Downregulation of NFAT3 Due to Lack of T-Box Transcription Factor TBX5 Is Crucial for Cytokine Expression in T Cells.

The NFAT family transcription factors play crucial roles in immunological and other biological activities. NFAT3 is rarely expressed in T cells, and the mechanisms and significance of the specific NFAT3 downregulation in T cells have been unknown. In human CD4+ T cells, overexpression of NFAT1 and NFAT3 enhanced and suppressed IL-2 expression, respectively. NFAT3 downregulation in Jurkat cells using RNA interference technology augmented IL-2 expression, whereas a knockdown of NFAT1, NFAT2, and NFAT4 suppressed it. The promoter/enhancer activity of the NFAT-binding site in the IL-2 gene was upregulated and downregulated by NFAT1 and NFAT3, respectively. A study employing NFAT1/NFAT3 chimeric molecules revealed that the region in NFAT3 responsible for NFAT promoter activity inhibition was located within its N-terminal transactivation domain, Ca2+-regulatory domain, and DNA-binding domain. Downregulation of NFAT3 expression in T cells is mediated by lower chromatin accessibility and enhancer activity in its promoter in comparison with aortic smooth muscle cells expressing endogenous NFAT3. The binding sites of T-box transcription factor TBX5 and NK-2 transcription factor-related locus 5 Nkx2.5, which were expressed at higher levels in aortic smooth muscle cells than in T cells, were located within the -387 to +97 NFAT3 promoter region, exhibiting the maximum enhancer activity. Mutating the binding site of TBX5 but not Nkx2.5 diminished the NFAT3 promoter activity, whereas the overexpression of TBX5 enhanced it. Introduction of TBX5 into CD4+ T cells enhanced the expression of NFAT3 and suppressed that of IL-2. TBX5 deficiency-mediated downregulation of NFAT3 is crucial for the high cytokine-producing activity of T cells.

Protein phosphatase 1 is involved in IL-2-induced IL-5 and IL-13 expression in human T cells.

IL-2 plays an important role in immunological and other biological functions. This cytokine directly induces the production of several cytokines, such as IL-5 and IL-13. The mechanisms of IL-2-mediated cytokine synthesis are mostly unclear; however, the involvement of IL-2 receptor (IL-2R)ß has been suggested. In this study, the signaling molecule downstream of IL-2Rß was investigated, employing a proteomic approach. Full-length IL-2Rß and its mutant in which the intracellular component was truncated were introduced in an IL-2Rα- and IL-2Rγ-stably transfected T cell hybridoma, S1. The differential phosphorylation profiles of protein tyrosine residues in these cells upon IL-2 stimulation were examined by two-dimensional gel electrophoresis. The candidate phosphoproteins of interest were re-covered, in-gel digested and mass spectrometry fingerprinted. Among proteins specifically phosphorylated in full-length IL-2Rß-expressing cells in response to IL-2 stimulation, protein phosphatase (PP)1ß and FK506-binding protein 4 were identified. Particularly, PP1ß augmented IL-5 and IL-13 expression stimulated by IL-2 but not by anti-CD3 antibody in human peripheral CD4+ T cells upon ectopic expression. IL-2-induced cytokine expression was suppressed by overexpression of PP1 regulatory subunit 2. A PP1 inhibitor, tautomycin, but not a PP2A inhibitor, okadaic acid, also inhibited the IL-2R-mediated responses. It was conclusively shown that PP1 is crucially involved in IL-2-mediated IL-5 and IL-13 synthesis in human T cells.

NFAT1 and NFAT2 differentially regulate IL-17A expression in human T cells.

BACKGROUND: The NFAT family transcription factors play crucial roles in T cell functions. Recently, NFAT has been implicated in the production of an inflammatory cytokine, IL-17A; however, functional differences among NFAT members in IL-17A synthesis have not been elucidated. In this study, the relative contribution of NFAT1 and NFAT2 to IL-17A expression in human T cells was investigated. METHODS: NFAT1 and NFAT2 were introduced in human cord blood CD4+ T cells by a lentiviral transduction system. Then, the expression of IL-17A mRNA was determined by quantitative real-time RT-PCR. The transient effects of NFAT1 and NFAT2 on IL-17A expression in Jurkat-Tag cells were also investigated. RESULTS: Stimulation-induced expression of IL-17A in human CD4+ T cells was augmented by the introduction of NFAT1 and more vigorously, NFAT2. IL-17A expression in Jurkat-Tag cells was also enhanced by NFAT1, whereas it was not affected by NFAT2. CONCLUSION: NFAT1 and NFAT2 facilitated IL-17A expression in human T cells, though distinct mechanisms might be involved in these effects.

Zinc finger protein, multitype 1, suppresses human Th2 development via downregulation of IL-4.

BACKGROUND: Among several GATA family transcription factor-associating proteins, zinc finger protein, multitype 1 (ZFPM1), at least that of murine origin, has been shown to modulate the activity of GATA-3. However, the functional role of human ZFPM1 in the immune system has not been elucidated. Therefore, we here investigated the contribution of ZFPM1 to human Th1/Th2 differentiation. METHODS: The cDNA of ZFPM1 was cloned and introduced into human cord blood CD4+ T cells by a lentiviral transduction system. Then, the expression of IL-4 and IFN-γ mRNA was determined by quantitative real-time RT-PCR. The effect of ZFPM1 on the promoter activity of IL-4 and IFN-γ in Jurkat cells was also investigated. RESULTS: Stimulation-induced expression of IL-4 and IFN-γ in human CD4+ T cells was suppressed and enhanced, respectively, by the introduction of ZFPM1. The transcriptional activity of IL-4 was also diminished by ZFPM1, whereas that of IFN-γ was not affected. CONCLUSION: ZFPM1 that facilitates human Th1 differentiation via the downregulation of IL-4 is a potential target for the treatment of allergic diseases.

Selective down-regulation of Th2 cytokines by C-terminal binding protein 2 in human T cells.

BACKGROUND: Among several C-terminal binding proteins (CtBPs), friend of GATA (FOG) has been implicated in the down-regulation of GATA-3-mediated Th2 cell differentiation. Here we investigated the role of CtBP2 in Th1 and Th2 cytokine expression in human T cells. METHODS: CtBP2 was introduced into human peripheral CD4+ T cells by a lentiviral transduction system. Subsequently, the expression of Th1 and Th2 cytokine mRNA was determined by quantitative real-time RT-PCR. RESULTS: CtBP2 significantly suppressed stimulation-induced expression of IL-4, IL-5 and IL-13 in human T cells. However, IFN-gamma expression was not affected by the introduction of CtBP2. CONCLUSION: CtBP2 selectively down-regulates Th2 cytokines, therefore it is a potential target for the treatment of allergic diseases.

T-box 21 transcription factor is responsible for distorted T(H)2 differentiation in human peripheral CD4+ T cells.

BACKGROUND: Regardless of T(H)1/T(H)2 theory, CD4(+) T cells of patients with allergic asthma, a typical T(H)2 disease, and those of healthy subjects expressed equivalent levels of IFN-gamma, even though T(H)2 cytokines were significantly upregulated in asthmatic patients. OBJECTIVE: The mechanisms underlying distorted T(H)2 cell polarization in human T cells were elucidated. METHODS: Cytokine-producing activity and the expression of T(H)1/T(H)2-specific transcription factors in naïve, T(H)1/T(H)2, or both CD4(+) T cells derived from human peripheral and cord blood were comparatively analyzed. The mechanisms of the differential expression of T-box 21 transcription factor (T-bet) in the cells were assessed by determining the chromatin accessibility at the TBX21 gene. The functional roles of T-bet and other transcription factors in human T(H)1/T(H)2 differentiation were further investigated. RESULTS: T(H)2 cells derived from naive CD4(+) T cells in peripheral blood but not in cord blood produced IFN-gamma. T-bet was expressed in peripheral, but not cord blood, resting naive T cells. Consistently, the accessibility at the proximal TBX21 gene promoter in peripheral naive T cells was higher than that in cord blood naive T cells. IFN-gamma-producing activity was induced in T(H)2-differentiated cord blood T cells by means of ectopic expression of T-bet. In addition, a reduction of T-bet in peripheral T cells suppressed IFN-gamma production. T-bet not only upregulated IFN-gamma but also downregulated IL-4 and IL-13 gene transcription, independently of the modification of T(H)1/T(H)2 balance. CONCLUSION: The expression of T-bet at a naive stage is crucial for the development of IFN-gamma-producing T cells in human peripheral blood, even in T(H)2-related diseases.

Suppressive role of C-terminal binding protein 1 in IL-4 synthesis in human T cells.

The functional role of C-terminal binding protein (CtBP)1, a transcriptional corepressor, in Th1 and Th2 cytokine expression in human T cells was investigated. Upon introduction of CtBP1 by lentiviral transduction system, IL-4 synthesis was suppressed but IFN-gamma was weakly up-regulated in human CD4(+) T cells. In contrast, a reduction of endogenously expressed CtBP1 in Jurkat T cells using RNAi technology selectively augmented IL-4 expression. The down-regulation of IL-4 by CtBP1 was achieved at the level of gene transcription. Deletion mutation analysis revealed that N-terminal approximately 200 amino acid and C-terminal approximately 50 amino acid residues are participated in CtBP1-mediated suppression of IL-4 expression. CtBP1 expressed in human CD4(+) T cells crucially contribute to Th1/Th2 differentiation via selective down-regulation of IL-4 synthesis.

Downregulation of IL-13 gene transcription by T-bet in human T cells.

BACKGROUND: Downregulation of a Th2 cytokine, IL-4, by a Th1-specific transcription factor, T-bet, has been demonstrated. However, the regulatory role of T-bet in another Th2 cytokine, IL-13, is not fully delineated. METHODS: IL-13 mRNA expression in Jurkat cells was examined by quantitative RT-PCR, while the transcriptional activity of 5'-flanking region in the IL-13 gene encompassing -1077 to +49 was investigated by fluorescence-based promoter reporter assay. The effect of T-bet was investigated by transfection of the cells with the T-bet expression vector. RESULTS: Stimulation with phorbol ester plus Ca2+ ionophore clearly induced IL-13 gene transcription in Jurkat cells. Ectopically expressed T-bet significantly suppressed the inducible mRNA expression and promoter activity of IL-13. CONCLUSION: IL-13 expression was downregulated by T-bet at the level of gene transcription, independently of the modulation of Th1/Th2 balance. T-bet is the potential key factor in the development of Th1/Th2-related diseases.

IL-4 gene transcription in human T cells is suppressed by T-bet.

UNLABELLED: T-bet is crucially implicated in Th1 differentiation due to its strong promoting activity for IFN-gamma gene transcription. However, the regulatory role of T-bet in Th2 cytokines is not fully delineated. METHODS: The effect of T-bet on mRNA expression as well as the promoter activity of IL-4 in human T cells was investigated by employing quantitative RT-PCR and fluorescence-based promoter reporter assay procedures. RESULTS: IL-4 mRNA expression as well as the transcriptional activity of 5'-flanking region in the IL-4 gene encompassing -1105 to +4 in Jurkat cells was clearly upregulated upon stimulation. The inducible mRNA expression and the promoter activity of IL-4 were significantly diminished by ectopic expression of T-bet. CONCLUSION: IL-4 gene transcription is inhibited by T-bet via the suppression of its promoter activity, independently of IFN-gamma. T-bet facilitates Th1 differentiation through not only upregulation of IFN-gamma, but also downregulation of IL-4 gene transcription.