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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 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, Ca-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.

Background: Mast cells (MCs) play a central role in allergic reactions through high-affinity IgE receptor (FcεRI)-mediated responses. Many attempts have been performed to investigate MC functions, though molecular bases of the intracellular signaling cascade through FcεRI, especially in human MCs, remain scant and unexplored. Methods: Human MCs were differentiated from CD34+ cells by culture with stem cell factor, IL-6 and IL-3. The differential phosphorylation profiles of protein tyrosine residues in the resulting MCs with or without FcεRI aggregation were examined by two-dimensional gel electrophoresis. The candidate phosphoproteins of interest were picked, in-gel digested and mass spectrometry fingerprinted. Results: Approximately 40 proteins in MCs were phosphorylated on their tyrosine residues in response to activation and some of them were identified. Particularly IL-31 receptor α, solute carrier family 39, syntaxin 5 and heterogeneous nuclear ribonucleoprotein are newly identified as phosphoproteins that are potentially involved in the MC signaling cascade through FcεRI. Conclusion: Our present phosphoproteome data may provide the clue to understand the molecular mechanisms for the activation of human MCs.

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.

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