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

Abstract

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.

Identification of novel interacting regions involving calcineurin and nuclear factor of activated T cells

“…The expression of IL‐4 and IL‐17 was enhanced by the introduction of NFATc2 and, more vigorously, by NFATc1 . In addition to the cytokine selectivity expected with the contribution of CNBR3, we have found that endogenously expressed NFATc4 plays a suppressive role in biological functions mediated by the other NFAT isoforms . Therefore, the pharmacological and adverse effects of drugs targeting CN‐Nc3/p8‐binding are possibly more and less potent, respectively, than those of nonselective CN and NFAT inhibitors.…”

“…Jurkat-TAg cells were transfected with pEF-NFATc1-CNBR3 expression vector by electroporation and cultured, as described previously. 19 Total RNA was extracted from 2 × 10 5 cells with or without stimulation with 1 μM IOM plus 5 nM phorbol 12-myristate 13-acetate (PMA) for 6 hours. Using 1 μg of total RNA, cDNA was synthesized by SuperScript III reverse transcriptase (Thermo Fisher Scientific).…”

“…Thus, NFATc4 downregulation in T cells using RNA interference technology results in augmented IL-2 expression, whereas knockdown of NFATc1, NFATc2, and NFATc3 suppresses it. 19 Consistently, in contrast to facilitating tumor invasion and metastasis in breast cancer by NFATc1 and NFATc2, NFATc4 act as a tumor suppressor. 29,30 Therefore, the specific regulation of some NFATc isoforms is promising for not only reducing side effects but also for augmenting clinical efficacy of NFAT-targeting agents.…”

“…Furthermore, we have found that NFATc4 has an opposite function to other NFATcs in terms of cytokine expression. Thus, NFATc4 downregulation in T cells using RNA interference technology results in augmented IL‐2 expression, whereas knockdown of NFATc1, NFATc2, and NFATc3 suppresses it . Consistently, in contrast to facilitating tumor invasion and metastasis in breast cancer by NFATc1 and NFATc2, NFATc4 act as a tumor suppressor …”

“…However, each NFAT family member exhibits distinct expression patterns in many tissues and is involved in different diseases. 10,18,19 In murine models of allergic diseases, NFATc1 preferentially promotes Th2 responses, while NFATc2 exhibits a complementary contribution to asthma phenotypes 20 or suppressive effects on Th1/Th17-type skin inflammation. 21 In terms of malignancy, NFATc1 induces the metastasis of colorectal cancer cells, 22 whereas NFATc2 expression is upregulated in the pulmonary cancer with a poor prognosis 23 and in advanced pancreatic cancer.…”