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The Journal of Immunology, 2007, 178: 3067-3075.
Copyright © 2007 by The American Association of Immunologists, Inc.

NF-AT-Mediated Expression of TGF-beta1 in Tolerant T Cells1

Naoko Nakano2, Hiroyuki Hosokawa3, Masako Kohyama4 and Nobumichi Hozumi

Research Institute for Biological Sciences, Tokyo University of Science, Chiba, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
During T cell development in the thymus, a certain population of self-reactive thymocytes differentiates into regulatory T cells that suppress otherwise harmful self-reactive T cells. In transgenic mice expressing both TCR that specifically recognizes moth cytochrome c and the moth cytochrome c ligand, a large proportion of CD4+ T cells expresses CD25 and secretes TGF-beta1 upon Ag stimulation. Because TGF-beta1 expression by these T cells can be decreased by cyclosporin A, a NF-AT inhibitor, NF-AT-mediated TGF-beta1 expression in T cells was addressed by characterizing a NF-AT response element in the TGF-beta1 promoter. Analysis of the mouse TGF-beta1 promoter (–1799 to +793) in transfection experiments in T cell 68-41 hybridoma cells detected NF-AT binding sites at positions +268 and +288 in the proximal promoter region. Binding of NF-AT to this region was detected only in tolerant CD4+ T cells, but not in fully activated CD4+ T cells by chromatin immunoprecipitation assays. Activation of these NF-AT sites was sufficient to induce TGF-beta1 promoter activity; however, additional signaling due to full Ag stimulation blocked NF-AT-mediated TGF-beta1 expression. This suppression of the TGF-beta1 promoter is mediated by the –1079 to –406 region, in which deletion of a GATA-binding motif at position –821 abrogates NF-AT-mediated activation of the TGF-beta1 promoter. Therefore, TGF-beta1 expression in T cells is controlled by multiple regulatory factors that have distinct functions in response to partial or full TCR activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The immune system establishes Ag-specific TCRs by gene rearrangement during T cell development in the thymus. Thymocytes expressing self-reactive TCRs are eliminated during maturation to avoid autoimmune responses. During this process, CD4+CD25+ regulatory T cells are generated as a distinct T cell lineage. Depletion of CD4+CD25+ regulatory T cells from the immune system causes autoimmune disorders, indicating that these T cells are required to maintain self-tolerance (1, 2). Regulatory T cells are also generated in the periphery. Orally administered Ags do not cause normal immune responses, because T cells that react with food Ags are either eliminated in the periphery or become regulatory T cells that suppress Ag-specific immune responses. These T cells are characterized as Th3 cells that secrete TGF-beta1 (Th3) or regulatory T cells that secrete IL-10. Although the expression of suppressive cytokines by tolerant T cells upon Ag stimulation is one of the hallmarks of regulatory T cells, it is not clear why tolerant T cells, but not normal T cells, express TGF-beta1 after Ag stimulation.

Tolerant T cells are also known to be anergic. Classically, engagement of TCRs without costimulatory molecule ligation induces T cell anergy (3). Similarly, weak stimulation of TCRs by recognizable altered peptide ligands also leads to T cell anergy (4, 5). Anergy induction, therefore, can be caused by partial activation of T cells, which may turn on genes that are not expressed in fully activated T cells. Various groups have extensively studied the induction of anergic T cells by sustained calcium signaling. Free calcium activates calcineurin, which in turn induces the translocation of NF-AT into the nucleus (6), resulting in expression of the ubiquitin ligases Itch, Cbl-b, and GRAIL (7). These ubiquitin ligases down-modulate signaling molecules that have been phosphorylated by TCR activation. Anergic T cells are impaired in activation of Zap70, ERK, JNK, and AP-1 (8). Decreased IL-2 gene expression is typical in anergic T cells; this can be explained by increased levels of active Rap-1, which suppresses Ras activity (9). In addition, Tob, an antiproliferative protein that is expressed in quiescent and anergic T cells, suppresses IL-2 transcription in association with Smad-2 and Smad-4 (10).

TGF-beta1 is a pleiotropic cytokine involved in the immune responses. TGF-beta1 modulates macrophage function, lymphocyte activation and apoptosis, functions of APCs, and lymphocyte differentiation (11). A wide range of lymphoid and nonlymphoid cells produces TGF-beta1. Viral or oncogene-induced transformation of cells or treatment with TGF-beta1 itself increases the transcription of TGF-beta1. Targeted disruption of the TGF-beta1 gene results in lethal multiple organ inflammatory disease. Such disease is dependent on CD4+ T cells because TGF-beta1-deficient mice carrying a MHC class II gene mutation have no autoimmune response (12). CD4+CD25+ regulatory T cells have been shown to express membrane-bound TGF-beta1, which functions to suppress autoreactive T cells (13). Although the involvement of TGF-beta1 in suppressive functions of regulatory T cells is still controversial, transgenic mice expressing a dominant-negative form of TGF-beta receptor II in T cells develop autoimmune lymphocyte infiltration in multiple organs (14, 15), suggesting that autoimmune responses are suppressed by TGF-beta1 signaling. Furthermore, it has been shown that TGF-beta signaling is required to maintain Foxp3 expression in CD25+ regulatory T cells (16). It is, therefore, of great importance to determine how TGF-beta1 expression is regulated in T lymphocytes when they recognize self Ags.

Initial characterization of the mouse TGF-beta1 gene detected two transcriptional start sites (17). Although a third transcription start site has now been reported in the TGF-beta1 promoter, there is no clear evidence for independent regulation of the different sites (17, 18), and two distinct TGF-beta1 transcripts are expressed in a 1:1 ratio in various mouse tissues. The mouse and human TGF-beta1 promoters lack TATA and CATT boxes, but are rather GC rich and include a number of SP-1 binding sites that may aid initiation. Minimal promoter constructs containing 113 or 104 bp upstream of the first and second transcription start sites, respectively, contributed to Ha-ras-mediated expression of TGF-beta1. Regions of the human TGF-beta1 promoter that are responsible for PMA- and TGF-beta1-mediated activation have been identified upstream of the first and second initiation start sites (19, 20).

Because TGF-beta1 gene expression in T cells has not been well studied, we analyzed the mouse TGF-beta1 promoter and the mechanisms by which stimulation of tolerant T cells leads to TGF-beta1 expression. Our results suggest that NF-AT-mediated TGF-beta1 expression is enabled in tolerant T cells, but is blocked in normal T cells.


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

Moth cytochrome c (MCC)5 88–103 peptide (ANERADLIAYLKQATK) was synthesized and purified to >90% by Sawady Technology.

Animals

AND transgenic mice expressing a TCR that specifically recognizes the C-terminal portion of cytochrome c peptide (21) were obtained from The Jackson Laboratory and backcrossed to B10.BR for >10 generations. Animals used in the experiments were 8–10 wk of age. Transgenic mice that express an altered peptide ligand with a single amino acid substitution from T to E at position 102 of MCC88–103 were described previously (22).

Computational analysis of the TGF-beta1 promoter

Putative recognition sites for regulatory factors were identified in the mouse (gi201947) and human (gi340526) TGF-beta1 promoters by searching the TRANSFAC database, and were verified by information from the literature. The two promoter sequences were aligned by BLAST under default settings.

Cloning of the mouse TGF-beta1 promoter

A C57BL/6 mouse genomic library ({lambda} DASH II; Stratagene) was screened with a short TGF-beta1 promoter fragment (+432 to +863) obtained by PCR amplification from mouse genomic DNA. A 6-kb SacI fragment covering the TGF-beta1 locus from –4731 to +1269, including the TGF-beta1 promoter, was subcloned into p-Bluescript SK– (Stratagene).

Reporter gene constructs

Truncated fragments of upstream TGF-beta1 promoter regions were generated by digestion with BamHI in combination with BstEII (–231), AatII (–406), SmaI (–1079), or PstI (–1799). A proximal promoter fragment (+55 to +793) was excised from a PstI (–1799) to PstI (+793) fragment and subcloned into pBluescript SK– by digestion with BamHI and PstI. All promoter fragments were subcloned into the pGL2 luciferase reporter vector (Promega). To delete the putative NF-AT and AP-1 binding sites, primers lacking the nucleotides AGGAA, TTTCC, TGAGAC, or AAGTCA were designed. Proximal promoter constructs lacking the putative NF-AT or AP-1 binding sites were synthesized by the QuickChange Site-Directed Mutagenesis Method using Pfu Turbo polymerase (Stratagene). Template DNAs were digested with the methylation-dependent restriction enzyme DpnI. Bacteria were then transformed with DpnI-digested DNA, and the cloned deletion constructs were confirmed by sequencing. Deletion mutants lacking either GATA or TGAC in the upstream promoter region were prepared by PCR using mutated primers.

Transfection experiments

T cell hybridoma 68-41 cells were electroporated with 10 µg of each reporter gene construct and 1 µg of SV40 promoter beta-galactosidase (beta-Gal) plasmid as a control. Forty hours after transfection, cells were stimulated for 6 h with 0.5 µM ionomycin (Sigma-Aldrich) ±50 ng/ml PMA (Sigma-Aldrich), and luciferase activity was measured under standard conditions. Promoter activities measured by the luciferase assay were normalized based on beta-Gal activity. All experiments were repeated at least three times. For the expression of constitutively active (CA)-NF-AT1, 10 µg of GFP-KV-DV-CA-NF-AT1 (23), GFP-KV-DV-CA-RIT-NF-AT1 (23), which is mutated to prevent binding to AP-1, or GFP-KV-DV (empty vector control) (23) was cotransfected with reporter constructs. To express AP-1, 1 µg each of c-Fos and c-Jun expression vectors was cotransfected. NF-AT1 expression vector was used where indicated.

RT-PCR

Total RNA was isolated from cells with Isogen. cDNA was then synthesized from 5 µg of total RNA using oligo(dT) priming and Super Script II (Invitrogen Life Technologies) at 42°C for 50 min and forwarded to amplification with specific primer sets. The sequences of 5' and 3' primers were as follows: TGF-beta1, 5'-AGATCTCCCTCGGACCTGCTGGCAGT-3', 5'-CACGGCACTTCGGAGAGCGGGAAC-3'; IL-2, 5'-TGATGGACCTACAGGAGCTCCTGAG-3', 5'-GCAATATCAGAGTAACTGTTGTAAAA-3'; hypoxanthine phosphoribosyltransferase (HPRT), 5'-CCAGCAAGCTTGCAACCTTAACCA-3', 5'-CACAGGGGGCAACTGACTAGTAATG-3'. To amplify the TGF-beta1 cDNA, PCR were performed using varying amounts (3-fold dilutions) of cDNA and 30 cycles of denaturing at 94°C for 45 s, annealing at 55°C for 45 s, and extension at 72°C for 1 min. Twenty-five cycles of denaturing at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min served to amplify the HPRT cDNA. All reactions were within the linear amplification range of PCR.

TGF-beta1 ELISA

Abs used for ELISA (capture and detection) were purchased from BD Pharmingen (23201D and 23212D). rTGF-beta1 was purchased from Genzyme/Techne. To detect TGF-beta1, culture supernatants were heated at 80°C for 10 min to convert TGF-beta1 into the active form, and ELISA was performed according to standard conditions. The detection limit for TGF-beta1 was 40 pg/ml.

Preparation of nuclear extracts

Splenocytes and lymph node cells were incubated with biotinylated mouse CD4 T lymphocyte enrichment mixture (BD Biosciences), and isolated CD4+ T cells were cultured with CD4 T cell-depleted splenocytes that had been pulsed with MCC. CD4+ T cells being cultured for 1–4 h were again isolated by eliminating other cells. In some experiments, splenocytes and lymph node cells were cultured with Ag for 48 h, cells were incubated with anti-CD4 MACS microbeads (Miltenyi Biotec) on ice, and CD4+ T cells were then separated using MACS columns. To isolate CD4+CD25+ and CD4+CD25– T cells, splenocytes were first incubated with biotinylated mouse CD4 T lymphocyte enrichment mixture, and negatively selected CD4+ T cells were then stained with FITC-labeled anti-CD25 Ab, followed by incubating with anti-FITC microbeads. CD4+CD25+ and CD4+CD25– cells were cultured with CD4 T cell-depleted splenocytes in the presence of 0.5 µM MCC for 48 h, and CD4+ T cells were isolated with anti-CD4 MACS microbeads. After washing in cold PBS, the cell pellet was resuspended in 100–200 µl of buffer A (10 mM HEPES (pH 7.9), 3 mM MgCl2, 10 mM NaCl, 0.1 mM EDTA, 300 mM sucrose, 0.5 mM DTT, and a mixture of protease inhibitors) and incubated on ice for 10 min. Nonidet P-40 was added to a final concentration of 0.1%, and cells were centrifuged at 800 x g for 1 min. The pellet was rinsed with the same volume of buffer A, resuspended in 15–30 µl of buffer B (20 mM HEPES (pH 7.9), 3 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT, and a mixture of protease inhibitors), and incubated on ice for 15 min. The suspension was mixed briefly and centrifuged at 16,000 x g for 5 min. Protein concentration was determined and extracts were stored at –70°C.

EMSAs

EMSAs were performed with 3 µg (1 µg for the assay using CD4+CD25+ and CD4+CD25– fractions) of nuclear extract incubated with 1 µg of poly(dI:dC) in 20 µl of binding buffer (10 mM HEPES (pH 7.9), 50 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 10% glycerol, 0.1% Nonidet P-40, 0.5 mg/ml BSA, 1 mM benzamidine, and 1 mM DTT) and 4 µl of 32P-labeled probe (~1 ng) for 20 min at room temperature. For cold competition, nuclear extracts were preincubated for 10 min on ice with 100 ng of double-stranded oligonucleotide resembling NF-AT or AP-1 binding sites before the addition of the 32P-labeled probe. For supershift experiments, 3 µg of nuclear extracts was preincubated for 30 min at 4°C with 1 µg of anti-NF-AT c1 (7A6) or c2 (4G6-G5) (Santa Cruz Biotechnology), and then the probe was added before continuing binding reactions. For detection of NF-AT and AP-1 binding, double-stranded oligonucleotides used in EMSAs were as follows: NF-AT, 5'-CTGGTGTAATAAAATTTTCCAATGTAAAC-3'; AP-1, 5'-TCGAAAGCATGAGTCAGACA-3'. Complexes were resolved on nondenaturing 6% polyacrylaminde gels in 0.5x Tris-Borate-EDTA for 3 h at 155 V. EMSAs with TGF-beta1 probes were performed using double-stranded oligonucleotides from the TGF-beta1 proximal promoter as follows: 5'-AGAGAAGAGGAAAAAAGTTTTGAGACTTTTCCGCTGC-3' and 5'-GCTACTGCAAGTCAGAGACGT-3'.

Chromatin immunoprecipitation (ChIP) assay

ChIP assays were performed according to the manufacturer’s instructions (Upstate Biotechnology). Briefly, 8 x 106 CD4+ T cells were fixed after Ag stimulation with 1% formaldehyde, washed with cold PBS, and lysed in buffer containing a mixture of protease inhibitors. Nuclei were sonicated to shear DNA, the lysates were centrifuged, and supernatants were diluted. Diluted lysates were precleared with salmon sperm DNA/protein A-agarose for 30 min before immunoprecipitation. A proportion (3%) of the diluted supernatant was kept as input to quantify genomic DNA. After immunoprecipitation with rabbit polyclonal anti-c-Jun Abs, anti-NF-ATc2 Abs, or normal rabbit IgG (Santa Cruz Biotechnology), the immune complexes were incubated with salmon sperm DNA/protein A-agarose, the protein-DNA complexes were eluted in 1% SDS/0.1 M NaHCO3, and cross-links were reversed at 65°C. DNA was recovered by phenol-chloroform extraction and ethanol precipitation before subjecting DNA to PCR analysis. PCR for the IL-2 gene and the TGF-beta1 gene were conducted for 35 cycles of 50 s of denaturation at 94°C, annealing for 1 min at 58°C (IL-2) or at 63°C (TGF-beta1), and extention for 1 min at 72°C. The reactions for the TGF-beta1 gene were performed in the presence of 5% DMSO. The PCR products were analyzed in 2% agarose gels and visualized by ethidium bromide staining. The sequences of 5' and 3' primers were as follows: the IL-2 promoter region, 5'-GCCACCTAAGTGTTGGGCTAA-3', 5'-ATATGGGGGTGTCACGATGT-3'; the TGF-beta1 promoter region, 5'-CGAGCTGGTTGAGAGAAGAGGAAA-3', 5'-TGTTCTTAAATAGGGGAGCTACTGC-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TGF-beta1 expression in self Ag-induced tolerant T cells

Using mice expressing both the AND TCR, which specifically recognizes a C-terminal peptide of MCC, and altered peptide ligands that have a single amino acid substitution at position 99 or 102 of MCC88–103, we have shown previously that a large portion of AND T cells differentiates into CD25+ regulatory T cells (22). AND T cells that developed in mice expressing a MCC peptide with a T to E replacement at position 102 (AND/102E) were analyzed for TGF-beta1 expression and secretion in response to Ag (MCC88–103; Fig. 1). TGF-beta1 and IL-2 mRNA levels in stimulated and unstimulated cells were determined by RT-PCR (Fig. 1A), and TGF-beta1 secreted into the medium by the same cells was measured by TGF-beta1-specific ELISA (Fig. 1B). Although low levels of the TGF-beta1 mRNA were detected in CD4+ T cells from both AND and AND/102E mice before stimulation, no TGF-beta1 protein was detected in the supernatants. Ag stimulation increased both the TGF-beta1 mRNA and protein levels significantly in AND/102E CD4+ T cells, but not in AND CD4+ T cells. In control experiments, CD4+ T cells from AND/102E mice expressed some IL-2 mRNA, but did not secrete detectable amounts of IL-2 upon Ag stimulation, whereas those from AND mice expressed high levels of IL-2 mRNA and secreted ~5 ng/ml IL-2 (data not shown), as would be expected from fully stimulated T cells. Approximately 20–30% of CD4+ T cells from AND/102E mice express CD25, and we further separated the CD4+ T cells into CD25+ and CD25– populations. TGF-beta1 expression after Ag stimulation was observed mainly in CD4+CD25+ cells (Fig. 1C), indicating that, among the tolerant CD4+ T cells from AND/102E mice, the CD4+CD25+ regulatory T cells expressed TGF-beta1. Thus, in AND/102E CD4+CD25+ T cells, stimulation of the TCR activates the TGF-beta1 promoter and leads to secretion of TGF-beta1.


Figure 1
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  		FIGURE 1. TGF-beta1 expression in Ag-stimulated regulatory T cells. A, Splenocytes and lymph node cells obtained from AND or AND/102E mice were stimulated with 0.5 µM MCC88–103 for 48 h. Cells were harvested and incubated with anti-CD4 microbeads for 15 min on ice. CD4+ T cells were separated using MACS columns. RNA was prepared from CD4+ T cells immediately after elution from the column, and cDNA was synthesized from 5 µg of RNA. PCR was performed using decreasing amounts of cDNA (3-fold dilutions) and TGF-beta1-, IL-2-, and HPRT-specific primer sets. B, ELISA was used to determine TGF-beta1 protein levels in the culture supernatants of the same cells used in A. C, CD4+ T cells were selected from AND/102E splenocytes by depleting other cells using a mouse CD4+ T lymphocyte enrichment mixture (BD Biosciences). Cells were then incubated with biotin-labeled anti-CD25 Ab, followed by PE-streptavidin. CD25+ cells were sorted using anti-PE microbeads. CD4+CD25+ and CD4+CD25– cells were cultured with CD4+ T cell-depleted splenocytes in the presence of 0.5 µM MCC for 48 h, total RNA was prepared from CD4+ T cells, and PCR was performed as in A. D, Nuclear extracts were prepared from CD4+ T cells from either normal (AND) or tolerant (AND/102E) mice, and were stimulated with Ag for 1, 2, and 4 h. Nuclear levels of NF-AT and AP-1 were measured by EMSA using specific probes. E, Expression of TGF-beta1 in Ag-stimulated AND/102E CD4+ T cells with or without CsA (1 µM) pretreatment for 15 min was measured by RT-PCR using decreasing amounts of cDNA (3-fold dilutions). In all RT-PCR experiments in A, C, and E, HPRT expression was tested as a control.

 
The differential regulation of TGF-beta1 and IL-2 argues for the activation of distinct transcription factors during cytokine gene regulation. In particular, NF-AT and AP-1 are activated upon Ag stimulation of T cells, and are important for the expression of cytokines. Therefore, nuclear extracts from AND and AND/102E CD4+ T cells were prepared and analyzed for the presence of NF-AT and AP-1 activation upon Ag stimulation in EMSA experiments using NF-AT- and AP-1-specific probes (Fig. 1D). Two bands were detected in the extracts from AND CD4+ T cells with a NF-AT probe containing an AP-1 binding site adjacent to a NF-AT site. The upper band corresponds to AP-1 plus NF-AT, and the lower one corresponds to NF-AT (24). Activation of NF-AT was observed in both AND/102E CD4+ T cells and AND CD4+ T cells; however, the lower band was mainly detected in the extracts from AND/102E CD4+ T cells (Fig. 1D). This could be due to lower activation levels of AP-1. These observations would be expected from previous reports that AP-1 is impaired in anergic T cells, which argues for a role of NF-AT in signaling mediated by partial TCR activation. AND/102E CD4+ T cells were treated with cyclosporin A (CsA) to block NF-AT activation (25). TGF-beta1 mRNA levels were determined by RT-PCR in Ag-stimulated AND/102E CD4+ T cells with or without CsA treatment (Fig. 1E). TGF-beta1 expression in CsA-treated cells was suppressed almost to basal levels. Along with the results from the EMSA experiments, these observations suggest that NF-AT activation, and not AP-1, may be a positive regulator of TGF-beta1 expression in Ag-activated tolerant T cells.

TGF-beta1 promoter regulation

Putative regulatory elements for important transcription factors such as NF-{kappa}B, AP-1, NF-AT, various GATA factors, and SP-1 can be identified in the TGF-beta1 promoter by computational means (Fig. 2A). To further explore NF-AT-mediated TGF-beta1 expression, we cloned a 6-kb fragment of the mouse TGF-beta1 gene that included the regions from –4731 to +1269. To identify regulatory elements in the promoter, we generated various luciferase reporter constructs driven by promoter fragments extending from –1799 to +55, including various deletions, and from +55 to +793 of the proximal promoter (Fig. 2A). These constructs were then electroporated into T cell hybridoma 68-41 cells, and the basal promoter activities in unstimulated cells were measured 40 h later (Fig. 2B). Of the five constructs, a construct containing the promoter region from –231 to +55 showed the strongest reporter activity; this is most likely mediated by clusters of SP-1 sites in this region, and is consistent with results obtained in mouse fibroblasts (17). Next, we studied NF-AT-mediated activation of the TGF-beta1 promoter in the same cell line following treatment with 0.5 µM ionomycin or ionomycin plus PMA, inducers that lead to NF-AT activation (25) (Fig. 2C). Strong induction of promoter activity was observed for the construct comprising the proximal promoter region from +55 to +793. Similar results were obtained by stimulating the cells with plate-bound anti-CD3 Abs, indicating that ionomycin plus PMA and TCR activation have the same effect (data not shown). However, there was no augmentation of the activity of the –231 to +55 promoter fragment upon stimulation (Fig. 2C), arguing that the basal promoter activity is modulated by regulatory elements in other promoter regions. In this study, ionomycin induced low activity from constructs containing the –231 to –1079 promoter fragment; this activity was not further enhanced by combined treatment with ionomycin plus PMA (Fig. 2C). Thus, the proximal promoter region from +55 to +793 seemed to play a dominant role in the induction of TGF-beta1 expression in Ag-stimulated T cells.


Figure 2
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  		FIGURE 2. TGF-beta1 promoter analysis. A, The principal structure of the mouse TGF-beta1 promoter is depicted. Predicted putative binding sites for AP-1 (*), NF-{kappa}B ({blacklozenge}), SP-1 ( Figure 2), GATA ({square}), and NF-AT (Figure 2) are shown. Regions of high homology to the human promoter are shown as boxes (% homology indicated below). Mouse TGF-beta1 promoter fragments used in the luciferase assays are shown below the promoter schematic. B, Each construct containing a different promoter fragment was electroporated into T cell hybridoma cells. Cells were cultured for 40 h, and luciferase activities were measured without stimulation. C, Cells transfected as in B were cultured for 40 h and then cultured for an additional 6 h in the presence of 0.5 µM ionomycin (Figure 2), 50 ng/ml PMA plus 0.5 µM ionomycin ({blacksquare}), or in the absence of any stimuli ({square}). All values were normalized based on beta-Gal. Representative results of more than four individual experiments are shown (B and C).

 
NF-AT binding sites in the proximal TGF-beta1 promoter

As shown in Fig. 2A, several putative NF-AT sites were predicted to exist in the proximal promoter; one or more of these sites may control the NF-AT-mediated activation of the proximal TGF-beta1 promoter. T cell hybridoma 68-41 cells were cotransfected with the reporter gene construct containing the +55 to +793 region of the TGF-beta1 promoter together with increasing amounts of an expression vector for mouse NF-AT1, and reporter activities were measured with or without ionomycin or ionomycin plus PMA stimulation (Fig. 3A). Increasing amounts of the NF-AT1 expression vector and ionomycin stimulation enhanced the activity of the proximal promoter; this could be further elevated by combined treatment with ionomycin plus PMA. CsA treatment, which blocks NF-AT activation, suppressed promoter activation by ionomycin and by ionomycin plus PMA (Fig. 3B), suggesting that the proximal TGF-beta1 promoter contains at least one functional NF-AT binding site. At least two mechanisms may explain the strong additional stimulation by PMA, as follows: PMA may act independently on the proximal promoter through AP-1 sites in the vicinity of the NF-AT sites, or PMA may enhance the effects of ionomycin on NF-AT. Two of the putative NF-AT binding sites are found at positions +268 (AGGAA) and +288 (TTTCC) in the proximal promoter, just upstream of the second transcription start site, and putative AP-1 binding sites exist at positions +281 (TGAGAC) and +304 (AAGTCA). There is another putative NF-AT binding site at position +761 (TTCCT); however, we focused on upstream NF-AT and AP-1 sites. Because the close proximity of these NF-AT and AP-1 sites suggests the possibility of combinatorial regulation, NF-AT and AP-1 may act synergistically by forming ternary protein complexes (26). To confirm the function of these putative binding sites, we deleted each site from the proximal promoter (+55 to +793) reporter construct (Fig. 3C), and used these new reporter constructs to determine the function of each site. Constructs lacking each NF-AT or AP-1 binding site were transiently coexpressed in T cell 68-41 hybridoma cells along with CA-NF-AT1, AP-1 (c-Jun plus c-Fos), or both, and reporter gene activities were determined (Fig. 3D). The wild-type promoter fragment was induced by either CA-NF-AT1 or AP-1 expression, and coexpression of CA-NF-AT and AP-1 led to stronger induction. Removal of the first putative NF-AT site (deletion N-1) affected CA-NF-AT1-induced promoter activity. However, a much stronger reduction in CA-NF-AT1-induced promoter activity was observed for when the second NF-AT site was removed (deletion N-2), suggesting that binding of NF-AT to the second NF-AT site may be critical. Deletion of the second AP-1 site (deletion A-2) significantly reduced AP-1-induced promoter activity, whereas deletion of the first AP-1 site (deletion A-1) had little effect. These results suggest that activation of the proximal promoter may be largely dependent on both NF-AT and AP-1 binding sites.


Figure 3
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  		FIGURE 3. NF-AT motifs in the proximal TGF-beta1 promoter. A, The proximal TGF-beta1 promoter reporter was cotransfected into T cell hybridoma cells with increasing amounts of a NF-AT1 expression vector. Promoter activities were measured, as described in Fig. 2. B, T cell hybridoma cells transfected with the TGF-beta1 proximal promoter were cultured for 40 h, and then incubated for 15 min in the presence (Figure 3) or absence ({blacksquare}) of CsA. Cells were further cultured with or without stimulation for 6 h before luciferase activities were measured. C, Putative NF-AT and AP-1 binding sites in the TGF-beta1 proximal promoter were deleted as indicated in the schematic. D, T cell hybridoma cells were transfected with the TGF-beta1 proximal promoter or deletion constructs together with empty vector (Mock), CA-NF-AT1 expression vector, c-Fos and c-Jun expression vectors (AP-1), or CA-NF-AT and the c-Fos and c-Jun expression vectors (CA-NF-AT1 + AP-1). Luciferase activities were measured 40 h after transfection. Mean values ± SD of triplicate assays from one representative experiment of three are shown.

 
The putative NF-AT and AP-1 binding sites in the proximal TGF-beta1 promoter were further analyzed by EMSA experiments. CD4+ T cells were isolated from the lymph nodes and spleens of AND (normal T cells) or AND/102E (tolerant T cells) transgenic mice, and were stimulated with 0.5 µM MCC88–103 for 48 h before isolation of nuclear extracts. A few proteins or protein complexes were apparently able to bind to the upstream promoter fragment in the nuclear extracts from both AND CD4+ and AND/102E CD4+ T cells (probe A; Fig. 4A). Two complexes indicated by arrows were disrupted by addition of an excess of NF-AT-specific probe. However, the addition of AP-1-specific probe had no effect on either of the two complexes, consistent with our results regarding the ratio of NF-AT and AP-1 in the nucleus (see Fig. 1D). Binding of NF-AT proteins to probe A was further analyzed in nuclear extracts from AND/102E CD4+ T cells by adding Abs against NF-ATc1 (NF-AT-2) and NF-ATc2 (NF-AT-1). Although a NF-AT oligonucleotide competitor disrupted both the upper and the lower complexes, only the lower complex was shifted by Abs against NF-ATc1. NF-ATc2 Abs seemed to affect the lower complex and the upper one to some extent. These results suggested that both NF-ATc1 and NF-ATc2 could bind to the NF-AT sites in this promoter region, and that the upper complex might contain NF-ATc2. The second promoter fragment (probe B), which contains a putative AP-1 binding site, was tested for differential binding of nuclear proteins using the same nuclear extracts (Fig. 4B, left). A few proteins or protein complexes bound to this promoter fragment (probe B), but none of these complexes were disrupted by an excess of AP-1-specific probe. Nuclear extracts obtained from CD25+ and CD25– fractions of AND/102E CD4+ T cells were further tested for binding to probes A and B. Similar binding patterns for probe A were observed both in CD25+ and CD25– fractions (data not shown). Because nuclear extracts from CD25+ cells, which are the major producers of TGF-beta1, exhibited less binding to probe B (Fig. 4B, right panel), this promoter region may not play an important role for the expression of TGF-beta1 in tolerant T cells. Binding of NF-AT and AP-1 in this promoter region was further tested by ChIP assays with anti-NF-ATc2 and anti-c-Jun Abs in Ag-stimulated AND and AND/102E CD4+ T cells (Fig. 4C). NF-ATc2 bound to the proximal TGF-beta1 promoter region containing NF-AT/AP-1-binding motifs was detected in Ag-stimulated AND/102E CD4+ T cells. However, binding of NF-ATc2 to this TGF-beta1 promoter region was not observed in Ag-stimulated AND CD4+ T cells, which contained both NF-ATc2 and c-Jun bound to the proximal IL-2 promoter. Although c-Jun was detected in the IL-2 promoter, it was absent in the proximal TGF-beta1 promoter region in AND CD4+ T cells. These results indicate that c-Jun does not activate TGF-beta1 promoter in fully activated T cells. Therefore, binding of NF-AT to the TGF-beta1 promoter in tolerant T cells can be a key factor for TGF-beta1 expression.


Figure 4
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  		FIGURE 4. NF-AT- vs AP-1-mediated activation of the TGF-beta1 promoter. EMSA probes used in the assay are proximal TGF-beta1 promoter probe A (A) and probe B (B). Nuclear extracts were prepared from CD4+ T cells that were isolated from the lymph nodes and spleens of either AND or AND/102E mice and stimulated with 0.5 µM MCC88–103 for 48 h. Binding of nuclear proteins to the TGF-beta1 promoter region were analyzed in the absence or presence of double-stranded competitors for NF-AT (5'-GCCCAAAGAGGAAAATTTGTTTCATACAG-3') or AP-1 (5'-TCGAAAGCATGAGTCAGACA-3') (A), and double-stranded competitors for AP-1 (B). Abs against NF-ATc1 (NF-AT2) or NF-ATc2 (NF-AT1) were used (A, right panel). CD4+ T cells from AND/102E mice were separated into CD25+ and CD25– before stimulation, and cells were then stimulated with 0.5 µM MCC88–103 for 48 h in the presence of APCs (B, right panel). C, ChIP analysis in Ag-stimulated AND and AND/102E CD4+ T cells of the proximal IL-2 and the proximal TGF-beta1 promoter regions. CD4+ T cells were isolated after Ag stimulation for 18 h. Cells were lysed, and proteins were cross-linked and immunoprecipitated with anti-NF-ATc2 Ab, anti-c-Jun Ab, or rabbit IgG. After reversing the cross-linking, PCR was performed. One of three separate experiments is shown.

 
In addition to the two NF-AT binding sites (discussed above) in the proximal promoter, a third putative NF-AT element was predicted further downstream at position +761. However, no NF-AT-specific binding could be demonstrated for this element in EMSA experiments similar to those discussed above (data not shown). Together, the promoter transfection and EMSA experiments strongly suggest that NF-AT binds predominately to the proximal TGF-beta1 promoter at two positions adjacent to the second transcriptional initiation site. Furthermore, these NF-AT binding sites may mediate an AP-1-dependent synergistic activation of the proximal TGF-beta1 promoter. To confirm whether activation of NF-AT in the absence of AP-1 is sufficient to induce TGF-beta1 expression, an expression vector for CA-NF-AT1 with mutations that abrogate association with AP-1 (27) was cotransfected into T cell 68-41 hybridoma cells along with reporter gene constructs containing various regions of the TGF-beta1 promoter (Fig. 5). As expected from our previous results, active NF-AT1 induced strong reporter activity from the reporter construct containing the proximal promoter (+55 to +793). This activation was in the same range as the activation observed when the entire promoter (–1799 to +793) was used, and this promoter induction was essentially as strong as that from a NF-AT reporter construct containing three NF-AT binding sites (3x NF-AT, positive control). Lower promoter activity was induced from some of the other truncated promoter fragments; this was reminiscent of the results observed following ionomycin stimulation (Fig. 2C). Therefore, TGF-beta1 expression required only NF-AT activation and seems to be regulated differently than other cytokines, whose expression requires both NF-AT and AP-1.


Figure 5
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  		FIGURE 5. NF-AT-dependent activation of the TGF-beta1 promoter. Reporter gene constructs were electroporated into T cell hybridoma cells together with either a vector expressing CA-NF-AT1 lacking AP-1 binding (Figure 5) or a mock vector ({square}). Luciferase activities were measured 40 h after transfection. Mean values ± SD of triplicate assays from one representative experiment of three are shown.

 
Suppression of the TGF-beta1 promoter in Ag-stimulated T cells

Activation of NF-AT was observed both in AND and AND/102E CD4+ T cells, although TGF-beta1 expression was only induced in AND/102E CD4+ T cells. This argues for additional mechanisms, probably involving repressor elements, that modulate the NF-AT-mediated activation of the TGF-beta1 promoter. We therefore analyzed whether other signals in T cells may suppress the NF-AT-mediated activation of the proximal promoter as indicated by the basal activity of promoter regions upstream of position –231. To examine a negative regulatory activity in the upstream promoter regions, the region from –231 to –1079 was ligated to the proximal promoter fragment (+55 to +793) in either the forward or reverse orientation. Induction from these reporter constructs was analyzed in transfected T cell 68-41 hybridoma cells after treatment with ionomycin or ionomycin plus PMA (Fig. 6A). Under these conditions, regulatory elements in this upstream promoter element were able to reduce the activity of the proximal promoter.


Figure 6
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  		FIGURE 6. Upstream promoter regions balance the proximal TGF-beta1 promoter activity. A, Promoter activity from the proximal promoter (+55 to +793) ligated to an upstream promoter fragment (–1079 to –231) in either the forward or reverse orientation was tested under the same assay conditions as described in Fig. 2. One representative result from more than three individual experiments is shown. B, Promoter activities from the proximal promoter (+55 to +793), the wild-type promoter (–1799 to +793), a deletion (–1079 to –406) mutant, and TGAC or GATA deletion mutants were measured as described in Fig. 2. Averages ± SD of three independent experiments are shown.

 
To further analyze which elements in the upstream regions of the TGF-beta1 promoter might function as suppressor binding sites, additional deletion mutations within the entire promoter (–1799 to +793) were prepared. Computational searches for putative binding sites in the TGF-beta1 promoter identified a putative AP-1 site at –801 and a putative GATA site at –821 (see Fig. 2). In transfection experiments in T cell 68-41 hybridoma cells, deletion of the region from –1079 to –406 virtually reversed suppression of ionocycin- or ionomycin plus PMA-induced activation (Fig. 6B). We next examined the putative AP-1 and GATA binding sites within the upstream region. Deleting the GATA motif at position –821 significantly enhanced promoter activity upon PMA plus ionomycin stimulation. Deleting the AP-1 site at position –801 enhanced activity to a much lesser extent. These results suggest that a GATA or GATA-associated factor may be required to balance the NF-AT-mediated activation of the TGF-beta1 promoter in T cells after complete TCR activation.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Characterization of regulatory T cells is an important aspect of understanding the processes involved in immune responses. In our transgenic mouse model, which expresses an Ag-specific TCR and its ligand, large numbers of TGF-beta1-secreting regulatory CD4+ T cells develop. The expression of TGF-beta1 is a unique feature of these regulatory T cells, and distinguishes them from normal T cells. Taking advantage of this mouse model, we have studied the regulation of TGF-beta1 gene expression in regulatory T cells.

Engagement of TCRs leads to the activation of various signaling pathways, the most prominent of which is calcium-induced activation of the phosphatase, calcineurin, which dephosphorylates NF-AT. After dephosphorylation, NF-AT translocates from the cytoplasm into the nucleus, where it activates target genes, including many cytokines (6). Underscoring the central role of NF-AT in T cell responses, T cells lacking NF-AT-1 and NF-AT-2 are incapable of cytokine production (28). In accordance with other observations, we detected active NF-AT in nuclear extracts from stimulated T cells at substantially higher levels than observed for AP-1. Treatment of these cells with CsA decreased the induction of TGF-beta1, indicating that NF-AT is most likely involved in TGF-beta1 expression. Indeed, transfection experiments identified NF-AT and AP-1 binding sites in the proximal TGF-beta1 promoter that induced NF-AT-mediated activation. These motifs are perfectly conserved in the mouse and human TGF-beta1 promoters, arguing for the importance of this region in the regulation of TGF-beta1 expression. NF-AT and AP-1 have been shown to physically interact at paired binding sites, where signal-specific activation of these two proteins facilitates cooperative transcriptional synergies that lead to greater than additive induction of gene expression. Deletion of either of the two putative NF-AT motifs within the proximal TGF-beta1 promoter reduced promoter activity upon the expression of CA-NF-AT1. The expression of c-Jun and c-Fos augmented the promoter activity of constructs with deletions of one or the other putative NF-AT-binding motif. The expression of c-Jun and c-Fos was also able to augment the activity of a promoter construct with deletions of both NF-AT sites, although the induction of this promoter by CA-NF-AT1 was diminished (data not shown). Deletions of both the N-2 NF-AT site and the A-2 AP-1 site reduced promoter activity significantly; however, the distance between these two sites is most likely too large for cooperative binding of NF-AT and AP-1. This view is supported by results obtained using the wild-type proximal promoter, in which coexpression of NF-AT and AP-1 did not lead to synergistic induction of the promoter. Therefore, AP-1 and NF-AT may function independently in this proximal promoter. Indeed, NF-AT is able to activate the TGF-beta1 promoter independently, as shown by experiments using a mutated form of NF-AT that is unable to interact with AP-1 (27). Moreover, AP-1 binding to the putative AP-1-binding motifs adjacent to the NF-AT-binding motifs was not clearly detected in nuclear extracts from Ag-stimulated AND or AND/102E CD4+ T cells. These observations may explain why TGF-beta1 can be expressed in stimulated regulatory T cells even though they have only low AP-1 levels. Macián et al. (23) recently demonstrated that activation of NF-AT1 plays a central role in gene expression in anergic T cells. Because T cells that develop in AND/102E mice are anergic, as shown by undetectable IL-2 protein levels (22), the expression of TGF-beta1 in these T cells may be similarly regulated by NF-AT1 activation.

One of the more striking observations in our model system is that TGF-beta1 was differentially expressed in tolerant T cells and normal effector T cells. Although activation of NF-AT was observed in both normal (AND) and regulatory T cells (AND/102E), binding of NF-AT to the proximal TGF-beta1 promoter region was detected only in AND/102E CD4+ T cells by ChIP assays, suggesting that the expression of TGF-beta1 may be regulated through modulating chromatin structure. It is currently unclear how the epigenetic modification in this region is induced in tolerant T cells and whether other signals may affect TGF-beta1 expression in fully activated T cells. For instance, inducible cAMP early repressor can bind specifically to NF-AT-AP-1 composite sites, and forms inactive complexes with NF-AT, as has been shown for IL-2 and IL-4 expression in human thymocytes (29). Furthermore, it has been shown that Foxp3 directly suppresses NF-AT function in Th cells (30), and Foxp3 may suppress the expression of cytokines by binding to a forkhead consensus motif next to the NF-AT binding site (31), where Foxp3 is proposed to disrupt the NF-AT-AP-1 protein complex by displacing AP-1 (32). The expression of Foxp3 plays a distinctive role in T cell development (33, 34), and forced expression of Foxp3 converts normal naive T cells into regulatory T cells. It was shown that Foxp3 in association with NF-AT represses IL-2 expression, whereas it up-regulates expression of CTLA4 and CD25 (32). Foxp3 may also regulate TGF-beta1 expression, as a putative forkhead-binding motif is found at position –1701 in the TGF-beta1 promoter. However, there does not seem to be a Foxp3 binding site adjacent to the NF-AT motif identified in the proximal TGF-beta1 promoter.

Our promoter analysis using upstream promoter fragments showed that there is a negative regulatory region (between –1079 and –406) that suppresses the proximal promoter activity upon Ag stimulation. Deletion of one GATA-binding motif significantly augmented reporter activity upon stimulation, suggesting that a GATA-binding factor might be involved in the repression. Two-hybrid screens identified friend of GATA in a Th2 cell-specific library; this protein was shown to interact with GATA-3 and block the GATA-3-mediated expression of several cytokines (35). Friend of GATA is expressed in naive T cells and is down-regulated in the Th1 and Th2 lineages. Deletion of an AP-1-binding motif adjacent to the GATA-binding motif had a partial effect on the release of repression. However, because the two sites are very close, we cannot exclude the possibility that the deletion mutations disrupted a larger regulatory region and abrogated the formation of a larger nuclear complex. The composition of such a complex is not known, but putative binding sites for YY1 and other GATA motifs can be found in this region. In addition, some transcription factors activated by calcium mobilization may also function as suppressors in this negative regulatory region because ionomycin stimulation induced only poor promoter activity on the construct containing the negative regulatory region, whereas expression of CA-NF-AT1 could induce strong promoter activity on the same construct.

Our data suggest a key role for NF-AT in the induction of the TGF-beta1 promoter; NF-AT most likely acts in a wider context with currently uncharacterized transcription factors that bind to upstream regions of the promoter. It can be speculated, therefore, that tolerant T cells may down-regulate factors needed to suppress TGF-beta1 expression. We have shown previously that weak activation of naive AND T cells by stimulation with alternative peptide ligands induces the expression of TGF-beta1 (5). These T cells give rise to anergic T cells that can express TGF-beta1 upon Ag stimulation, indicating that TGF-beta1 is induced by weak T cell stimulation. The state of activation of downstream components of the TCR signaling pathway in normal T cells after weak stimulation might be similar to that in Ag-stimulated tolerant T cells, in which strong signals are blocked due to their anergic status. Ligation of CTLA-4 expressed in regulatory T cells, which induces negative signals, might play a similar role in reducing integrated signaling levels and promoting the expression of TGF-beta1 (36). Further studies of gene regulation in these T cells will greatly help our understanding of the mechanisms underlying regulatory T cell function.


   Acknowledgments
 
We are grateful to Dr. R. Mizuta for providing us with a genomic C57BL/6 mouse library, and to Drs. F. Macián and A. Rao for providing us with the retroviral expression vectors GFP-KV-DV-CA-NF-AT1, GFP-KV-DV-CA-RIT-NF-AT1, and GFP-KV-DV. Dr. M. Kubo provided the pGL2 reporter gene containing three NF-AT binding sites, and a NF-AT1 expression vector. Dr. T. Tokuhisa provided c-Fos and c-Jun expression vectors. We are very grateful to Drs. A. Rao and H. Kikutani for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 the Ministry of Education, Science, Sports and Culture of the Government of Japan (to N.N.). Back

2 Address correspondence and reprint requests to Dr. Naoko Nakano, Research Institute for Biological Sciences, Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan. E-mail address: naoko{at}rs.noda.tus.ac.jp Back

3 Current address: Department of Immunology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan. Back

4 Current address: Department of Pathology and Center of Immunology, Washington University School of Medicine, Howard Hughes Medical Institute, St. Louis, Missouri 63110. Back

5 Abbreviations used in this paper: MCC, moth cytochrome c; beta-Gal, beta-galactosidase; CA, constitutively active; ChIP, chromatin immunoprecipitation; CsA, cyclosporin A; HPRT, hypoxanthine phosphoribosyltransferase. Back

Received for publication December 12, 2005. Accepted for publication December 14, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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