NFATc1 Regulation of TRAIL Expression in Human Intestinal Cells

TNF-related apoptosis-inducing ligand (TRAIL; Apo2) has been shown to promote intestinal cell differentiation. Nuclear factor of activated T cells (NFAT) participates in the regulation of a variety of cellular processes, including differentiation. Here, we examined the role of NFAT in the regulation of TRAIL in human intestinal cells. Treatment with a combination of phorbol 12-myristate 13-acetate (PMA) plus the calcium ionophore A23187 (Io) increased NFAT activation and TRAIL expression; pretreatment with the calcineurin inhibitor cyclosporine A (CsA), an antagonist of NFAT signaling, diminished NFAT activation and TRAIL induction. In addition, knockdown of NFATc1, NFATc2, NFATc3, and NFATc4 blocked PMA/Io increased TRAIL protein expression. Expression of NFATc1 activated TRAIL promoter activity and increased TRAIL mRNA and protein expression. Deletion of NFAT binding sites from the TRAIL promoter did not significantly abrogate NFATc1-increased TRAIL promoter activity, suggesting an indirect regulation of TRAIL expression by NFAT activation. Knockdown of NFATc1 increased Sp1 transcription factor binding to the TRAIL promoter and, importantly, inhibition of Sp1, by chemical inhibition or RNA interference, increased TRAIL expression. These studies identify a novel mechanism for TRAIL regulation by which activation of NFATc1 increases TRAIL expression through negative regulation of Sp1 binding to the TRAIL promoter

Interferon Regulatory Factor 2 Binding Protein 2 Is a New NFAT1 Partner and Represses Its Transcriptional Activity▿

The nuclear factor of activated T cells (NFAT) family of transcription factors is expressed in a wide range of cell types and regulates genes involved in cell cycle, differentiation, and apoptosis. NFAT proteins share two well-conserved regions, the regulatory domain and the DNA binding domain. The N- and C-terminal ends are transactivation sites and show less sequence similarity, whereas their molecular functions remain poorly understood. Here, we identified a transcriptional repressor, interferon regulatory factor 2 binding protein 2 (IRF-2BP2), which specifically interacts with the C-terminal domain of NFAT1 among the NFAT family members. IRF-2BP2 was described as a corepressor by inhibiting both enhancer-activated and basal transcription. Gene reporter assays demonstrated that IRF-2BP2 represses the NFAT1-dependent transactivation of NFAT-responsive promoters. The ectopic expression of IRF-2BP2 in CD4 T cells resulted in decreased interleukin-2 (IL-2) and IL-4 production, supporting a repressive function of IRF-2BP2 for NFAT target genes. Furthermore, NFAT1 and IRF-2BP2 colocalized in the nucleus in activated cells, and the mutation of a newly identified nuclear localization signal in the IRF-2BP2 rendered it cytoplasmic, abolishing its repressive effect on NFAT1 activity. Collectively, our data demonstrate that IRF-2BP2 is a negative regulator of the NFAT1 transcription factor and suggest that NFAT1 repression occurs at the transcriptional level