NFAT1 C-Terminal Domains Are Necessary but Not Sufficient for Inducing Cell Death

<div><p>The proteins belonging to the nuclear factor of activated T cells (NFAT) family of transcription factors are expressed in several cell types and regulate genes involved in differentiation, cell cycle and apoptosis. NFAT proteins share two conserved domains, the NFAT-homology region (NHR) and a DNA-binding domain (DBD). The N- and C-termini display two transactivation domains (TAD-N and TAD-C) that have low sequence similarity. Due to the high sequence conservation in the NHR and DBD, NFAT members have some overlapping roles in gene regulation. However, several studies have shown distinct roles for NFAT proteins in the regulation of cell death. The TAD-C shows low sequence similarity among NFAT family members, but its contribution to specific NFAT1-induced phenotypes is poorly understood. Here, we described at least two regions of NFAT1 TAD-C that confer pro-apoptotic activity to NFAT1. These regions extend from amino acids 699 to 734 and 819 to 850 of NFAT1. We also showed that the NFAT1 TAD-C is unable to induce apoptosis by itself and requires a functional DBD. Furthermore, we showed that when fused to NFAT1 TAD-C, NFAT2, which is associated with cell transformation, induces apoptosis in fibroblasts. Together, these results suggest that the NFAT1 TAD-C includes NFAT death domains that confer to different NFAT members the ability to induce apoptosis.</p> </div

Apoptosis induced by CA-NFAT1 in NIH3T3 fibroblasts is dependent on DNA binding.

<p>(A) Schematic representation of the primary structure of CA-NFAT1 MutDBD. The mutated residues are indicated in the Figure. (B, E, F, G) NIH3T3 cells were transduced with empty vector or retrovirus expressing CA-NFAT1 or CA-NFAT1 MutDBD. (B) The total lysate of transduced NIH3T3 cells was obtained for analysis of NFAT1 and GAPDH expression levels by Western Blot. The molecular weights are indicated in kilodaltons (kDa). (C) The ability of the NFAT1 DBD and NFAT1 MutDBD to bind to DNA was tested by EMSA. The NFAT1 DBD and NFAT1 MutDBD peptides were incubated with oligonucleotides corresponding to the NFAT responsive element in the IL-2 promoter. (D) Jurkat cells were transfected with expression vector (empty vector or vector containing the CA-NFAT1 or CA-NFAT1 MutDBD cDNAs), luciferase reporter vector pGL4.30 and the Renilla luciferase expression vector pRL-TK. After 24 hours, the luciferase activity was measured by the release of luminescence resulting from the oxidation of its substrate (luciferin), normalized with the Renilla vector and expressed as relative light units (RLU). (E) Proliferation was assessed by incorporation of crystal violet. The cells were plated in triplicate and analyzed for 120 hours. This graph is representative of three independent experiments. (F, G) NIH3T3 cells were stained with propidium iodide (PI) and analyzed by flow cytometry for cell death. (F) Analysis of cell death 48 hours after plating. The graph represents the average percentage of cells in sub-G0 in three independent experiments. (G) Representative graph of the cell death analysis shown in (F). The percentage of cells in sub-G0 is shown in the graph.</p

CA-NFAT1 expression induces cell cycle arrest and apoptosis in NIH3T3 fibroblasts.

<p>NIH3T3 cells were transduced with empty vector or retrovirus expressing CA-NFAT1 and subjected to proliferation, cell death and cell cycle assays. (A) Proliferation was assessed by incorporation of crystal violet. The cells were plated in triplicate and analyzed for proliferation for 120 hours. This graph is representative of three independent experiments. (B, C, D) NIH3T3 cells were stained with propidium iodide (PI) and analyzed by flow cytometry for cell cycle and death. (B) Cell cycle was analyzed by the incorporation of propidium iodide (PI). The cells were plated in triplicate and analyzed 24 hours after plating. The percentage of cells in each phase of the cell cycle is indicated in the graphs. (C) Analysis of cell death 48 hours after plating. The percentage of cells in sub-G0 is shown in the graph. (D) The graph shows the average percentage of cells in sub-G0 from three independent experiments. (E) Analysis of pyknotic nuclei by DAPI staining. Cells were stained 48 hours after plating and visualized by fluorescence microscopy. (F) Analysis of phosphatidylserine exposure 24 hours after plating. The cells were stained with APC-conjugated annexin-V and analyzed by flow cytometry. The graph showing annexin-V staining and EGFP fluorescence is representative of three independent experiments.</p

The overexpression of NFAT1 691-927 does not prevent the induction of apoptosis by CA-NFAT1.

<p>(A) Schematic representation of the primary structure of NFAT1 and NFAT1 691-927. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047868#pone-0047868-g002" target="_blank">Figure 2A</a> for detailed information. (B, C, D) NIH3T3 cells were transduced with empty vector or retrovirus expressing CA-NFAT1 or NFAT1 691-927. (B) Proliferation was assessed by incorporation of crystal violet. NIH3T3 cells were plated in triplicate and analyzed for 120 hours. This graph is representative of three independent experiments. (C, D) NIH3T3 cells were stained with propidium iodide (PI) and analyzed for cell death by flow cytometry. (C) Analysis of cell death 48 hours after plating. The graph shows the average percentage of cells in sub-G0 determined in three independent experiments. (D) Representative graph of cell death analysis shown in (C). The percentage of cells in sub-G0 is shown in the graph. (E, F) NIH3T3 cells transduced with empty vector or retrovirus expressing NFAT1 691-927 were re-infected with empty vector or retrovirus expressing CA-NFAT1 and subjected to proliferation and cell death assays. (E) Proliferation was assessed by incorporation of crystal violet. The cells were plated in triplicate and analyzed for 120 hours. This graph is representative of three independent experiments. (F) Cell death analysis 48 hours after plating. NIH3T3 cells were stained with propidium iodide (PI) and analyzed by flow cytometry for cell death. The percentage of cells with sub-G0 DNA content is shown in the graph.</p

The fusion of NFAT1 TAD-C to CA-NFAT2 reverses the phenotype induced by CA-NFAT2.

<p>(A) Schematic representation of the primary structures of CA-NFAT1, CA-NFAT2 and CA-NFAT2 TAD-C NFAT1. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047868#pone-0047868-g002" target="_blank">Figure 2A</a> legend for details. (B, C, D, E) NIH3T3 cells were transduced with empty vector or retrovirus expressing CA-NFAT1, CA-NFAT2 or CA-NFAT2 TAD-C NFAT1. (B) The total lysate of transduced NIH3T3 cells was obtained for analysis of NFAT expression levels and molecular weight by Western Blot using anti-NFAT1 and anti-NFAT2 antibodies. Levels of the housekeeping protein GAPDH were also analyzed as a loading control. The molecular weights are indicated in kilodaltons (kDa). (C) Proliferation was assessed by incorporation of crystal violet. The cells were plated in triplicate and analyzed for 144 hours. This graph is representative of three independent experiments. (D, E) NIH3T3 cells were stained with propidium iodide (PI) and analyzed for cell death by flow cytometry. (D) Analysis of cell death 48 hours after plating. The graph shows the average percentage of cells in sub-G0 observed in three independent experiments. (E) Representative graph of cell death analysis shown in (D). The percentage of cells in sub-G0 is shown in the graph.</p

Dual Roles for NFAT Transcription Factor Genes as Oncogenes and Tumor Suppressors ▿ †

Nuclear factor of activated T cells (NFAT) was first described as an activation and differentiation transcription factor in lymphocytes. Several in vitro studies suggest that NFAT family members are redundant proteins. However, analysis of mice deficient for NFAT proteins suggested different roles for the NFAT family of transcription factors in the regulation of cell proliferation and apoptosis. NFAT may also regulate several cell cycle and survival factors influencing tumor growth and survival. Here, we demonstrate that two constitutively active forms of NFAT proteins (CA-NFAT1 and CA-NFAT2 short isoform) induce distinct phenotypes in NIH 3T3 cells. Whereas CA-NFAT1 expression induces cell cycle arrest and apoptosis in NIH 3T3 fibroblasts, CA-NFAT2 short isoform leads to increased proliferation capacity and induction of cell transformation. Furthermore, NFAT1-deficient mice showed an increased propensity for chemical carcinogen-induced tumor formation, and CA-NFAT1 expression subverted the transformation of NIH 3T3 cells induced by the H-rasV12 oncogene. The differential roles for NFAT1 are at least partially due to the protein C-terminal domain. These results suggest that the NFAT1 gene acts as a tumor suppressor gene and the NFAT2 short isoform acts gene as an oncogene, supporting different roles for the two transcription factors in tumor development

The amino acids 699 to 850 of TAD-C are sufficient to provide to CA-NFAT1 the ability to induce apoptosis.

<p>NIH3T3 cells were transduced with empty vector or retrovirus expressing CA-NFAT1 or CA-NFAT1 DD699-850. (A, B) NIH3T3 cells were stained with propidium iodide (PI) and analyzed by flow cytometry for cell death. (A) Analysis of cell death 48 hours after plating. The graph shows the average levels of cell death observed in three independent experiments. This graph was normalized by setting the percentage of cells with sub-G0 DNA content induced by CA-NFAT1 to 100%. The cell death index shown is the ratio of the percentage of cells in sub-G0 induced by empty vector or the indicated CA-NFAT1 construct and the percentage of cells in sub-G0 induced by full-length CA-NFAT1. A schematic of full-length CA-NFAT1 and CA-NFAT1 DD699-850 proteins is shown. (B) Representative graph of sub-G0 DNA content of NIH3T3 cells transduced with empty vector or retrovirus expressing CA-NFAT1 or CA-NFAT1 DD699-850. The percentage of cells in sub-G0 is shown in the graph. (C) Proliferation was assessed by incorporation of crystal violet. The cells were plated in triplicate and analyzed for 120 hours. This graph is representative of three independent experiments.</p

The alpha-synuclein oligomers activate nuclear factor of activated T-cell (NFAT) modulating synaptic homeostasis and apoptosis

Abstract Background Soluble oligomeric forms of alpha-synuclein (aSyn-O) are believed to be one of the main toxic species in Parkinson’s disease (PD) leading to degeneration. aSyn-O can induce Ca2+ influx, over activating downstream pathways leading to PD phenotype. Calcineurin (CN), a phosphatase regulated by Ca2+ levels, activates NFAT transcription factors that are involved in the regulation of neuronal plasticity, growth, and survival. Methods Here, using a combination of cell toxicity and gene regulation assays performed in the presence of classical inhibitors of the NFAT/CN pathway, we investigate NFAT’s role in neuronal degeneration induced by aSyn-O. Results aSyn-O are toxic to neurons leading to cell death, loss of neuron ramification and reduction of synaptic proteins which are reversed by CN inhibition with ciclosporin-A or VIVIT, a NFAT specific inhibitor. aSyn-O induce NFAT nuclear translocation and transactivation. We found that aSyn-O modulates the gene involved in the maintenance of synapses, synapsin 1 (Syn 1). Syn1 mRNA and protein and synaptic puncta are drastically reduced in cells treated with aSyn-O which are reversed by NFAT inhibition. Conclusions For the first time a direct role of NFAT in aSyn-O-induced toxicity and Syn1 gene regulation was demonstrated, enlarging our understanding of the pathways underpinnings synucleinopathies

NFAT2 isoforms differentially regulate gene expression, cell death, and transformation through alternative n-terminal domains

SimThe NFAT (nuclear factor of activated T cells) family of transcription factors is composed of four calcium-responsive proteins (NFAT1 to -4). The NFAT2 (also called NFATc1) gene encodes the isoforms NFAT2 and NFAT2 that result mainly from alternative initiation exons that provide two different N-terminal transactivation domains. However, the specific roles of the NFAT2 isoforms in cell physiology remain unclear. Because previous studies have shown oncogenic potential for NFAT2, this study emphasized the role of the NFAT2 isoforms in cell transformation. Here, we show that a constitutively active form of NFAT2 (CANFAT2 ) and CA-NFAT2 distinctly control death and transformation in NIH 3T3 cells. While CA-NFAT2 strongly induces cell transformation, CA-NFAT2 leads to reduced cell proliferation and intense cell death through the upregulation of tumor necrosis factor alpha (TNF- ). CA-NFAT2 also increases cell death and upregulates Fas ligand (FasL) and TNF- in CD4 T cells. Furthermore, we demonstrate that differential roles of NFAT2 isoforms in NIH 3T3 cells depend on the N-terminal domain, where the NFAT2 -specific N-terminal acidic motif is necessary to induce cell death. Interestingly, the NFAT2 isoform is upregulated in Burkitt lymphomas, suggesting an isoform-specific involvement of NFAT2 in cancer development. Finally, our data suggest that alternative N-terminal domains of NFAT2 could provide differential mechanisms for the control of cellular functions

Molecular Modeling and In Vitro Evaluation of Piplartine Analogs against Oral Squamous Cell Carcinoma

Cancer is a principal cause of death in the world, and providing a better quality of life and reducing mortality through effective pharmacological treatment remains a challenge. Among malignant tumor types, squamous cell carcinoma-esophageal cancer (EC) is usually located in the mouth, with approximately 90% located mainly on the tongue and floor of the mouth. Piplartine is an alkamide found in certain species of the genus Piper and presents many pharmacological properties including antitumor activity. In the present study, the cytotoxic potential of a collection of piplartine analogs against human oral SCC9 carcinoma cells was evaluated. The analogs were prepared via Fischer esterification reactions, alkyl and aryl halide esterification, and a coupling reaction with PyBOP using the natural compound 3,4,5-trimethoxybenzoic acid as a starting material. The products were structurally characterized using 1H and 13C nuclear magnetic resonance, infrared spectroscopy, and high-resolution mass spectrometry for the unpublished compounds. The compound 4-methoxy-benzyl 3,4,5-trimethoxybenzoate (9) presented an IC50 of 46.21 µM, high selectively (SI > 16), and caused apoptosis in SCC9 cancer cells. The molecular modeling study suggested a multi-target mechanism of action for the antitumor activity of compound 9 with CRM1 as the main target receptor