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Originally published In Press as doi:10.1074/jbc.M511759200 on February 2, 2006

J. Biol. Chem., Vol. 281, Issue 16, 10682-10690, April 21, 2006
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Proteolytic Regulation of Nuclear Factor of Activated T (NFAT) c2 Cells and NFAT Activity by Caspase-3*

Wenfang Wu{ddagger}, Ravi S. Misra{ddagger}, Jennifer Q. Russell{ddagger}, Richard A. Flavell§, Mercedes Rincón{ddagger}, and Ralph C. Budd{ddagger}1

From the {ddagger}Immunobiology Program, Department of Medicine, The University of Vermont College of Medicine, Burlington, Vermont 05405 and §Section of Immunobiology, Yale University School of Medicine, and Howard Hughes Medical Institute, New Haven, Connecticut 06510

Received for publication, October 31, 2005 , and in revised form, January 18, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The nuclear factor of activated T (NFAT) cell family of transcription factors is important in regulating the expression of a broad array of genes, including cytokines, T cell surface receptors, and other transcription factors. NFATc1 and NFATc2 are two principal NFAT members that are expressed in peripheral T cells. Levels of NFAT expression in T cells are partly transcriptionally regulated, but less is understood regarding their post-transcriptional control. We show here that NFATc1 and NFATc2 are rapidly degraded in apoptotic T cells. NFATc2 is highly sensitive to cleavage by caspase-3, whereas NFATc1 is only weakly sensitive to caspase-3 or caspase-8. Two potential caspase-3 cleavage sites were identified in the N-terminal transactivation domain. These sites were confirmed by in vitro caspase cleavage assays. Abolition of NFATc2 cleavage by mutation of these two cleavage sites resulted in augmented NFAT transcriptional activity. Furthermore, NFAT activity could be augmented in wild-type effector T cells by inhibition of caspase activity. Of particular interest was that non-apoptotic T cells from cellular FLIP long transgenic (c-FLIPL-Tg) mice that manifest elevated caspase activity have greatly reduced levels of NFATc2 protein and NFAT transcriptional activity. Our findings reveal a new post-transcriptional regulation of NFATc2 that operates, not only during apoptosis, but also in non-apoptotic effector T cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis is a genetically programmed, morphologically distinct form of cell death that can be triggered by a variety of stimuli, including irradiation, cytokine withdrawal, hormones, and viruses. The common biochemical pathway of these processes is the initiation of downstream effector caspase, a family of cysteine-dependent aspartate-directed proteases (1, 2). Numerous caspase targets have been identified that disrupt important cellular metabolic and structural components, contributing to the stereotypic morphological and biochemical changes that characterize apoptotic cell death (3). More recently it has been appreciated that caspase activity is also necessary for certain types of cell development and proliferation (4). In these situations, it is less clear what initiates the caspase cascade and what are the substrates of caspases in actively proliferating, non-apoptotic cells. This is particularly true in T lymphocytes, where caspase-8 and caspase-3 are activated following T cell activation (5, 6–8). Thus, identifying caspase substrates in T cells is central to defining their role in cell growth and death.

A potential candidate for the regulation of caspase activity during T cell activation is the death receptor inhibitor, c-FLIPL2 (cellular FLICE inhibitory protein-long). c-FLIPL is a caspase-8 homolog that lacks enzymatic activity due to mutations in the C-terminal caspase domain (9). However, c-FLIPL can heterodimerize with caspase-8 via its mutual death effector domains and caspase-like domain and activate caspase-8 by repositioning an activation loop of caspase-8 without any proteolytic processing (10). Increased expression of c-FLIPL in T cells leads to augmented caspase activation (6, 11). In this capacity, c-FLIPL may thus be a principal regulator of caspase-8 activity and other downstream caspases in effector T cells. This could result in cleavage of selective substrates that might be critical for proper effector T cell function, as in cytokine production.

The NFAT family of transcription factors has crucial roles in T cell development and function by regulating the transcription of a broad array of cytokines, T cell surface receptors, and other transcription factors (12). The NFAT family consists of the five members NFATc1 through NFATc5. NFATc1 and NFATc2 are the principal NFAT members in mature T cells (13). The phenotypes of NFAT-deficient mice confirm a strong influence of this transcriptional family on cytokine expression patterns. Thus, NFATc1-deficient mice exhibit diminished production of Th2 cytokines (14, 15). By contrast, mice lacking NFATc2 manifest increased and prolonged IL-4 expression (16), which is further augmented in NFATc2–/– x NFATc3–/– mice (17, 18). Mice deficient in both NFATc1 and NFATc2 in T cells manifest profoundly impaired production of many cytokines, including IL-2, -4, and -10, IFN-{gamma}, GM-CSF (granulocyte macrophage-colony stimulating factor), as well as expression of CD40L and FasL (19). NFATc2 has also been demonstrated to be involved in cell cycle control. NFATc2 inhibits CDK4 expression by directly binding and repressing its transcription through the recruitment of histone deacetylases (20). Consistent with that observation, T cells from NFATc2-deficient mice are hyperproliferative and manifest increased levels of several cyclins (21).

Here we have shown that the N-terminal transactivation domain of NFATc2 contains two highly sensitive caspase-3 cleavage sites and is processed, not only in apoptotic T cells, but also in non-apoptotic T cells from c-FLIPL-Tg mice that have elevated caspase activity. This results in dramatically reduced levels of NFATc2 and NFAT activity in c-FLIPL-Tg T cells. Inhibition of caspase activity results in restoration of uncleaved NFATc2 in c-FLIPL-Tg T cells and enhances NFAT transcriptional activity. A noncleavable NFATc2 mutant also increases NFAT activity. The findings demonstrate a previously unknown caspase-3 substrate and propose a new mechanism for the post-translational regulation of NFATc2 in effector T cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
CD4+ T Cell Purification and Culture—Spleen cells were hemolyzed and combined with lymph node cells followed by negative selection to enrich the population for CD4+ cells. The cells were incubated with antibodies to CD8 (Tib105), major histocompatibility complex class II (3F12), NK1.1 (PK136), and CD11b (BD Biosciences) for 30 min. The samples were washed and then incubated with goat anti-rat/mouse IgG-labeled magnetic beads (Polysciences, Inc.) for 45 min followed by magnetic field separation to remove CD8+ cells, B cells, NK cells, and macrophages. CD4+ T cell purity was confirmed by flow cytometry and was typically >90%. The cells were activated with plate-bound anti-CD3 (10 µg/ml, 145–2C11) and anti-CD28 ascites (1:500) plus recombinant human IL-2 (50 units/ml, Cetus) and cultured in complete medium (RPMI 1640 supplemented with 25 mM HEPES, 2.5 mg/ml glucose (Sigma), 10 µg/ml folate (Invitrogen), 110 µg/ml pyruvate (Invitrogen), 5 x 10–5 M beta-mercaptoethanol (Sigma), 292.3 µg/ml glutamine (Invitrogen), 100 units/ml penicillin-streptomycin (Invitrogen), and 5% fetal bovine serum). On day 2 after stimulation, T cells were removed from anti-CD3-coated plates and propagated in IL-2-containing medium.

Induction of Apoptosis—Anti-CD3- and anti-CD28-stimulated day 4 effector CD4+ T cells were resuspended in complete medium at 10 x 106/ml. Apoptosis was induced by incubating the cells with 200 ng/ml FLAG-tagged FasL (Apotech Corporation) and 4 µg/ml of cross-linking anti-FLAG antibody (Sigma).

Caspase Activity Assay—Cellular caspase activity was determined by the Apo-ONETM Caspase-3/7 assay kit (Promega Corp.) according to the manufacturer's instructions. Cells were resuspended in 100 µl of medium and mixed with 100 µl of caspase substrate. After incubation at room temperature, fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

Plasmid Constructs and Site-directed Mutagenesis—FLAG-tagged NFATc2 and FLAG-tagged NFATc1 were kindly provided by Dr. Sean Diehl and Dr. Melissa Brown, respectively. Asp-29 and Asp-66 of NFATc2 were mutated to Ala using the primers 5'-CCCAAGACGAGCTGGCCTTTTCCATCCTCTTC-3' and 5'-GGATGATGTCCTGGCCTATGGCCTCAAGCC-3' and their complementary oligonucleotides that overlap the target sites. Mutants were made using the QuikChange® II XL site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The mutations were confirmed by sequencing.

In Vitro Caspase Cleavage Assay—Wild-type, D29A NFATc2, D66A NFATc2, or D29A/D66A NFATc2 plasmids were transcribed and translated using the TNT quick-coupled transcription/translation systems (Promega) in the presence of [35S]methionine. 2.5 µl of in vitro translation products were incubated in 50 µl of assay buffer (20 mM PIPES, 100 mM NaCl, 10 mM dithiothreitol, 1 mM EDTA, 0.1% (w/v) CHAPS, 10% sucrose, pH 7.2) with caspase-3 or caspase-8 at 37 °C. Reactions were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were analyzed by autoradiography and immunoblotting.

Biotin-VAD-fmk Caspase Precipitation Assay—Whole cell lysates were made from cells that were preincubated with 10 µM biotin-VAD-fmk (MP Biomedicals) at 37 °C for 15 min. 600 µg of whole cell lysate protein was first precleared with Sepharose 6B (Sigma) for 2 h to remove nonspecific binding, followed by overnight incubation with streptavidin-Sepharose beads (Zymed Laboratories Inc.. After being washed to remove unbound proteins, the streptavidin-Sepharose-bound complex was resuspended in sample loading buffer and boiled. Supernatants were analyzed by immunoblotting.

Immunoblot Analysis—Whole cell lysates were made as follows. Cells were washed once with phosphate-buffered saline and lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 2 mM dithiothreitol, 2 mM sodium vanadate, and protease inhibitor mixture (CompleteTM, Roche Applied Science)) followed by centrifugation. The antibodies used were NFATc1 (Affinity Bioreagents, Inc.), NFATc2 (Affinity Bioreagents, Inc.), NFATc2 (Santa Cruz Biotechnology), P44/42 mitogen-activated protein kinase (ERK1/ERK2) (New England Biolabs), actin (Santa Cruz Biotechnology), and FLAG (Sigma). Proteins were detected with horseradish peroxidase-conjugated secondary antibodies and developed by chemiluminescence.

Gel Mobility Shift and Supershift Binding Assays for NFAT—Nuclear extracts were obtained as previously described (22). Binding reactions were done using 2 µg of nuclear protein in the presence of 10,000 counts/min of the specific 32P-end-labeled double-stranded oligonucleotide 5'-GTAATAAAATTTTCCAATGTAAA-3' for 20 min at 4 °C. For supershift analysis, 1 µg of anti-NFATc1 antibody (Affinity Bioreagents, Inc.) or anti-NFATc2 antibody (Affinity Bioreagents, Inc.) was incubated with the binding reaction mix.

NFAT-Luciferase Reporter Mice and Luciferase Activity—NFAT-luciferase transgenic mice contain the firefly luciferase gene driven by a 200-bp fragment of the IL-2 minimal promoter (–326 to –294 and –72 to +47) with NFAT-binding sites inserted in an XhoI site between these fragments (23). Purified CD4+ T cells were activated with anti-CD3 and anti-CD28. At 48 h following activation, the cells were harvested, washed with phosphate-buffered saline, and lysed. The lysates from 0.5 x 106 cells were then analyzed using luciferin and measured in a luminometer for 10 s. Two measurements were made for each sample. Results are presented as the average of two measurements with background subtracted. For experiments with caspase inhibitor, T cells were first activated with anti-CD3 and anti-CD28 for 48 h and then cultured for an additional 24 h in the absence or presence of 50 µM Z-VAD-fmk before being lysed and analyzed for luciferase activity.

Cell Culture, Transient Transfection, and Luciferase Assay—293T cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum. The cells were transfected with LipofectamineTM 2000 (Invitrogen) according to the manufacturer's instructions. 18 h after transfection, the cells were either unstimulated or stimulated with phorbol 12-myristate 13-acetate (10 ng/ml) plus ionomycin (500 ng/ml) for 6 h before being lysed for luciferase activity measurement.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Selective Degradation of NFATc1 and NFATc2 during Fas-induced Apoptosis—During apoptosis, caspases are activated and cleave various vital proteins, including certain transcription factors that are critical to normal cell function (24). Scanning the protein sequence of NFATc2, we identified two caspase-3 consensus cleavage sites (26DELD29, 63DVLD66) in the N-terminal transactivation domain (Fig. 1A). A DELD consensus site has been defined as a caspase cleavage site in several proteins, including acinus, D4-GDI, MstII, and FOXO3a, whereas DVLD is a caspase cleavage site in RAD51 (3, 25–28).

This prompted us to examine the fate of NFATc2 following Fas-induced apoptosis. Day 4 effector T cells were treated with Fas ligand (FasL) for different times, and whole cell lysates were analyzed by immunoblot for NFATc2 and NFATc1, the major NFAT family members that are expressed in mature T cells. For both NFATc2 and NFATc1, the latter of which includes three isoforms, NFATc1A, -B, and -C (29), most of the full-length protein disappeared within 3 h of FasL treatment (Fig. 1B). The loss of full-length NFAT was prevented by the addition of the caspase inhibitor Z-VAD-fmk (Fig. 1C). In addition, no cleavage fragments of NFATc2 or NFATc1 could be detected at these time points at the level of sensitivity of these assays. This suggests that either the molecules were rapidly degraded or the epitopes of the cleaved fragments were not detectable by the antibodies used. During apoptotic cell death, only a fraction of the cellular proteome is cleaved by caspases (30, 31). To show that the caspase-dependent cleavage of NFAT was selective, immunoblot for an unrelated signaling molecule, ERK, revealed that it remained intact for at least 3 h of FasL treatment. The levels of the loading control actin were also unchanged during the treatment (Fig. 1B). Measurement of caspase-3 activity by the DEVD-rhodamine fluorogenic assay revealed the expected increase in caspase activity over the same time period after FasL treatment (Fig. 1D).


Figure 1
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  		FIGURE 1.
Degradation of NFATc1 and NFATc2 following FasL stimulation. A, schematic representation of NFATc2 transactivation domain indicating the location of the potential caspase-3 cleavage sites. B, day 4 CD4+ effector T cells (aCD3/CD28 + IL-2) were treated with soluble FasL (sFasL, 200 ng/ml) cross-linked by anti-FLAG (4 µg/ml) for the indicated times. Whole cell lysates of treated cells were analyzed with immunoblot. ERK was included to show that FasL-induced cleavage was selective. C, effector T cells were pretreated with 50 µM Z-VAD-fmk or Me2SO vehicle control for 30 min, followed by FasL treatment for 3 h. Whole cell lysates were analyzed with immunoblot. D, caspase activity of the cells treated as described for A was determined using a DEVD-rhodamine fluorogenic assay. AFU, arbitrary fluorescence units.

 
NFATc1 Is Weakly Sensitive to Cleavage by Caspase-3 and Caspase-8—In addition to caspases, other proteases that contribute to the cleavage of various cellular proteins (32) are also activated during apoptosis. To investigate whether degradation of NFATc1 and NFATc2 during apoptosis was mediated by caspases, the potential cleavage of NFATc1 and NFATc2 was examined using an in vitro caspase cleavage assay. Caspase-8 and caspase-3 were chosen, because they represent, respectively, an upstream initiator caspase and a downstream executioner caspase that are both activated during FasL-induced apoptosis as well as during T cell receptor-mediated activation (8). N-terminal FLAG-tagged NFATc1 or NFATc2 was transcribed and translated in vitro in the presence of [35S]methionine and incubated with recombinant caspase-3 or caspase-8. Reactions were separated on SDS-PAGE and analyzed by autoradiography and immunoblotting. In the presence of as much as 100 ng of caspase-3 or 200 ng of caspase-8, only a small proportion of NFATc1 was cleaved after 2 h, as revealed by both autoradiograph and immunoblot (Fig. 2A), indicating that NFATc1 was only weakly sensitive to caspase-3 and caspase-8 in vitro. Agreeing with this, there is no typical caspase cleavage site in the NFATc1 amino acid sequence. To confirm the activity of the caspases in vitro, c-FLIPL was used as a positive control substrate, because c-FLIPL is a known substrate of both caspase-3 and caspase-8 (9). In contrast to NFATc1, most of c-FLIPL was rapidly cleaved within 30 min by 50 ng of caspase-3 or caspase-8 (Fig. 2B).

NFATc2 Is a Substrate of Caspase-3 but Not of Caspase-8—The in vitro cleavage assay showed that NFATc2 was totally resistant to caspase-8. In the presence of as much as 200 ng of caspase-8, no cleavage of NFATc2 was detected after 2 h of incubation (Fig. 3A). By contrast, the autoradiograph revealed that NFATc2 was highly sensitive to cleavage by caspase-3 (Fig. 3B). At a caspase-3 dose as low as 6.3 ng, virtually all of full-length NFATc2 was cleaved to a slightly lower molecular weight. Only at higher doses of caspase-3 was this initial cleavage product further processed to at least three fragments. A similar rapid loss of detectable full-length NFATc2 was apparent using an anti-FLAG antibody that reacts to the N-terminal FLAG tag of NFATc2 (Fig. 3B, second panel). No new cleavage fragments were detected by the anti-FLAG immunoblot, suggesting that there might be a caspase-3 cleavage site close to the N terminus. In addition, the cleavage of NFATc2 was reversed in the presence of caspase inhibitor Z-VAD-fmk, confirming that cleavage was due to caspase activity and not other proteolytic activity.


Figure 2
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  		FIGURE 2.
NFATc1 is weakly sensitive to caspase-3 and caspase-8. A, 35S-labeled FLAG-tagged NFATc1 was incubated with buffer control or the indicated amount of caspase-3 or caspase-8 for 2 h. The reaction products were separated by SDS-PAGE followed by autoradiography and immunoblotting. B, cleavage of 35S-labeled c-FLIPL as a positive control for the activities of caspase-3 and caspase-8 (50 ng each for 2 h).

 


Figure 3
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  		FIGURE 3.
NFATc2 is cleaved by caspase-3 but is not a substrate of caspase-8. 35S-labeled NFATc2 was incubated with buffer control or the indicated amount of caspase-8 (A) or caspase-3 (B) for 2 h. Z-VAD-fmk (400 µM) or Me2SO (DMSO) vehicle control was included in some samples. The reaction products were analyzed by 4–15% gradient SDS-PAGE gel followed by autoradiography and immunoblot. Immunoblot was performed using two different anti-NFATc2 antibodies, one that reacts to NFATc2-(51–69) and the other reacts to -(433–567).

 
To further define the sites of NFATc2 cleavage by caspase-3, we examined the cleavage products with two monoclonal anti-NFATc2 antibodies that react to different epitopes. The first antibody reacts to amino acids 51–69 of NFATc2 (Fig. 1A). This antibody revealed the same pattern of loss of full-length NFATc2 as did the anti-FLAG immunoblot, with no appearance of a cleaved NFATc2 fragment (Fig. 3A). The second anti-NFATc2 antibody reacts to amino acids 433–567. This antibody revealed an immunoblot pattern similar to the autoradiograph, in that an NFATc2 fragment appeared that was only slightly lower in size than full-length NFATc2 (Fig. 3B). The cleaved NFATc2 fragment was detectable with anti-NFATc2-(433–567) at low dose caspase-3 and was still detectable at higher doses. In addition, this anti-NFATc2 antibody also detected further cleavage fragments at higher doses of caspase-3, nearly identical to the pattern seen in the autoradiograph.

Caspase-3 Cleaves NFATc2 at the Consensus Sites 26DELD29 and 63DVLD66—Collectively, the above studies suggested that NFATc2 possesses a highly sensitive caspase-3 cleavage site(s) very near the N terminus, with possible additional less-sensitive sites in other parts of the molecule. To determine whether caspase-3 could cleave either of the two candidate sites in the N terminus of NFATc2, alanine substitutions were introduced at Asp-29 (D29A NFATc2) and Asp-66 (D66A NFATc2) or at both sites (D29A/D66A NFATc2). These were in vitro translated and subjected to caspase-3 cleavage. Based on both the autoradiograph and the immunoblot, the cleavage of NFATc2 by 50 ng of caspase-3 was completely prevented in the double mutant D29A/D66A NFATc2 (Fig. 4B). A closer inspection of the cleavage products of the single mutant NFATc2 molecules suggested that 26DELD29 was somewhat more sensitive to caspase-3 than 63DVLD66, because full-length D29A NFATc2 was slightly protected from cleavage by 50 ng of caspase-3 (Fig. 4B, lane 4), whereas full-length NFATc2D66A was still efficiently cleaved at the 26DELD29 site (Fig. 4B, lane 6). It is interesting that the single mutation of D66A totally abolished the recognition of NFATc2 by the anti-NFATc2-(51–69) antibody (Fig. 4B, lanes 5–8). This is consistent with the known specificity of this monoclonal antibody to amino acids 51–69 of NFATc2. It also likely explains why this antibody could not detect any cleavage fragments after wild-type NFATc2 was cleaved by caspase-3 (see Fig. 3B).


Figure 4
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  		FIGURE 4.
Mutation of the two potential NFATc2 cleavage sites inhibits cleavage of NFATc2 by caspase-3. A, schematic representation of full-length NFATc2 and C-terminal cleavage products after potential cleavage sites. White arrowheads indicate full-length NFATc2; gray arrowheads indicate the C-terminal cleavage fragment after cleavage site 26DELD29; black arrowheads indicate the C-terminal cleavage fragment after cleavage site 63DVLD66. B, 35S-labeled FLAG-tagged NFATc2 and its mutants were incubated with buffer alone (–) or with 50 ng of caspase-3 for 2 h(+). The reaction products were analyzed by SDS-PAGE followed by autoradiography and immunoblot. WT, wild-type.

 
Higher Caspase-3 Activity and Decreased NFATc2 Expression in c-FLIPL-Tg CD4+ T Cells—According to the in vitro cleavage assay, NFATc2 is extremely sensitive to caspase-3 and is rapidly cleaved following Fas ligation. However, caspase activation also has recently been demonstrated in viable effector T cells (8). We thus considered that NFATc2 might be subject to caspase cleavage in non-apoptotic situations when caspase activity is mildly elevated in T cells. The level of caspase activity in effector T cells is likely regulated to a large extent by the initial heterodimerization of caspase-8 with c-FLIPL. c-FLIPL contains a loop in its C terminus that binds and activates the enzymatic pocket of caspase-8 (10). This leads to activation of downstream caspases in effector T cells, including caspase-3 (8).

We thus wanted to investigate NFATc2 expression and NFAT activity in cycling effector T cells bearing normal levels of caspase activity versus elevated levels. The latter occurs in c-FLIPL-Transgenic (c-FLIPL-Tg) mice, in which increased expression of c-FLIPL in T cells provokes augmented caspase activity and proliferation (6, 11). Caspase activity was initially determined by the DEVD-rhodamine assay and showed that both freshly isolated and day 4 effector c-FLIPL-Tg CD4+ T cells possessed more caspase activity than equivalent T cell populations from normal littermate controls (Fig. 5A). Because this assay reflects, not only caspase-3 activity, but also other caspases, we precipitated selectively the active caspase fraction from effector T cells by first incubating them with biotin-VAD and then subjecting cell lysates to precipitation with streptavidin-Sepharose. As anticipated, more c-FLIPL was present in the biotin-VAD precipitate from c-FLIPL-Tg CD4+ T cells, consistent with the model that c-FLIPL heterodimerizes with and activates caspase-8. Importantly, more cleaved active caspase-3 was revealed in the c-FLIPL-Tg CD4+ T cells (Fig. 5B).

Having established that c-FLIPL-Tg CD4+ T cells have more caspase activity, especially more active caspase-3, than wild-type CD4+ T cells, we examined the level of NFATc2 expression and NFAT activity in CD4+ T cells of both groups. Immunoblot of whole cell lysates for NFATc1 showed little difference in expression of the three NFATc1 isoforms A, B, and C (Fig. 6A, upper panel). By contrast, levels of NFATc2 protein were profoundly reduced in c-FLIPL-Tg CD4+ T cells (Fig. 6A, second and third panels). This was apparent both in naïve resting T cells as well as in days 1 and 2 effector T cells. However, mRNA levels of NFATc2 were not significantly different between wild-type and c-FLIPL-Tg CD4+ T cells (Fig. 6B).

NFAT DNA binding was also examined by electrophoretic mobility shift assay. This revealed that nuclear extracts from c-FLIPL-Tg CD4+ T cells had less NFAT binding activity than non-transgenic littermate controls at days 1 and 2 following activation (Fig. 6C). Supershift analysis further revealed that NFATc2 binding was less apparent in nuclear extracts from c-FLIPL-Tg CD4+ T cells (Fig. 6D). To assess the level of NFAT transcriptional activity, c-FLIPL-Tg mice were crossed to NFAT-luciferase transgenic mice. These reporter mice contain the luciferase gene driven by the NFAT promoter (23). CD4+ T cells were purified from c-FLIPL-Tg x NFAT-luciferase double-transgenic mice or from NFAT-luciferase single-transgenic controls and stimulated for 2 days with anti-CD3 plus anti-CD28. Similar to the NFAT DNA binding results, lower NFAT transcriptional activity was found in CD4+ T cells from c-FLIPL-Tg mice (Fig. 6E). Collectively, the findings are consistent with the view that the augmented caspase-3 activity in the resting and effector c-FLIPL-Tg CD4+ T cells promotes the cleavage of NFATc2.


Figure 5
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  		FIGURE 5.
c-FLIPL-Tg CD4+ T cells have more caspase-3 activity. A, fresh CD4+ T cells and day 4 effector CD4+ T cells from c-FLIPL-Tg (FLIP) mice or non-transgenic littermate control (NLC) mice were tested for caspase activity by a DEVD-rhodamine release. B, day 4 effector CD4+ T cells were incubated for 15 min with 10 µM biotin-VAD and then lysed. The active caspases were precipitated with avidin-Sepharose and immunoblotted for c-FLIPL, caspase-8, and caspase-3. AFU, arbitrary fluorescence units; WCL, whole cell lysate.

 


Figure 6
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  		FIGURE 6.
Decreased NFAT activity and NFATc2 in c-FLIPL-Tg CD4+ T cells. A, CD4+ T cells from c-FLIPL-Tg mice (F) or non-transgenic littermates (N) were either not stimulated (NS) or stimulated with anti-CD3/CD28 for the times indicated. Whole cell lysates were probed for expression of NFATc1 or NFATc2 by immunoblot. B, RNA was prepared from freshly isolated CD4+ T cells and analyzed with real-time PCR for NFATc2 and NFATc1 expression. Statistical analysis revealed no significant difference between c-FLIPL-Tg (FLIP) CD4+ T cells and their control for both NFATc1 (p = 0.1132) and NFATc2 (p = 0.078). NLC, non-transgenic littermate control. C, nuclear extracts were prepared and tested by electrophoretic mobility shift assay for NFAT binding activity. D, specific NFATc1 or NFATc2 binding activity measured by supershift assay in nuclear extracts from day 2 effector CD4+ T cells. Black arrowhead indicates NFAT binding complex. White arrowheads identify the supershifted complexes containing the indicated NFAT antibody (NFAT Ab) bound to either anti-NFATc1 (c1) or anti-NFATc2 (c2) antibodies. E, CD4+ T cells from c-FLIPL-Tg x NFAT-luciferase mice or control NFAT-luciferase mice (NLC) were stimulated for 2 days. Cells were lysed and measured for luciferase activity. p < 0.0001 for c-FLIPL-Tg; ALU, arbitrary light units.

 


Figure 7
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  		FIGURE 7.
Inhibition of NFATc2 cleavage conserves its transcriptional activity. A, 293T cells were cotransfected with the NFAT-luciferase plasmid, in the presence of control vector or expression plasmids encoding the wild-type NFATc2, D29A/D66A mutant NFATc2 (DM). Cells were stimulated with or without ionomycin plus phorbol 12-myristate 13-acetate (P+I) and lysed for luciferase activity measurement. Results are representative of three similar experiments. B, NFAT-luciferase-transgenic T cells were first stimulated for 2 days with anti-CD3 and anti-CD28 followed by treatment with 50 µM Z-VAD-fmk or Me2SO vehicle control for 1 day. Cells were then lysed and measured for luciferase activity. ALU, arbitrary light units. C, T cells from c-FLIPL-Tg mice (FLIP), their non-transgenic littermate controls (NLC), and caspase-3 deficient mice were treated as described for B, and NFATc2 proteins levels were examined with immunoblot using both anti-NFATc2-(51–69) and anti-NATc2-(433–567). White arrowheads indicate full-length NFATc2; gray arrowheads indicate the C-terminal cleavage fragment after cleavage site 26DELD29.

 
Inhibition of NFATc2 Cleavage Conserves Its Transcriptional Activity—Because the two caspase-3 cleavage sites are located in the N-terminal transactivation domain of NFATc2, we further examined whether the cleavage of NFATc2 at these two sites would affect its transcriptional activity using a luciferase reporter assay. Wild-type NFATc2 or D29A/D66A NFATc2, together with an NFAT-luciferase reporter plasmid, were transfected into 293T cells, and 24 h later, the cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin for 6 h. Interestingly, D29A/D66A NFATc2 showed 2-fold higher transcriptional activity than wild-type NFATc2 (Fig. 7A). The DEVD-rhodamine caspase assay revealed caspase activity in cycling 293T cells (data not shown). It is possible that the active caspase in cycling 293T cells cleaves the wild-type NFATc2 and diminishes its transcriptional activity.

The possible contribution of caspase activity to the regulation of NFAT transcriptional activity in T cells was examined using NFAT-luciferase reporter mice (23). Day 2 effector T cells from NFAT-luciferase-transgenic mice were incubated with Z-VAD-fmk or its vehicle control for 24 h, lysed, and luciferase activity measured. This time interval was chosen, because caspase-3 activation is not detectable in stimulated T cells until day 2, and earlier addition of Z-VAD-fmk would also block early caspase-8 activation, which is required for T cell activation (5, 8). The inhibition of caspase activity greatly enhanced the NFAT transcriptional activity (Fig. 7B), strongly suggesting that NFATc2 cleavage occurs in effector T cells. To further examine this possibility, effector T cells from c-FLIPL-Tg mice, their wild-type control mice, and caspase-3–/– mice were treated in the same manner, and whole cell lysates were immunoblotted for NFATc2. The cleavage of NFATc2 was observed in both c-FLIPL-Tg T cells and wild-type control cells, and the caspase inhibitor Z-VAD-fmk reversed the NFATc2 cleavage, partly in wild-type T cells, and almost completely in c-FLIPL-Tg T cells. More strikingly, caspase-3–/– T cells showed total block of NFATc2 cleavage. Proliferation of caspase-3–/– T cells was similar to wild-type effector T cells, and Z-VAD-fmk did not inhibit proliferation of wild-type effector T cells (data not shown). These results indicate that NFATc2 cleavage occurs in cycling T cells, and the cleavage is mediated by caspase-3 in vivo.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The current findings demonstrate by an in vitro caspase cleavage assay that NFATc1 is less sensitive than NFATc2 to caspase-3-mediated cleavage and that both NFAT members are rather insensitive to caspase-8-induced degradation. These differences in NFAT sensitivity to in vitro caspase cleavage were closely paralleled by the degree of NFAT degradation observed in vivo in wild-type T cells with low caspase activity, c-FLIPL-Tg T cells bearing intermediate caspase activity, and Fas ligand-treated T cells manifesting high caspase activity. Thus, compared with naïve wild-type T cells, c-FLIPL-Tg T cells had considerably less NFATc2 protein but comparable NFATc1, whereas FasL treatment of T cell blasts caused a rapid loss of both NFAT members. Site-directed mutagenesis confirmed the two caspase-3 cleavage sites in the N-terminal transactivation domain of NFATc2. The levels of caspase activity generated during resting, effector, and apoptotic stages of T cells are likely to strongly influence the amount of NFAT activity.

c-FLIPL is an enzymatically inactive homolog of caspase-8 that blocks apoptosis by death receptors (9). Besides its anti-apoptotic activity, c-FLIPL can also activate procaspase-8 by heterodimerization (10). The DEVD-rhodamine assay and biotin-VAD precipitation assay demonstrated that both resting and effector c-FLIPL-Tg CD4+ T cells have more caspase activity, especially caspase-3, than equivalent populations of wild-type T cells. This is a likely cause of the loss of NFATc2 in c-FLIPL-Tg CD4+ T cells.

The selective decrease of NFATc2 in T cells from c-FLIPL-Tg mice may also be relevant to the cytokine bias of these mice. We have previously reported that activated CD4+ T cells from c-FLIPL-Tg mice produce increased amounts of Th2 cytokines (IL-4, -5, -10, and -13) and decreased amounts of Th1 cytokines (IFN-{gamma}). This correlated with an enhanced sensitivity to allergic airway hypersensitivity (33). These cytokine patterns are closely paralleled by observations in NFAT-deficient mice. NFATc1-deficient mice manifest decreased production of IL-4 (14, 15). NFATc2-deficient mice, by contrast, manifest elevated and prolonged IL-4 gene transcription and are more susceptible to infection with Leishmania major (16). NFATc2–/– x NFATc3–/– mice have dramatic increases of Th2 cytokines and elevated serum levels of IgG1 and IgE (17). The decreased NFATc2 protein levels observed in c-FLIPL-Tg CD4+ T cells could therefore be a contributing factor to the Th2 bias of these cells. In a similar manner, CD4+ T cells from death receptor 6-deficient mice are also Th2 biased, due in part to increased levels of nuclear NFATc1 (34). The loss of NFATc2 in c-FLIPL-Tg CD4+ T cells might result in a dominance of NFATc1 activity as in DR6-deficient mice and NFATc2-deficient mice, which would favor Th2 cytokine expression. Consistent with this model, preliminary studies show that caspase-3–/– T cells not only maintain uncleaved NFATc2, they manifest a Th1 cytokine profile, opposite that of c-FLIP -Tg T cells.3 Taken together with our observations, this suggests that death receptor-dependent and -independent activation of caspases may affect the expression of specific NFAT family members, the ratio of which may profoundly influence CD4+ T cell cytokine patterns.

The in vitro caspase cleavage assay showed that NFATc2 is highly sensitive to caspase-3, whereas NFATc1 is only weakly sensitive to caspase-3 and -8. Subsequently, two caspase-3 cleavage sites were identified in N-terminal transactivation domain NFATc2. We further immunoblotted the apoptotic cell whole lysates with the anti-NFATc2-(433–567) antibody and did not detect the C-terminal cleavage product that was revealed by the in vitro caspase-3 cleavage assay. This suggests that this C-terminal fragment might be very unstable and degraded rapidly by other proteases after initial caspase processing during apoptosis. However, the C-terminal cleavage product after 26DEVD29 was observed in non-dying, cycling effector T cells that have only moderate caspase-3 activity. In agreement with this possibility, both the transfection with caspase-3-resistant D29A/D66A NFATc2 mutant, as well as primary cells cultured with the caspase inhibitor Z-VAD-fmk, showed that prevention of NFATc2 cleavage promoted enhanced NFATc2 transcriptional activity.

It is well established that caspase activity is required for T cell activation (35). The first evidence for this came from murine T cells either deficient in FADD (Fas-associating death domain) or expressing a dominant negative form of FADD, which were found to be hypoproliferative in response to activation via the T cell antigen receptor (36, 37). Direct inhibition of caspase activity also blocked CD3-induced proliferation and IL-2 production by human T cells (5). In addition, loss-of-function caspase-8 mutations in humans was linked to defects in the activation of T, B, and NK cells, resulting in an immunodeficiency syndrome (38). This was corroborated in mice in which targeted caspase-8 disruption in T cells caused defects in the T cell receptor-induced expansion of peripheral T cells (39). We observed in this study that non-dying cycling T cells manifest both active caspase-8 and caspase-3. Presumably this level of caspase-3 activity is sufficient to cleave NFATc2, as evidenced by the lack of NFATc2 cleavage in caspase-3–/– T cells. Thus, although NFATc2 expression is increased in activated T cells, active caspase-3 may restrict the level of increased NFATc2 and hence serve to regulate overall NFAT activity.

At present it is unknown which molecule(s) is targeted by caspases and how it contributes to T cell activation. Recent studies have shown that NFATc2 represses the expression of CDK4, which is an important G0/G1 restriction point kinase that allows cells to exit the resting state and enter the cell cycle (20). Correspondingly, NFATc2-deficient lymphocytes are hyperproliferative after antigen stimulation and manifest increased levels of several cyclins (21). Interestingly, increased T cell proliferation in vivo was also observed with c-FLIPL-Tg mice (11). Conceivably, caspase-mediated cleavage of NFATc2 may be partly responsible for the increased cell cycling of c-FLIPL-Tg T cells (11). A similar process may regulate NFATc2 levels and cell cycling in wild-type T cells.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants AI36333 (to R. C. B.) and AI45666 (to R. C. B. and M. R.) and the Howard Hughes Medical Institute (to R. A. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Immunobiology Program, The University of Vermont College of Medicine, Given Medical Bldg., D305, 89 Beaumont Dr., Burlington, VT 05405-0068. Tel.: 802-656-2286; Fax: 802-656-3854; E-mail: ralph.budd{at}uvm.edu.

2 The abbreviations used are: c-FLIPL, cellular FLIP long; FLIP, FLICE inhibitory protein; FLICE, FADD-like IL-1beta-converting enzyme; FADD, Fas-associating death domain; Tg, transgenic; Z-, b enzyloxycarbonyl; fmk, fluoromethyl ketone; IL, interleukin; IFN, interferon; PIPES, 1,4-piperazinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ERK, extracellular signal-regulated kinase; aa, amino acid(s). Back

3 W. Wu, unpublished observations. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Cohen, G. M. (1997) Biochem. J. 326, 1–16[Medline] [Order article via Infotrieve]
  2. Nicholson, D. W., and Thornberry, N. A. (1997) Trends Biochem. Sci. 22, 299–306[CrossRef][Medline] [Order article via Infotrieve]
  3. Fischer, U., Janicke, R. U., and Schulze-Osthoff, K. (2003) Cell Death Differ. 10, 76–100[CrossRef][Medline] [Order article via Infotrieve]
  4. Garrido, C., and Kroemer, G. (2004) Curr. Opin. Cell Biol. 16, 639–646[CrossRef][Medline] [Order article via Infotrieve]
  5. Kennedy, N. J., Kataoka, T., Tschopp, J., and Budd, R. C. (1999) J. Exp. Med. 190, 1891–1896[Abstract/Free Full Text]
  6. Dohrman, A., Russell, J. Q., Cuenin, S., Fortner, K., Tschopp, J., and Budd, R. C. (2005) J. Immunol. 175, 311–318[Abstract/Free Full Text]
  7. Dohrman, A., Kataoka, T., Cuenin, S., Russell, J. Q., Tschopp, J., and Budd, R. C. (2005) J. Immunol. 174, 5270–5278[Abstract/Free Full Text]
  8. Misra, R. S., Jelley-Gibbs, D. M., Russell, J. Q., Huston, G., Swain, S. L., and Budd, R. C. (2005) J. Immunol. 174, 3999–4009[Abstract/Free Full Text]
  9. Irmler, M., Thome, M., Hahne, M., Schneider, P., Hofmann, K., Steiner, V., Bodmer, J. L., Schroter, M., Burns, K., Mattmann, C., Rimoldi, D., French, L. E., and Tschopp, J. (1997) Nature 388, 190–195[CrossRef][Medline] [Order article via Infotrieve]
  10. Micheau, O., Thome, M., Schneider, P., Holler, N., Tschopp, J., Nicholson, D. W., Briand, C., and Grutter, M. G. (2002) J. Biol. Chem. 277, 45162–45171[Abstract/Free Full Text]
  11. Lens, S. M., Kataoka, T., Fortner, K. A., Tinel, A., Ferrero, I., MacDonald, R. H., Hahne, M., Beermann, F., Attinger, A., Orbea, H. A., Budd, R. C., and Tschopp, J. (2002) Mol. Cell. Biol. 22, 5419–5433[Abstract/Free Full Text]
  12. Rao, A., Luo, C., and Hogan, P. G. (1997) Annu. Rev. Immunol. 15, 707–747[CrossRef][Medline] [Order article via Infotrieve]
  13. Macian, F. (2005) Nat. Rev. Immunol. 5, 472–484[CrossRef][Medline] [Order article via Infotrieve]
  14. Yoshida, H., Nishina, H., Takimoto, H., Marengere, L. E., Wakeham, A. C., Bouchard, D., Kong, Y. Y., Ohteki, T., Shahinian, A., Bachmann, M., Ohashi, P. S., Penninger, J. M., Crabtree, G. R., and Mak, T. W. (1998) Immunity 8, 115–124[CrossRef][Medline] [Order article via Infotrieve]
  15. Ranger, A. M., Hodge, M. R., Gravallese, E. M., Oukka, M., Davidson, L., Alt, F. W., de la Brousse, F. C., Hoey, T., Grusby, M., and Glimcher, L. H. (1998) Immunity 8, 125–134[CrossRef][Medline] [Order article via Infotrieve]
  16. Kiani, A., Viola, J. P., Lichtman, A. H., and Rao, A. (1997) Immunity 7, 849–860[CrossRef][Medline] [Order article via Infotrieve]
  17. Ranger, A. M., Oukka, M., Rengarajan, J., and Glimcher, L. H. (1998) Immunity 9, 627–635[CrossRef][Medline] [Order article via Infotrieve]
  18. Rengarajan, J., Tang, B., and Glimcher, L. H. (2002) Nat. Immunol. 3, 48–54[CrossRef][Medline] [Order article via Infotrieve]
  19. Peng, S. L., Gerth, A. J., Ranger, A. M., and Glimcher, L. H. (2001) Immunity 14, 13–20[CrossRef][Medline] [Order article via Infotrieve]
  20. Baksh, S., Widlund, H. R., Frazer-Abel, A. A., Du, J., Fosmire, S., Fisher, D. E., De-Caprio, J. A., Modiano, J. F., and Burakoff, S. J. (2002) Mol. Cell 10, 1071–1081[CrossRef][Medline] [Order article via Infotrieve]
  21. Caetano, M. S., Vieira-de-Abreu, A., Teixeira, L. K., Werneck, M. B., Barcinski, M. A., and Viola, J. P. (2002) FASEB J. 16, 1940–1942[Abstract/Free Full Text]
  22. Phillips, R. J., and Ghosh, S. (1997) Mol. Cell. Biol. 17, 4390–4396[Abstract]
  23. Rincon, M., and Flavell, R. A. (1997) Mol. Cell. Biol. 17, 1522–1534[Abstract]
  24. Earnshaw, W. C., Martins, L. M., and Kaufmann, S. H. (1999) Annu. Rev. Biochem. 68, 383–424[CrossRef][Medline] [Order article via Infotrieve]
  25. Sahara, S., Aoto, M., Eguchi, Y., Imamoto, N., Yoneda, Y., and Tsujimoto, Y. (1999) Nature 401, 168–173[CrossRef][Medline] [Order article via Infotrieve]
  26. Flygare, J., Armstrong, R. C., Wennborg, A., Orsan, S., and Hellgren, D. (1998) FEBS Lett. 427, 247–251[CrossRef][Medline] [Order article via Infotrieve]
  27. Lee, K. K., Murakawa, M., Nishida, E., Tsubuki, S., Kawashima, S., Sakamaki, K., and Yonehara, S. (1998) Oncogene 16, 3029–3037[CrossRef][Medline] [Order article via Infotrieve]
  28. Na, S., Chuang, T. H., Cunningham, A., Turi, T. G., Hanke, J. H., Bokoch, G. M., and Danley, D. E. (1996) J. Biol. Chem. 271, 11209–11213[Abstract/Free Full Text]
  29. Chuvpilo, S., Jankevics, E., Tyrsin, D., Akimzhanov, A., Moroz, D., Jha, M. K., Schulze-Luehrmann, J., Santner-Nanan, B., Feoktistova, E., Konig, T., Avots, A., Schmitt, E., Berberich-Siebelt, F., Schimpl, A., and Serfling, E. (2002) Immunity 16, 881–895[CrossRef][Medline] [Order article via Infotrieve]
  30. Robaye, B., Doskeland, A. P., Suarez-Huerta, N., Doskeland, S. O., and Dumont, J. E. (1994) Electrophoresis 15, 503–510[CrossRef][Medline] [Order article via Infotrieve]
  31. Amess, B., and Tolkovsky, A. M. (1995) Electrophoresis 16, 1255–1267[CrossRef][Medline] [Order article via Infotrieve]
  32. Lockshin, R. A., and Zakeri, Z. (2004) Oncogene 23, 2766–2773[CrossRef][Medline] [Order article via Infotrieve]
  33. Wu, W., Rinaldi, L., Fortner, K. A., Russell, J. Q., Tschopp, J., Irvin, C., and Budd, R. C. (2004) J. Immunol. 172, 4724–4732[Abstract/Free Full Text]
  34. Liu, J., Na, S., Glasebrook, A., Fox, N., Solenberg, P. J., Zhang, Q., Song, H. Y., and Yang, D. D. (2001) Immunity 15, 23–34[CrossRef][Medline] [Order article via Infotrieve]
  35. Budd, R. C. (2002) J. Clin. Investig. 109, 437–441[CrossRef][Medline] [Order article via Infotrieve]
  36. Newton, K., Harris, A. W., Bath, M. L., Smith, K. G., and Strasser, A. (1998) EMBO J. 17, 706–718[CrossRef][Medline] [Order article via Infotrieve]
  37. Yeh, W. C., Pompa, J. L., McCurrach, M. E., Shu, H. B., Elia, A. J., Shahinian, A., Ng, M., Wakeham, A., Khoo, W., Mitchell, K., El-Deiry, W. S., Lowe, S. W., Goeddel, D. V., and Mak, T. W. (1998) Science 279, 1954–1958[Abstract/Free Full Text]
  38. Chun, H. J., Zheng, L., Ahmad, M., Wang, J., Speirs, C. K., Siegel, R. M., Dale, J. K., Puck, J., Davis, J., Hall, C. G., Skoda-Smith, S., Atkinson, T. P., Straus, S. E., and Lenardo, M. J. (2002) Nature 419, 395–399[CrossRef][Medline] [Order article via Infotrieve]
  39. Salmena, L., Lemmers, B., Hakem, A., Matysiak-Zablocki, E., Murakami, K., Au, P. Y., Berry, D. M., Tamblyn, L., Shehabeldin, A., Migon, E., Wakeham, A., Bouchard, D., Yeh, W. C., McGlade, J. C., Ohashi, P. S., and Hakem, R. (2003) Genes Dev. 17, 883–895[Abstract/Free Full Text]

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