NFAT-mediated transcription in lymphocytic and monocytic cell lines. RNase protection assays showed that 2-acetoxy-4-trifluoromethylbenzoic acid (triflusal) inhibited, in a dose-dependent manner, mRNA expression of several cytokine genes, most of which are NFAT-regulated and cyclosporin A (CsA)-sensitive. In Jurkat cells, the expression of IL-3, GM-CSF, TNF-{alpha}, TGF-{beta}1, IL-2, lymphotactin, MIP-1{alpha}, and MIP-1{beta} was inhibited to different extents. In THP-1 cells, inhibition of the expression of M-CSF, G-CSF, stem cell factor, IFN-{gamma}, TNF-{alpha}, TGF-{beta}1, lymphotoxin-{beta}1, MIP-1{alpha}, MIP-1{beta}, and IL-8 was observed. Sodium salicylate and aspirin only showed significant effects at 5 mM. The transcriptional activity of two genes that contain NFAT sites, a GM-CSF full promoter and a T cell-specific enhancer from the IL-3 locus, was also inhibited by salicylates. Transactivation experiments performed with several NFAT-dependent and AP-1-dependent reporter genes showed that triflusal strongly inhibited NFAT-dependent transcription at concentrations as low as 0.25 mM. Sodium salicylate and aspirin were less potent. The triflusal inhibitory effect was reversible and synergized with suboptimal doses of CsA. Experiments to address the mechanism of action of salicylates in the NFAT activation cascade disclosed a mechanism different from that of CsA, because salicylates inhibited DNA-binding and NFAT-mediated transactivation without affecting phosphorylation or subcellular localization of NFAT. In summary, these data describe a new pharmacological effect of salicylates as inhibitors of NFAT-dependent transcription.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Because abnormal gene expression is a common cause of disease, pharmacological modulation of transcription factor activity can be an important therapeutic approach to various pathological disorders (1). One of the best known examples of modulation of transcriptional activity by drugs is the inhibition of the NFAT transcription factor by cyclosporin A (CsA)3 and FK506, potent immunosuppressants that are widely used for treatment after organ transplantation (2, 3, 4). NFAT proteins contain a conserved DNA binding domain with some homology to the NF-{kappa}B binding site (2, 3). The NFAT family comprises five members: NFAT1 (also known as NFATc2 or NFATp), NFAT2 (also known as NFATc1 or NFATc), NFAT3 (also known as NFATc4), NFAT4 (also known as NFATc3 or NFATx), and NFAT5/TonEBP. NFAT is normally located at the cytoplasm in resting cells; its activity is tightly regulated by the Ca2+/calmodulin-dependent phosphatase calcineurin (2, 3). Upon activation, NFAT translocates to the nucleus, where it binds cooperatively with AP-1 transcription factors to composite NFAT:AP-1 sites found in the regulatory regions of inducible genes. NFAT dephosphorylation and consequent nuclear translocation and binding to DNA are inhibited by CsA and FK506 (reviewed in Ref. 4). NFAT proteins, which are expressed in several immune and nonimmune cells, play an important role in the immune and inflammatory responses by regulating the transcription of a large number of inducible genes encoding cytokines and cell surface receptors, such as IL-2, IL-3, IL-4, IL-5, IL-13, GM-CSF, TNF-{alpha}, and CD40L (2, 3, 4). NFAT regulatory sites have also been found to modulate the promoter of cyclooxygenase-2 (COX-2) (5), an enzyme that plays a relevant role in inflammation.

Salicylates are widely used anti-inflammatory agents. For decades, their therapeutic effects have entirely been attributed to the inhibition of COX activity (6, 7); however, it has been suggested that additional mechanisms underlie some of their anti-inflammatory actions (reviewed in Ref. 8). For instance, inhibition of ERK activation required for integrin-mediated responses may account for COX-independent effects of salicylates on neutrophil adhesion (9). Moreover, interference with the activation of transcription factors such as NF-{kappa}B has been reported to be relevant (10). In a murine air-pouched model of inflammation, it has been shown that the anti-inflammatory effects of salicylates are largely mediated by adenosine independently of inhibition of PG synthesis by COX-1 or COX-2 and of NF-{kappa}B (11). In addition, some effects of salicylates have been explained through actions on C/EBP{beta} (12) and STAT-6 (13) transcription factors. Finally, it was recently found that aspirin inhibits IL-4 gene transcription in CD4+ human T cells by a mechanism independent of NF-{kappa}B inhibition and PG synthesis (14). Thus, it is plausible that additional pharmacological properties of salicylates remain to be discovered.

Trifluoromethylated derivatives of salicylates such as 2-acetoxy-4-trifluoromethylbenzoic acid (triflusal) and its main metabolite 2-hydroxy-4-trifluoromethylbenzoic acid (HTB) have platelet antiaggregatory properties and are used for treatment of thromboembolic disease (15, 16). Triflusal and HTB have been reported to be potent inhibitors of NF-{kappa}B activation in HUVECs (17), rat peritoneal macrophages (17), human astrocytoma cells (18), and glial cells (19). These drugs have also been shown to inhibit NF-{kappa}B activation and COX-2 expression in both human peripheral blood monocytic cells and rat inflammatory models (17, 20).

The involvement of NFAT in the regulation of various genes relevant to the immune and inflammatory responses prompted us to investigate the possible contribution of NFAT transcriptional activity to the anti-inflammatory effects of salicylates. To identify the cytokine and chemokine genes targeted by salicylates, RNase protection assays (RPA) were conducted. Transactivation experiments with several NFAT reporter genes showed inhibition of NFAT-mediated transcription by salicylates. To examine the effect of salicylates on the NFAT activation cascade, experiments to study phosphorylation, subcellular localization, and DNA binding of NFAT were performed. Our data suggest a novel pharmacological effect of salicylate derivatives through the inhibition of NFAT-dependent transcription and the subsequent down-regulation of cytokines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

The luciferase reporter plasmid NFAT3x-Luc, which contains three copies of the distal NFAT:AP-1 site of the murine IL-2 promoter, the GAL4-mNFAT1(1–415) and GAL4-mNFAT1(1–144) plasmids, which contain the NH2-terminal transactivation domain of NFAT1, and the GAL4 luciferase reporter plasmid (GAL4-Luc), have been previously described (21, 22). The luciferase reporter plasmids pXPG-GM627, which contains a GM-CSF full promoter (23), and pCLE, containing the IL-3 enhancer (24), were provided by Dr. P. Cockerill (University of Leeds, Leeds, U.K.). pFA-c-Jun, which encodes a fusion protein of the GAL4 DNA binding domain and c-Jun, was previously described (25). The use of an IL13-Luc construct was previously reported (26). pM3-VP16, which encodes a fusion of the GAL4 DNA binding domain and the VP16 transactivation domain, was obtained from BD Clontech (Palo Alto, CA). The Renilla luciferase gene reporter plasmid, pRL-TK, and dual luciferase kits were obtained from Promega (Madison, WI). Sodium salicylate, aspirin, ionomycin, PMA, and CsA were purchased from Sigma-Aldrich (St. Louis, MO). Triflusal and HTB were obtained from Uriach (Barcelona, Spain). The in vitro translation kit, RiboQuant multiprobe kits (hCK1, hCK3, hCK4, and hCK5), mouse anti-human CD3, mouse anti-human CD28, rat anti-mouse IgG2a, mouse anti-human CD4, and recombinant human IL-2 were purchased from BD Pharmingen (San Diego, CA). TRIzol reagent and cell culture media were purchased from Invitrogen Life Technologies (Gaithersburg, MD). 5'-[{alpha}-32P]UTP, triethylammonium salt (3000 Ci/mmol, 10 mCi/ml), ECL kit, Ficoll solution, and Cy3-conjugated goat anti-rabbit IgG were purchased from Amersham Biosciences (Arlington Heights, IL). The Bradford protein assay reagent was obtained from Bio-Rad (Hercules, CA).

Cell culture and transient transfections

Jurkat (human T cell line) and Cl.7W2 (murine T cell line) (27) cells were cultured in RPMI 1640 medium supplemented with 10% FCS. THP-1 cells (human monocytic cell line) were cultured in RPMI 1640 medium supplemented with 5% FCS. Human peripheral mononuclear cells were isolated from blood donors by Ficoll centrifugation, and cultured in RPMI 1640 medium supplemented with 10% FCS and 20 ng/ml human rIL-2 for 6 days. Then, cells were incubated in serum-free medium with anti-human CD4 for 15 min at 37°C and later incubated with magnetic beads (Miltenyi Biotec, Auburn, CA). Isolation of CD4+ T cells was performed by MACS on an autoMACS apparatus (Miltenyi Biotec). Jurkat cells were transiently transfected by electroporation as previously described (22). Briefly, 12 x 106 Jurkat cells in 330 µl of serum-free RPMI 1640 medium were transfected with 2 µg of reporter plasmid (firefly luciferase-expressing plasmid), 0.5 µg of pRL-TK (Renilla luciferase-expressing plasmid), and empty vector to a total of 10 µg of plasmid DNA. Electroporation was performed with pulses of 0.25 V and 975 µF using a gene pulser (Bio-Rad) and 0.4-cm gap cuvettes.

RNase protection assay

Jurkat and THP-1 cells were stimulated with PMA plus ionomycin for 4 h. Total cellular RNA was extracted with TRIzol reagent according to the manufacturer’s protocol, and its concentration was estimated by measuring the absorbance at 260 nm. Typically, 3–5 µg of total RNA was used. RPA was performed as described previously (26). Cytokine transcripts were analyzed using RiboQuant multiprobe kits containing human DNA templates: hCK1, hCK3, hCK4, and hCK5. The 32P-radiolabeled probe set was hybridized in molar excess to target RNA in solution, followed by digestion with RNases A and T1. RNase-protected probes were resolved on denaturing polyacrylamide gels. Undigested RNA probes were also resolved on each gel to verify their integrity and to serve as size markers. Transcript levels were quantified by autoradiography and densitometric scanning using a Bio-Rad personal molecular imager FX. RNA loading was estimated by measuring the intensities of the protected fragments of the housekeeping genes L32 and GAPDH.

Transactivation experiments

Twenty-four hours after transfection, cells were treated with drug vehicle solution or different salicylates for 30–45 min before stimulation. Where indicated, 3 µM CsA was added 15 min before stimulation. Both pharmacological (50 nM PMA and 3 µM ionomycin) and TCR stimulation (0.5 µg/ml anti-CD3 and 5 µg/ml anti-CD28 cross-linked with IgG2a) were performed. After 8–12 h of stimulation, cells were harvested and lysed. Firefly and Renilla luciferase activities were assayed following the manufacturer’s instructions by quantification of the corresponding luminescence signals in a microplate luminometer equipped with a dual injector system (Berthold Technologies, Oak Ridge, TN). Renilla luciferase activity values, which are independent of cell activation, were used to normalize results. In experiments designed to study the possible reversibility of the effects of drugs, cells were pretreated with salicylates or vehicle for 45 min, then centrifuged and washed to remove drugs, and later stimulated in the presence or the absence of drugs.

SDS whole cell lysates and Western blotting

After the various treatments, cells (1 x 106/lane) were resuspended in 30 µl of buffer (40 mM Tris-HCl (pH 7.8), 60 mM sodium pyrophosphate, and 10 mM EDTA) and lysed by addition of an equal volume of 20% SDS, followed by boiling for 10 min in Laemmli buffer. The lysates were analyzed by 6% SDS-PAGE, followed by Western blotting with rabbit anti-67.1 Ab against NFAT1 (c2, p) as previously described (21). The bands were visualized using ECL.

Immunocytochemistry

Cells (5 x 104) were plated on uncoated coverslips and cultured overnight. Cells were preincubated with the indicated drugs for 45 min at 37°C, and stimulated with ionomycin or vehicle for 30 min. Immediately after stimulation, cells were fixed with 3% paraformaldehyde for 20 min at room temperature and permeabilized by washing three times in wash buffer (PBS supplemented with 0.5% Nonidet P-40). Nonspecific binding was blocked by incubation with wash buffer plus 10% FCS. NFAT1 was detected with rabbit anti-67.1 Ab (1/1000 dilution, 1 h), followed by Cy3-conjugated goat anti-rabbit IgG (1/2000 dilution, 45 min). Immunofluorescence confocal microscopy images were obtained with a Radiance 2100 laser-scanning system from Bio-Rad.

Cytoplasmic and nuclear extracts

Cell extracts were prepared as previously described (28). Briefly, 20 x 106 cells were resuspended in buffer RSB (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl2, and 0.5 mM DTT) supplemented with protease inhibitors (0.1 mM EGTA, 2 mM leupeptin, 20 µg/ml aprotinin, and 1 mM PMSF), incubated on ice for 30 min, then permeabilized with 0.5% Nonidet P-40 and centrifuged. Cytoplasmic extracts were saved, and the nuclear pellets were extracted in 60 µl of buffer C (20 mM HEPES (pH 7.4), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 25% glycerol), supplemented with protease inhibitors as described above. Nuclear extracts were diluted with an equal volume of buffer D (20 mM HEPES (pH 7.4), 50 mM KCl, 0.2 mM EDTA, and 20% glycerol). The total protein content of cytoplasmic and nuclear extracts was determined by the Bradford protein assay.

EMSAs

NFAT-DNA binding reactions were performed as previously described (26). Reactions were conducted in a buffer containing 10 mM HEPES (pH 7.5), 120 mM NaCl, 10% glycerol, 20 µg/ml poly(dI)-poly(dC), 0.8 mg/ml BSA, and 0.25 mM DTT in a total volume of 20 µl. Approximately 20,000 cpm of 32P-end-labeled ARRE2 probe corresponding to the distal NFAT site of the murine IL-2 promoter (29) and 15 µg of total protein from nuclear extracts were used in each binding reaction. After 20 min of incubation on ice, DNA-protein complexes were separated from the free probe by electrophoresis on a 4% polyacrylamide gel. Dried gels were exposed to film for autoradiography or quantified using a Bio-Rad personal molecular imager FX.

Statistical analysis

Results are expressed as the mean ± SD. Data were analyzed by unpaired t test using PRISM version 4 (GraphPad, San Diego, CA). Differences were considered statistically significant at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhibition of cytokine gene expression by salicylates

Because NFAT is involved in the transcriptional regulation of various proinflammatory genes (2), and salicylates are widely used as anti-inflammatory agents, we addressed the possible modulation of salicylates on NFAT-mediated transcription in both lymphocytic and monocytic cells. To identify proinflammatory genes targeted by salicylates, RPAs were performed. In Jurkat cells, stimulation up-regulated IL-3 and GM-CSF (Fig. 1A), as well as IL-2 (Fig. 1D), but not M-CSF, G-CSF, LIF, or stem cell factor (SCF; Fig. 1A). When cells were stimulated with either ionomycin or PMA alone, only partial induction was observed (data not shown). Triflusal inhibited the expression of the IL-3 and GM-CSF transcripts, which was also observed with CsA (Fig. 1A). mRNA expression induction of TNF-{alpha} and TGF-{beta}1 was observed upon stimulation, and partial inhibition by CsA as well as by trifluoromethyl derivatives was observed (Fig. 1C, left panel). Triflusal inhibited gene expression in a dose-dependent manner, and its effects were noticeable at a concentration of 0.5 mM (Fig. 1, A and C). Sodium salicylate and aspirin were less potent than triflusal; their effects were only observed at concentrations of 2–5 mM (Fig. 1A). A different pattern of gene expression was observed in THP-1 cells compared with Jurkat cells, because mRNA expression of M-CSF, G-CSF, LIF, and SCF, but not that of IL-3 or GM-CSF, was observed upon activation. Cytokine expression was sensitive to both CsA and triflusal treatment (Fig. 1B). Triflusal also inhibited other CsA-sensitive, NFAT site-containing genes, such as IFN-{gamma} and TNF-{alpha} (Fig. 1C, right panel). The expression of TGF-{beta}1 showed a weak induction, which was slightly inhibited by both CsA and triflusal. Aspirin and sodium salicylate showed inhibition at concentrations of 2–5 mM. mRNA expression of IL-2, a well-known NFAT-regulated gene, was partially inhibited by triflusal and was completely inhibited by CsA in Jurkat cells (Fig. 1D, left panel). The same effect was observed when RNA from primary human CD4+ T cells was analyzed (Fig. 1D, right panel). Taken together, these results show that even though different patterns of cytokine expression are elicited by ionomycin and PMA in T and monocytic cells, salicylates inhibit the expression of some CsA-sensitive and NFAT-regulated cytokine genes.



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  		FIGURE 1. Salicylates inhibit the expression of cytokine genes. Jurkat (A, C, and D), THP-1 (B and C), and primary human CD4+ T cells (D) were pretreated with the corresponding drugs or vehicle for 45 min, then stimulated for 4 h with 50 nM PMA and 3 µM ionomycin (Iono). Cytokine mRNA expression was determined with the RiboQuant multiprobe hCK4 (A and B) containing IL-3, IL-7, GM-CSF, M-CSF, G-CSF, IL-6, LIF, SCF, oncostatin M, L32, and GADPH probes, the RiboQuant multiprobe hCK3 (C) containing TNF-{beta}, Ltn-{beta}, TNF-{alpha}, IFN-{gamma}, IFN-{beta}, TGF-{beta}3, TGF-{beta}2, TGF-{beta}1, L32, and GAPDH probes; and the RiboQuant multiprobe hCK1 (D) containing IL-5, IL-4, IL-10, IL-14, IL-15, IL-9, IL-2, IL-13, L32, and GAPDH probes. One experiment representative of three independent experiments is shown.

 
As shown in Fig. 2A, stimulation of Jurkat cells with PMA plus ionomycin induced marked expression of C-C chemokines, such as lymphotactin (Ltn), MIP-1{alpha}, and MIP-1{beta}, as well as induction of C-X-C chemokines, such as IL-8. mRNA expression was sensitive to CsA treatment (Fig. 2A). Triflusal inhibited mRNA induction of Ltn, MIP-1{alpha}, and MIP-1{beta} (Fig. 2A). Sodium salicylate and aspirin showed only a partial inhibitory effect at a concentration of 5 mM. With the exception of RANTES expression, THP-1 monocyte cells showed a similar pattern of chemokine gene expression as that of Jurkat cells (Fig. 2B). mRNA expression of MIP-1{alpha}, MIP-1{beta}, and IL-8 was only partially sensitive to CsA, suggesting that NFAT is not the main regulator of their gene expression in this cell type, and that other transcription factors, such as C/EBP{beta} or NF-{kappa}B, might be involved (30). Triflusal treatment also resulted in partial inhibition of RANTES, MIP-1{alpha}, MIP-1{beta}, and IL-8 expression, whereas aspirin and sodium salicylate showed weak or no inhibition (Fig. 2B). Taken together, these data show that salicylates inhibit the expression of some chemokine genes involved in early stages of inflammation.



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  		FIGURE 2. Salicylates inhibit the expression of chemokine genes. Jurkat and THP-1 cells were treated and processed as described in Fig. 1. Chemokine mRNA expression was determined with the RiboQuant multiprobe hCK5 containing Ltn, RANTES, IFN-inducible protein-10, MIP-1{beta}, MIP-1{alpha}, MCP-1, IL-8, L32, and GAPDH probes. The data are from one experiment representative of three independent experiments.

 
Salicylates inhibit GM-CSF and IL-3 gene promoters

Because the expression of GM-CSF and IL-3 was inhibited by salicylates, we addressed the effects of these drugs on the NFAT-containing promoters of these genes. GM-CSF is expressed in lymphocytes and monocytes; its gene is regulated by an inducible tissue-specific enhancer containing several NFAT sites (31). When a GM-CSF full promoter was used in transactivation assays, inhibition by salicylates was observed in Jurkat cells (Fig. 3A), consistent with inhibition of GM-CSF mRNA expression. IL-3 expression is restricted primarily to T cells, and its locus contains several NFAT sites and NFAT/OCT elements that confer T cell specificity (24). Transactivation experiments using a reporter construct driven by the IL-3 enhancer/promoter showed transcriptional inhibition by triflusal, aspirin, and sodium salicylate (Fig. 3B). These results show that salicylates inhibit T cell-specific enhancers that contain NFAT binding sites.



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  		FIGURE 3. Salicylates inhibit transcriptional activity of GM-CSF and IL-3 promoters. Jurkat cells were cotransfected with reporter constructs containing the GM-CSF full promoter (A) or the IL-3 enhancer (B). Transfected cells were treated and processed as described in Materials and Methods. The results represent the mean ± SD of three independent experiments, expressed as the percent inhibition of transcriptional activity observed upon stimulation after preincubation with vehicle alone. *, p < 0.05 compared with vehicle-treated cells.

 
Effect of salicylates on NFAT-mediated transcription

To address the possible effect of salicylates on NFAT transcriptional activity, transactivation experiments were performed in Jurkat cells transfected with a reporter construct containing the firefly luciferase gene driven by the distal NFAT site of the IL-2 promoter (NFAT3x-Luc) and a Renilla luciferase reporter, pRL-TK. To produce complete activation of NFAT-dependent transcription, cells were stimulated with either PMA and ionomycin (Fig. 4, A and C) or anti-CD3 and anti-CD28 (Fig. 4B). Results from luciferase assays showed that triflusal strongly inhibited NFAT-dependent transactivation in a dose-dependent manner (Fig. 4, A and B). Interestingly, similar levels of inhibition were observed in cells stimulated with either a physiological (Fig. 4B) or a pharmacological (Fig. 4A) stimulus. The degree of inhibition by triflusal was higher than that on IL-2 mRNA expression (Fig. 1D), suggesting that its effect on IL-2 gene transcription cannot be solely explained by inhibition of individual factors, but may be the sum of modulations by several ones. Sodium salicylate also inhibited, although it was less potent than the trifluoromethylated compounds (Fig. 4, A and B). Aspirin only showed significant effects at 2 mM (data not shown). Similar results were obtained in 293-T cells transiently transfected with NFAT3x-Luc reporter and NFAT plasmids (data not shown). The reversibility of triflusal effect was evaluated by removing triflusal from cells before stimulation. Triflusal acted in a reversible manner, and similar results were observed at various time points of NFAT activation (Fig. 4C). Suboptimal doses of CsA, i.e., 1000–2000 times lower that those producing optimal inhibition of NFAT-dependent transcription, achieved further inhibition when used in combination with triflusal (Fig. 4D).



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  		FIGURE 4. Inhibition of NFAT transcriptional activity by salicylates. Jurkat cells were transfected with different reporter constructs: NFAT3x-Luc containing the distal NFAT site of the IL-2 promoter (A–D), a combination of GAL4-Luc reporter and GAL4-mNFAT1(1–415) (E, left panel), GAL4-mNFAT1(1–114) (E, central panel), GAL4-VP16 (E, right panel), GAL4-c-Jun constructs (G), and IL-13 Luc reporter (F). Transfected cells were pretreated with the corresponding drugs or vehicle and stimulated for 8–12 h with either 50 nM PMA and 3 µM ionomycin (A and C–G) or anti-CD3/anti-CD28 cross-linking (B). In reversibility studies, cells were pretreated with 1 mM triflusal, washed, and stimulated for the indicated times (C). Cell lysates were assayed for luciferase activity. Data are shown as the percent inhibition of the NFAT transcriptional activity observed after preincubation with vehicle alone. Results in A–C and E–G represent the mean ± SD of three to five independent experiments. The data in D are representative of three experiments. *, p < 0.05 compared with vehicle-treated cells. Tri, triflusal; Asp, aspirin; Sal, sodium salicylate.

 
In most cases, NFAT-dependent transactivation requires the interaction of NFAT with AP-1, a heterodimer of Fos and Jun proteins (2). Because it has been reported that aspirin inhibits AP-1 activity (32), transactivation experiments were performed with AP-1-independent NFAT reporters to address whether the effect of salicylates on NFAT-dependent transactivation (Fig. 4, A and B) is due to interference with NFAT rather than AP-1. For this purpose, cells were cotransfected with GAL4-mNFAT1(1–415), a constitutively nuclear GAL4 fusion protein containing the NH2-terminal transactivation domain of NFAT1 and the DNA binding domain of GAL4, and with the GAL4-Luc reporter plasmid (GAL4-Luc) (21, 22). Salicylates significantly inhibited NFAT1-dependent transactivation (Fig. 4E, left panel), although to a lesser extent than that observed with an AP-1-dependent NFAT promoter (Fig. 4, A and B). Similar results were obtained using another AP-1-independent promoter, a GAL4-NFAT1(1–144) (22) containing the strong acidic NH2-terminal transactivation domain of NFAT1 (Fig. 4E, central panel). Data obtained, expressed as fold induction, are the following: vehicle, 9.2 ± 0.2; 1 mM triflusal, 5.2 ± 1.3; 5 mM aspirin, 5.4 ± 0.9; and sodium salicylate, 5.7 ± 0.8. The specificity of the effect of salicylates on the GAL4 system was tested with a GAL4-VP16 control plasmid, the transactivation of which was not affected by these drugs (Fig. 4E, right panel). To demonstrate the specificity of inhibition of NFAT, we performed transactivation experiments with an IL-13-Luc reporter, which contains a promoter that relies on NFAT, but not on AP-1. As shown in Fig. 4F, IL-13 transactivation was also inhibited by 1 mM triflusal, 5 mM aspirin, and 5 mM sodium salicylate. Taken together, these results suggest that the observed effects may be accounted for at least in part by inhibition of NFAT transcriptional activity.

The effect of triflusal on AP-1 activity was tested with a GAL4-c-Jun reporter construct. Inhibition by 1 mM triflusal, 5 mM aspirin, and 5 mM sodium salicylate was observed (Fig. 4G), although to a lesser extent than with GAL4-NFAT constructs (Fig. 4E). These results are in agreement with previous reports showing inhibition of AP-1 activities by aspirin (32, 33), and collectively indicate that triflusal inhibits both NFAT and AP-1 activities when GAL4 constructs are used.

Mechanism of action of salicylates on NFAT function

Given that many of the genes targeted by salicylates are CsA-sensitive genes, and because CsA inhibits calcineurin, thus preventing dephosphorylation and translocation of NFAT, we investigated the effect of salicylates on these two steps of the NFAT activation cascade. The phosphorylation state of NFAT was investigated by determining the changes in its mobility on SDS-polyacrylamide gels. In keeping with previous reports (reviewed in Ref. 4), CsA inhibited the NFAT dephosphorylation induced by ionomycin stimulation. However, no effect of triflusal on the phosphorylation state of NFAT was observed in Jurkat cells (Fig. 5A), even at concentrations producing maximal inhibition of NFAT transcriptional activity. Similar results were observed during treatment with either aspirin or sodium salicylate (Fig. 5A). When experiments were performed with Cl.7W2, a murine T cell line previously used to study NFAT1 dephosphorylation and translocation (34), the same effects were observed (Fig. 5B). Likewise, no effect of salicylates on the NFAT phosphorylation state in either Jurkat or Cl.7W2 resting cells was observed (data not shown), suggesting that CsA and salicylates exhibit different mechanisms of inhibition of NFAT activation.



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  		FIGURE 5. Salicylates have no effect on NFAT phosphorylation state. Jurkat (A) and Cl.7W2 (B) cells were treated with vehicle, 1 mM triflusal, 5 mM aspirin, 5 mM salicylate, or 3 µM CsA and stimulated with 3 µM ionomycin. SDS total cell lysates were obtained and used for Western blot analysis. The data shown are representative of at least three separate experiments.

 
The possible effect of salicylates on NFAT translocation was tested by immunocytochemistry in Cl.7W2 cells. As shown in Fig. 6A, ionomycin induced translocation of NFAT to the nucleus in most cells observed. No effect of salicylates on NFAT translocation was observed, even at concentrations that showed maximal inhibition of NFAT transactivation, whereas CsA did inhibit translocation, as previously reported (4). Similar results were obtained in Jurkat cells (data not shown). Likewise, no effect of salicylates on NFAT subcelullar localization was observed in resting cells treated with drugs (data not shown). Consistent with these results, a lack of effect of salicylates on NFAT subcellular localization was also observed by Western blot analysis of cytoplasmic and nuclear extracts from Cl.7W2 cells (Fig. 6B). Taken together, these results suggest that salicylates affect a step downstream of NFAT nuclear translocation in the activation cascade.



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  		FIGURE 6. Salicylates have no effect on NFAT subcellular localization. A, Cl.7W2 cells were attached to coverslips; treated with vehicle, 1 mM triflusal, 5 mM aspirin, 5 mM salicylate, or 3 µM CsA; and stimulated with 3 µM ionomycin (Iono). NFAT localization was analyzed by immunocytochemistry and confocal microscopy. Images shown are representative of at least three separate experiments. B, Cells were treated as described above, and cytosolic and nuclear extracts were analyzed by Western blot as described in Materials and Methods. The data shown are representative of at least three separate experiments.

 
The effect of salicylates on NFAT-DNA binding was tested in EMSAs by incubating nuclear extracts from activated Cl.7W2 cells with a radioactively labeled ARRE2 probe from the distal NFAT site of the murine IL-2 promoter. Triflusal inhibited DNA binding in a dose-dependent manner (Fig. 7A) consistent with results obtained in transactivation experiments and RPA. Aspirin and sodium salicylate inhibited only partially at a concentration of 5 mM, in agreement with the results presented in this study and with their reported effect on NF-{kappa}B (8). Similar results were observed in Jurkat cells (Fig. 7C). When salicylates were added directly to the binding reaction, partial inhibition of NFAT binding to DNA was observed (Fig. 7, B and D). These results indicate that, in contrast to CsA, salicylates inhibit NFAT function by a mechanism interfering with DNA binding and NFAT transactivation, but not with NFAT phosphorylation or subcellular localization.



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  		FIGURE 7. Inhibition of NFAT binding to DNA by salicylates. Cl.7W2 and Jurkat cells were treated as described in Fig. 5. Binding activity was assayed by incubating nuclear extracts from stimulated cells pretreated with drugs with a murine IL-2 promoter 32P-end-labeled ARRE-2 probe (A and B). Equivalent amounts of NFAT were used in each binding reaction. The probe indicates the control reaction in the absence of nuclear extract. The specificity of binding was determined by using a 100-fold molar excess of unlabeled probe as competitor (comp). Binding reactions using nuclear extracts from stimulated cells and not pretreated with drugs were conducted in the presence of the corresponding drug added to the binding reaction (C and D). The data shown are representative of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the last few years, the concept of a single mechanism of action for salicylates has been challenged by several studies showing that these drugs display anti-inflammatory and antiproliferative effects independent of COX activity and PG synthesis inhibition, by inhibiting NF-{kappa}B and AP-1 transcription factors (reviewed in Ref. 8). To explore new mechanisms underlying the actions of these drugs, and taking into account the important role of NFAT in the transcriptional regulation of numerous genes involved in the immune and inflammatory responses, the possible pharmacological modulation of NFAT-mediated transactivation by salicylate and its trifluoromethylated derivatives was addressed with different reporter gene constructs. The data obtained, showing inhibition of transcriptional activity of the distal NFAT site of the IL-2 promoter, provide the first description of an effect of salicylates on NFAT-mediated transcription. This effect was dependent on the dose, was observed with either TCR or pharmacological stimulation, and was reversible. NFAT-dependent transactivation usually requires cooperative binding of NFAT with members of the AP-1 family of transcription factors, as it has been demonstrated for the promoter/enhancer regions of many genes (2). Because AP-1 transactivation activity is inhibited by aspirin (32, 33), and AP-1 inhibition may account for the results described above, the actual effect of salicylates on NFAT-dependent transcription in our model was confirmed with AP-1 independent NFAT reporters (21, 22, 26). The effect of triflusal on AP-1 activity was tested using a GAL4-c-Jun reporter construct. Results with the GAL4 constructs suggested that triflusal inhibits both NFAT and AP-1 activities in the context of GAL4, which may explain why the inhibitory effect on the GAL4 NFAT system is less than that observed with a reporter containing a composite NFAT:AP-1 site.

Triflusal inhibited NFAT-mediated transcription at 0.5 mM, a therapeutically relevant concentration (15) also reported to inhibit NF-{kappa}B activation (17, 18, 19). Aspirin and sodium salicylate inhibited NFAT transactivation only at doses of 2–5 mM, i.e., higher concentrations than those needed to inhibit PG synthesis (10–5–10–4 M). Concentrations of 2–5 mM have also been used for NF-{kappa}B and AP-1 inhibition (reviewed in Ref. 8). Because relatively high concentrations are used to achieve these effects, their pharmacological relevance in vivo has been questioned. However, the antitumor activity of nonsteroidal anti-inflammatory drugs related to induction of apoptosis (9) and inhibition of angiogenesis (35) has only been observed at concentrations 100- to 1000-fold higher than those needed to inhibit PG synthesis. Chronic inflammatory diseases are treated with higher doses of aspirin than those required to inhibit PG synthesis (reviewed in Ref. 8), and a recent study has disclosed that aspirin shows a protective effect on colorectal adenoma at doses significantly higher than those recommended for the prevention of cardiovascular disease (36). Moreover, because suboptimal concentrations of CsA produced greater inhibition when used in combination with triflusal, these results might have clinical relevance to diminish untoward side effects of high doses of CsA.

The data presented in this report show that salicylates inhibit the expression of several cytokine genes, many of which are CsA-sensitive and NFAT site-containing. Because inhibition of both mRNA expression and NFAT transcriptional activity were observed, the results suggest interference with transcription, rather than with mRNA stability. CsA is a genuine inhibitor of NFAT-dependent transcription (2), but it has also been shown to be an uncompetitive inhibitor of proteasome activity, preventing NF-{kappa}B activation (37). Therefore, CsA sensitivity does not rule out the involvement of NF-{kappa}B in the regulation of these cytokine genes. In fact, pharmacological modulation by salicylate and trifluoromethylated derivatives might be due to a combination effect, as described for other anti-inflammatory drugs. For instance, N-substituted benzamides, which are anti-inflammatory, antipsychotic, and antitumoral agents, have been reported to inhibit both NF-{kappa}B and NFAT activities (38). Rolipram and other phosphodiesterase inhibitors have also been shown to suppress the functional responses of many inflammatory cells and to inhibit both NF-{kappa}B and NFAT activation (39).

The set of genes targeted by gene expression-modulating drugs depends on the cell type, the stimulus, and the array of transcription factors involved in their regulation (1). As shown in the present study, the pattern of cytokine gene expression was different in lymphocyte and monocyte cell lines, and the potency of drugs varied depending on the cell type. The binding of specific factors to specific gene promoters may also be determined by the type of cell. For instance, NFAT has been shown to bind to a distal IL-4 enhancer in a cell type-restricted manner (40). Similarly, T cell-specific expression of IL-3 is in part controlled through the cooperative interaction of proteins of the OCT and NFAT families to the enhancer (24). In our experiments, salicylates showed marked suppression of IL-3 gene expression, which could be explained by the inhibition of either the enhancer or the promoter itself. Transactivation experiments using a reporter gene driven by an IL-3 enhancer containing several NFAT/OCT sites, which confer T cell specificity, indicate that composite NFAT/OCT sites might be the targets of the action of salicylates, although additional effects on the promoter cannot be ruled out.

CsA regulates NFAT function at the levels of phosphorylation, subcellular localization, DNA binding activity, and transactivation (2, 4). In contrast, salicylates seem to inhibit NFAT binding to DNA and NFAT transactivation. The fact that some inhibition is observed when salicylates are directly added to the binding reaction suggests that the effect could involve direct interference with NFAT binding to DNA. The inhibition of NFAT-mediated transcriptional activity may involve the interaction with the family of transcriptional coactivators CREB-binding protein/p300 (21) or the association with other proteins described to increase NFAT transactivation, such as Ca2+/calmodulin-dependent kinase IV (22), although these possibilities remain to be studied. Recently, other molecules have been reported to act at the level of NFAT binding to DNA. For instance, inhibition of IL-4 production in CD4+ T cells by peroxisome proliferator-activated receptor-{gamma} ligands has been explained by both inhibition of NFAT-DNA interactions and competitive recruitment of transcription integrators such as p300 (41). The N-terminal transactivation domain of NFAT, the activity of which is inhibited by salicylates in reporter assays, has been found to contain an inducible phosphorylation site required for full transactivation (34). In addition, this domain has been reported to be phosphorylated in vitro by protein kinase C{zeta} (PKC{zeta}) (42). Moreover, Cot kinase (43) and PKC{zeta} (42) have been reported to increase NFAT-mediated transactivation in a CsA-independent manner. Whether some of the pharmacological properties of salicylates involve Cot kinase or PKC{zeta} is an exciting possibility that deserves additional examination.

In summary, the present study shows inhibition of NFAT-dependent transcription by salicylates and defines a set of inflammatory mediators, the expression of which is suppressed by these drugs. The mechanism of action of salicylates on NFAT function differs from that described for CsA. These data provide new insight for understanding the actual mechanisms of action of this family of anti-inflammatory drugs.


   Acknowledgments
 
We are grateful to Drs. Fernando Macián and Anjana Rao for technical training and advice, to Drs. M. Fresno and F. García-Cózar for plasmids, and to Dr. Cockerill for plasmids and useful discussion. We thank Dr. Miguel Ángel Gijón Porta for invaluable help with confocal microscopy and critical reading of the manuscript. We are also thankful to R. Alonso and Dr. M. E. Fernández for invaluable help with isolation of primary human CD4+ T cells.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from Fondo de Investigaciones Sanitarias (FIS 01/1600), Plan Nacional de Salud y Farmacia (SAF 2001/0506), and Junta de Castilla León (VA016/03). A.D. is the recipient of a grant from the Instituto de Salud Carlos III, Spain. C.G.-R. is under contract within the Ramon y Cajal Program of the Ministerio de Ciencia y Tecnología of Spain. Back

2 Address correspondence and reprint requests to Dr. Carmen García-Rodríguez, Instituto de Biología y Genética Molecular, Facultad de Medicina, Avda. Ramón y Cajal 7, 5°, 47005 Valladolid, Spain. E-mail address: cgarcia{at}ibgm.uva.es Back

3 Abbreviations used in this paper: CsA, cyclosporin A; COX-2, cyclooxygenase-2; HTB, 2-hydroxy-4-trifluoromethylbenzoic acid; Ltn, lymphotactin; PKC{zeta}, protein kinase C{zeta}; RPA, RNase protection assay; SCF, stem cell factor; triflusal, 2-acetoxy-4-trifluoromethylbenzoic acid. Back

Received for publication March 5, 2004. Accepted for publication August 19, 2004.


   References
Top
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 Introduction
 Materials and Methods
 Results
 Discussion
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