Requirement for transcription factor NFAT in interleukin-2 expression

Abstract

The nuclear factor of activated T cells (NFAT) transcription factor is implicated in expression of the cytokine interleukin-2 (IL-2). Binding sites for NFAT are located in the IL-2 promoter. Furthermore, pharmacological studies demonstrate that the drug cyclosporin A inhibits both NFAT activation and IL-2 expression. However, targeted disruption of the NFAT1 and NFAT2 genes in mice does not cause decreased IL-2 secretion. The role of NFAT in IL-2 gene expression is therefore unclear. Here we report the construction of a dominant-negative NFAT mutant (dnNFAT) that selectively inhibits NFAT-mediated gene expression. The inhibitory effect of dnNFAT is mediated by suppression of activation-induced nuclear translocation of NFAT. Expression of dnNFAT in cultured T cells caused inhibition of IL-2 promoter activity and decreased expression of IL-2 protein. Similarly, expression of dnNFAT in transgenic mice also caused decreased IL-2 gene expression. These data demonstrate that NFAT is a critical component of the signaling pathway that regulates IL-2 expression.

FIG. 1

The PxIxIT domain acts as a dominant inhibitor of NFAT transcription activity. (A) Schematic representation of the NH₂-terminal region of NFAT transcription factors. The conserved SP boxes (A, B, and C), TAD, PxIxIT motif, YRE motif, and SRR are indicated. Mutation of the PxIxIT motif by replacement of the Pro, Ile, and Thr residues with Ala (AxAxAA) is indicated by a cross. The deletion mutations correspond to the NFAT3 isoform. (B) Expression of the NH₂-terminal NFAT homology region inhibits NFAT-mediated transcription activity. Various NFAT3 deletion mutants (residues 1 to 450, 1 to 365, and 1 to 160) were coexpressed with full-length NFAT2 and an NFAT-luciferase reporter plasmid in BHK cells. Luciferase activity was measured in cultures incubated without (Untreated) or with ionomycin (2 μM) and PMA (100 nM) (I+P). The data are presented as fold activation compared to an untreated control. (C) The PxIxIT motif is responsible for the dominant-negative activity of the NH₂-terminal NFAT homology region. The effects of NFAT3 deletion mutants (residues 1 to 160, 1 to 130, and 1 to 112) on NFAT2-mediated transcription activity were examined by using an NFAT-luciferase reporter plasmid in BHK cells. The effect of mutation of the PxIxIT motif by replacement of the Pro, Ile, and Thr residues with Ala (AxAxAA) was investigated. Luciferase activity was measured in cultures incubated without (Untreated) or with ionomycin (2 μM) and PMA (100 nM) (I+P). The data are presented as fold activation compared to an untreated control. (D) Epitope-tagged Flag-NFAT3 proteins were expressed in COS cells, and detected by protein immunoblotting of cell lysates with MAb M5, specific to the Flag epitope (Sigma). Sizes are indicated in kilodaltons.

FIG. 2

dnNFAT inhibits transcription activity of all four NFAT isoforms. NFAT proteins were expressed in BHK cells together with dnNFAT (NFAT3 amino acids 1 to 130). The effect of mutation of the PxIxIT motif by replacement of the Pro, Ile, and Thr residues with Ala (AxAxAA) was investigated. Cotransfection assays in BHK cells using an NFAT-luciferase reporter plasmid and NFAT1 (A), NFAT2 (B), NFAT3 (C), and NFAT4 (D) were performed. Luciferase activity was measured in cultures treated without (open bar) and with (filled bar) PMA and ionomycin. The data are presented as fold activation compared to an untreated control.

FIG. 3

AP-1 and NF-κB transcription activities are not inhibited by dnNFAT. NFAT, AP-1, and NF-κB transcription activities were measured by using luciferase reporter plasmids cotransfected in Jurkat T cells without (Control) and with dnNFAT. Luciferase activity was measured in cultures incubated with ionomycin (2 μM) and PMA (100 nM). The data are presented as relative percentage activity compared to a control without dnNFAT.

FIG. 4

Mechanism of dominant inhibitory activity of dnNFAT. (A) Transcription activity mediated by the COOH-terminal region of NFAT is not affected by dnNFAT. Full-length NFAT4 and the NFAT4 COOH-terminal region (NFAT4 Rel; residues 365 to 708) were expressed together with an NFAT-luciferase reporter plasmid in BHK cells without (Control) and with dnNFAT. Luciferase activity was measured in cultures incubated with ionomycin (2 μM) and PMA (100 nM). The data are presented as relative percentage activity compared to a control without dnNFAT. (B) Transcription activity mediated by the NH₂-terminal activation domain of NFAT is not affected by dnNFAT. GAL4-NFAT4 fusion proteins were expressed in BHK cells together with a GAL4-luciferase reporter plasmid and dnNFAT. Luciferase activity was measured in cultures incubated with ionomycin (2 μM) and PMA (100 nM). The data are presented as relative percentage activity compared to a control without dnNFAT. The effect of replacement of the phosphorylation sites Ser-163 and Ser-165 with Ala is shown. DBD, DNA binding domain. (C) Regulation of the subcellular distribution of NFAT proteins by dnNFAT. NFAT1 and NFAT2 were coexpressed with dnNFAT in BHK cells. Immunofluorescence analysis was performed on cells treated without or with ionomycin (2 μM, 30 min). NFAT proteins (red) and the nucleus (blue) were visualized. Arrowheads indicate the nuclei of cells expressing transfected proteins. (D) Overexpression of calcineurin opposed the inhibitory effect of dnNFAT. Various amounts of calcineurin expression vector (50 and 100 ng) were coexpressed with dnNFAT in BHK cells. Immunofluorescence analysis was performed to examine the subcellular distribution of NFAT1 and NFAT2 proteins in the absence or presence of ionomycin (2 μM, 30 min). One hundred transfected cells were examined. The percentage of cells with NFAT in the nucleus is presented.

FIG. 4

Mechanism of dominant inhibitory activity of dnNFAT. (A) Transcription activity mediated by the COOH-terminal region of NFAT is not affected by dnNFAT. Full-length NFAT4 and the NFAT4 COOH-terminal region (NFAT4 Rel; residues 365 to 708) were expressed together with an NFAT-luciferase reporter plasmid in BHK cells without (Control) and with dnNFAT. Luciferase activity was measured in cultures incubated with ionomycin (2 μM) and PMA (100 nM). The data are presented as relative percentage activity compared to a control without dnNFAT. (B) Transcription activity mediated by the NH₂-terminal activation domain of NFAT is not affected by dnNFAT. GAL4-NFAT4 fusion proteins were expressed in BHK cells together with a GAL4-luciferase reporter plasmid and dnNFAT. Luciferase activity was measured in cultures incubated with ionomycin (2 μM) and PMA (100 nM). The data are presented as relative percentage activity compared to a control without dnNFAT. The effect of replacement of the phosphorylation sites Ser-163 and Ser-165 with Ala is shown. DBD, DNA binding domain. (C) Regulation of the subcellular distribution of NFAT proteins by dnNFAT. NFAT1 and NFAT2 were coexpressed with dnNFAT in BHK cells. Immunofluorescence analysis was performed on cells treated without or with ionomycin (2 μM, 30 min). NFAT proteins (red) and the nucleus (blue) were visualized. Arrowheads indicate the nuclei of cells expressing transfected proteins. (D) Overexpression of calcineurin opposed the inhibitory effect of dnNFAT. Various amounts of calcineurin expression vector (50 and 100 ng) were coexpressed with dnNFAT in BHK cells. Immunofluorescence analysis was performed to examine the subcellular distribution of NFAT1 and NFAT2 proteins in the absence or presence of ionomycin (2 μM, 30 min). One hundred transfected cells were examined. The percentage of cells with NFAT in the nucleus is presented.

FIG. 5

dnNFAT causes a dose-dependent inhibition of NFAT activity in Jurkat T cells. Increasing amounts (1, 3, 6, and 9 μg) of a dnNFAT expression vector were transfected in Jurkat T cells. The transcription activity of endogenous NFAT was detected with an NFAT-luciferase reporter plasmid. The effect of mutation of the PxIxIT motif by replacement of the Pro, Ile, and Thr residues with Ala (AxAxAA) was investigated. Luciferase activity was measured in cultures incubated without (−) or with (+) ionomycin (2 μM) and PMA (100 nM) (I+P). The data are presented as fold activation compared to an untreated control.

FIG. 6

dnNFAT inhibits IL-2 production. (A) The activity of the IL-2 promoter is inhibited by dnNFAT. Jurkat T cells were cotransfected with an IL-2 promoter-luciferase reporter plasmid and various amounts (1 and 10 μg) of dnNFAT expression vector. The effect of mutation of the PxIxIT motif by replacement of the Pro, Ile, and Thr residues with Ala (AxAxAA) was investigated. Luciferase activity was measured in cultures incubated without (−) or with (+) ionomycin (2 μM) and PMA (100 nM) (I+P). The data are presented as fold activation compared to an untreated control. (B) IL-2 secretion is inhibited by dnNFAT. Jurkat T cells were cotransfected with expression vectors for GFP and dnNFAT (either wild-type PxIxIT or mutated AxAxAA). Transfected cells expressing GFP were selected by flow cytometry and treated without (Untreated) or with ionomycin (2 μM) and PMA (100 nM) (I+P), and the amount of IL-2 secreted in the culture medium was measured. (C) IL-2 expression is inhibited by dnNFAT. Jurkat T cells were cotransfected without (Control) and with expression vectors for GFP and dnNFAT (either wild-type PxIxIT or mutated AxAxAA). The cells were treated without (thin line) or with (thick line) ionomycin (2 μM) and PMA (100 nM) (I+P). The intracellular IL-2 and GFP was measured by flow cytometry. IL-2 expression (mean fluorescence intensity) of the transfected GFP positive (+) and untransfected GFP negative (−) cells in each culture is shown. (D) Thymocytes from dnNFAT transgenic mice have reduced IL-2 expression. Thymocytes were isolated from dnNFAT transgenic mice (Tg+) and control nontransgenic littermates (NLC). Expression of dnNFAT was detected by protein immunoblot analysis using MAb M2, specific to the Flag epitope. Cells were stimulated with ionomycin (2 μM) and PMA (100 nM), and the amount of IL-2 secreted in the culture medium was measured. hGH, human growth hormone.