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. 2012;7(10):e47868.
doi: 10.1371/journal.pone.0047868. Epub 2012 Oct 26.

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

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Free PMC article

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

Douglas V Faget et al. PLoS One. .
Free PMC article

Abstract

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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. CA-NFAT1 expression induces cell cycle arrest and apoptosis in NIH3T3 fibroblasts.
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.
Figure 2
Figure 2. The deletion of amino acids 699 to 850 of TAD-C prevents the induction of apoptosis by CA-NFAT1.
NIH3T3 cells were transduced with empty vector or retrovirus expressing CA-NFAT1 or the indicated CA-NFAT1 truncated proteins. (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 the truncated CA-NFAT1 proteins is shown. The crosshatched bar represents TAD-N (N-terminal transactivation domain), the grey bar represents NHR (NFAT-homology region), the black bar represents the DBD (DNA-binding domain), and the white bar represents the TAD-C (C-terminal transactivation domain). (B) Representative graph of sub-G0 DNA content of NIH3T3 cells transduced with empty vector or retrovirus expressing CA-NFAT1, CA-NFAT1 Δ699-927 or CA-NFAT1 Δ699-850. 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 144 hours. This graph is representative of three independent experiments.
Figure 3
Figure 3. The amino acids 699 to 850 of TAD-C are sufficient to provide to CA-NFAT1 the ability to induce apoptosis.
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.
Figure 4
Figure 4. The overexpression of NFAT1 691-927 does not prevent the induction of apoptosis by CA-NFAT1.
(A) Schematic representation of the primary structure of NFAT1 and NFAT1 691-927. See Figure 2A 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.
Figure 5
Figure 5. Apoptosis induced by CA-NFAT1 in NIH3T3 fibroblasts is dependent on DNA binding.
(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.
Figure 6
Figure 6. The fusion of NFAT1 TAD-C to CA-NFAT2 reverses the phenotype induced by CA-NFAT2.
(A) Schematic representation of the primary structures of CA-NFAT1, CA-NFAT2 and CA-NFAT2 TAD-C NFAT1. See Figure 2A 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.

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Grant support

This work was supported by grants to J.P.B.V. from the International Centre for Genetic Engineering and Biotechnology (CRP/BRA09-01), Conselho Nacional de Desenvolvimento Científico e Tecnológico (478780/2010-9), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (102.357/2009) and Instituto Nacional de Ciência e Tecnologia em Câncer (573806/2008-0 and 170.026/2008). D.V.F. and B.K.R. were supported by a fellowship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior and P.I.L. was supported by a fellowship from the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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