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Originally published online as doi:10.1189/jlb.1207829 on July 1, 2008

Published online before print July 1, 2008
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(Journal of Leukocyte Biology. 2008;84:1183-1191.)
© 2008 by Society for Leukocyte Biology

Signaling through ephrin-A ligand leads to activation of Src-family kinases, Akt phosphorylation, and inhibition of antigen receptor-induced apoptosis

Halvor L. Holen*, Mohsen Shadidi{dagger}, Kristina Narvhus*, Oddveig Kjøsnes{ddagger}, Anne Tierens{ddagger} and Hans-Christian Aasheim*,1

{dagger} Departments of Immunology and
{ddagger} Pathology, Institute for Cancer Research, Rikshospitalet-Radiumhospitalet Medical Center, Oslo, Norway; and
* Department of Medical Genetics, Ullevaal University Hospital, Oslo, Norway

1 Correspondence: Department of Medical Genetics, Ullevaal University Hospital, 0407 Oslo, Norway. E-mail: hansas{at}ulrik.uio.no


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ABSTRACT
 
Eph receptor tyrosine kinases and ephrins play important roles in diverse biological processes such as migration, adhesion, and angiogenesis. Forward and reverse signaling has been reported in receptor- and ligand-bearing cells. The ligands can be divided into the transmembrane ephrin-B family and the GPI-anchored ephrin-A family. Here, we show expression of ephrin-A ligands on CD4+ T cells cultured in medium with human serum and the T cell line Jurkat TAg and on cells isolated from patients with T cell lymphomas and T cell leukemias. Functional role and identification of proteins involved in ephrin-A signaling were investigated here in the T cell line Jurkat TAg. Signaling through ephrin-A induces phosphorylation of several proteins, including the Src kinases Lck and Fyn. In addition, PI-3K is activated, shown by induced phosphorylation of the Akt kinase. An ephrin-A signaling complex could be isolated, containing several phosphorylated proteins including Lck and Fyn. Interestingly, we show that signaling through ephrin-A in Jurkat TAg cells, initiated by interaction with the EphA2 receptor, leads to inhibition of activation-induced cell death. To conclude, ephrin-A signaling in Jurkat TAg cells leads to induced phosphorylation of several proteins including Lck, Fyn, and Akt. A consequence of ephrin-A signaling is inhibition of antigen receptor-induced apoptosis.

Key Words: Jurkat • CD3 • cell death • tyrosine kinases


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INTRODUCTION
 
Ephrins are cell surface-bound ligands binding to the Eph receptor tyrosine kinases [1 ]. These ligands can be divided into two subgroups: five ephrin-A and three ephrin-B members. In general, ephrin-A ligands bind EphA receptors, and ephrin-B ligands bind EphB receptors [2 ]. One major difference between the two subgroups is that ephrin-A members are linked to the plasma membrane through a GPI anchor, and ephrin-B members are transmembrane proteins. The interaction between ephrins and Eph receptors is important in neural and vascular development, morphogenesis, tissue patterning, angiogenesis, and neural plasticity [2 3 4 5 6 ]. In addition, its role in stem cell biology, immune function, blood clotting, and glucose homeostasis is beginning to be characterized [7 , 8 ].

To date, much information is available regarding function and signaling through Eph receptors. A number of associated proteins and downstream signaling molecules have been identified [9 ]. So far, reverse signaling through ephrin-B members is best understood. Transmembrane ephrin-B signaling is important in axon guidance, cell migration, midline fusion, plasticity, and synaptogenesis [10 11 12 13 ]. It has been shown that activated (clustered) ephrin-B ligands can be phosphorylated on tyrosine by Src family kinase members (Src and Fyn) [14 15 16 ]. This can then lead to interaction with adaptor proteins such as Grb4, followed by activation of signaling pathways, ultimately leading to changes in the actin cytoskeleton and focal adhesions [17 ].

Signaling through ephrin-A members has been demonstrated to have effects on adhesion and morphology [18 , 19 ] and on insulin secretion from pancreatic β cells [8 ]. Few molecules involved in ephrin-A reverse signaling have been identified so far. The Src-kinase family member Fyn and the MAPKs Erk1/2 have been shown to be activated after signaling through overexpressed ephrin-A5 [19 , 20 ]. The activity of the Rho GTPase member Rac1 is stimulated after ephrin-A signaling in pancreatic β cells [8 ]. Signaling through the members of the ephrin-A family is still far less understood than ephrin-B in relation to functional effects and downstream signaling molecules involved.

In this study, ephrin-A members were identified on T cells, Jurkat TAg cells, and T cell lymphomas. Functional effect and identification of molecules involved in reverse ephrin-A signaling are presented. Our data indicate that ephrin-A signaling inhibits activation-induced cell death (AICD) in Jurkat TAg cells.


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MATERIALS AND METHODS
 
Antibodies and inhibitors
The antibodies used in this study were biotinylated anti-human ephrin-A4, anti-ephrin-A4 (R&D Systems, Minneapolis, MN, USA), FITC-labeled anti-CD45RA, anti-Fyn, HRP-labeled rabbit anti-goat, anti-CD3 [clone SK7; allophycocyanin (APC)- and PerCP-labeled], anti-CD4 (clone SK3, APC- and FITC-labeled), anti-CD45 (clone 2D1, PerCP-labeled; BD Biosciences, San Jose, CA, USA), HRP-linked antiphosphotyrosine (clone 4G10, Upstate Biotechnology, Lake Placid, NY, USA), antiphospho-Src family (Tyr416), antiphospho-Zap70 (Tyr319)/Syk (Tyr352), antiphospho-Akt (Ser473), antiphospho-Akt (Thr308; Cell Signaling, Denver, MA, USA), anti-Lck, anti-Fyn, anti-Syk, anti-Akt1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), FITC-labeled anti-CD45RO, polyclonal goat anti-rabbit Igs/HRP and polyclonal rabbit anti-mouse Igs/HRP (DakoCytomation, Carpinteria, CA, USA), goat anti-mouse Ig-R-PE (Ig-RPE; Southern Biotechnology Associates, Birmingham, AL, USA), mouse {gamma}-globulin (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), and human {gamma}-globulin (Sigma-Aldrich, St. Louis, MO, USA). The Src kinase inhibitors Damnacanthal and SU6656 (Calbiochem, Darmstadt, Germany) were used in this study.

Cell lines, separation, and incubation procedures
The T cell line Jurkat TAg, a stable Jurkat transfectant line expressing SV40 large T antigen, and the human embryonic kidney (HEK) cell line HEK293T (ATCC CRL 1573) were used in this study. Cell lines were grown in RPMI-1640 medium (PAA Laboratories, Pasching, Austria) supplemented with 10% FCS (PromoCell, Heidelberg, Germany) at 37°C in a humidified atmosphere with 5% CO2.

CD4+ T cells were isolated using anti-CD4-coated beads (Dynal, Oslo, Norway). T cells were grown in RPMI-1640 medium (PAA Laboratories), supplemented with 10% human pooled serum (PAA Laboratories) at 37°C in a humidified atmosphere with 5% CO2.

For isolation of tonsillar endothelial cells used as control in expression experiments, tonsils were minced and washed before adding a collagenase solution (Collagenase/Dispase/DNaseI, Boehringer Mannheim, Mannheim, Germany), followed by incubation for 15 min at 37°C. The solution was discarded, and fresh solution was added to the minced tissue for an additional 2 h at 37°C. The resulting cell suspension was purified further by Lymphoprep (Nycomed Pharma, Oslo, Norway) density gradient centrifugation. Endothelial cells were then positively isolated with anti-CD34-coated magnetic beads (Dynal) [21 ].

Single-cell suspensions from a part of the patient lymph node tumor tissues involved by T cell lymphoma were prepared using a tumor disaggregation system (79200 Medimachine, Dako). Cell suspensions were subsequently frozen at –70ºC in RPMI 1640 containing 10% FCS and 5% DMSO until further use. The tissues were used for this study in correspondence with institutional and national ethical requirements.

HEK293T cells were transfected, using Lipofectamine (Invitrogen, Carlsbad, CA, USA), with an ephrin-A4 cDNA [22 ] expression vector to obtain expressing cells for control lysates.

Small interfering (si)RNA transfection
CD4+ T cells were transfected with siRNA, directed to ephrin-A1 or ephrin-A4 (ON-TARGETplus SMARTpool, Dharmacon, Chicago, IL, USA), using the AMAXA (Cologne, Germany) nucleofector, according to the manufacturer’s description. Briefly, 5 x 106 cells were transfected with 0.125 nmol siRNA. The cells were then grown in RPMI-1640 medium with 10% human serum for 5 days. Jurkat TAg cells were transfected by using an ECM 830 Electro Square Porator (BTX Inc., San Diego, CA, USA) using a 200 low-voltage setting and 70 ms at room temperature in a 0.4-cm electroporation cuvette (BTX, Genetronics, San Diego, CA, USA). Cells were washed twice with RPMI-1640 medium without any additives before transfection. Cells (5x106/ml) were transfected with 0.25 nmol siRNA and were allowed to grow for 2 days before analysis.

Fusion protein generation
Soluble fusion proteins EphA2-Fc (Fc is the Fc part, including hinge, of mouse IgG2b), EphA4-Fc, ephrin-A1-Fc, and control-Fc were produced as described previously [21 22 23 ]. Proteins were produced in HEK293T cells transfected with fusion protein vectors using Lipofectamine (Invitrogen). Soluble fusion proteins were purified from culture supernatant by affinity chromatography on a protein-A column (Pharmacia, Uppsala, Sweden) as described previously [22 ].

Flow cytometry
CD4+ and Jurkat TAg cells were incubated with fusion proteins (30 µg/ml) at 4°C for 30 min before staining with RPE-linked goat anti-mouse. Further phenotypical separation of CD4+ cells was accomplished through staining with FITC-linked anti-CD45RA or anti-CD45RO (5 µg/ml) after blocking with mouse {gamma}-globulin (1.1 mg/ml).

Single-cell suspensions of lymph node tissues containing T cell lymphoma cells were stained with combinations of anti-CD3, anti-CD4, anti-CD45, and EphA2-Fc to gate T cells and measure their expression of ephrin-A by EphA2-Fc binding.

Analysis of surface protein expression was performed on a FACSCalibur flow cytometer (Becton Dickinson, Heidelberg, Germany). Flow data were analyzed using the computer programs CellQuest (BD Biosciences, San Jose, CA, USA) and FlowJo (Tree Star, Ashland, OR, USA).

Real-time PCR
Quantitative real-time RT-PCR was performed by using the ABI Prism 7900 HT sequence detection system (Applied Biosystems, Foster City, CA, USA). The Assays-on-Demand products purchased from Applied Biosystems contained Taqman minor groove binder probes (6-FAM dye-labeled) combined with the primers for the genes of interest: ephrinA1 (Hs00358886_m1), ephrin-A2 (Hs001154858), ephrin-A3 (Hs00191913), ephrin-A4 (Hs00193299_m1), and ephrin-A5 (Hs00157342_m1). An Assay-on-Demand product for eukaryotic GAPDH (Hs99999905_m1) was used as endogenous control.

Total RNA was isolated from ~10 million cells using the RNAqueous small-scale phenol-free total RNA isolation kit (Applied Biosystems; Ambion, Austin, TX, USA) and quantified using the ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA (1 µg) was reverse-transcribed using a high-capacity cDNA RT kit (Applied Biosystems), and quantitative RT-PCR was performed using TaqMan Universal Master Mix (Applied Biosystems). Reactions were done in triplicate in MicroAmp Optical 384-well plates covered by Optical Adhesive Film (Applied Biosystems). The PCR program was 50°C for 2 min, 95°C for 10 min before 40 cycles of 95°C for 15 s, and 60°C for 1 min. Relative mRNA levels were obtained by the comparative threshold (Ct) method (2-{Delta}{Delta}Ct method; User Bulletin No. 2, Applied Biosystems) using 40 cycles (no detection) as calibration {Delta}Ct. Variations in input of RNA amounts were compensated for by normalizing with GAPDH RNA levels. The data were analyzed with Applied Biosystems RQ Manager, Version 1.2.

DNA synthesis assay
Jurkat TAg cells were cultured (1x104 cells per well) in EphA2-Fc- or control-Fc-precoated (30 µg/ml in 50 mM Tris-HCl, pH 9.5), 96-well, round-bottomed plates in the presence or absence of anti-CD3-coated beads (Dynal). The cell cultures were pulsed with 1 µCi methyl-[3H]thymidine (American Radiolabeled Chemicals, St. Louis, MO, USA) for 24 h after 3 days of incubation. [3H]Thymidine incorporation was measured in the scintillation counter TopCount microplate scintillation counter (Packard Instrument Co., Downers Grove, IL, USA).

Apoptosis assay
Jurkat TAg cells were cultured (2x104 cells per well) in EphA2-Fc- or control-Fc-precoated (30 µg/ml in 50 mM Tris-HCl, pH 9.5), 96-well, round-bottomed plates in the presence or absence of anti-CD3-coated beads (Dynal). Twelve wells were set up for each test condition. After 24 h of incubation, cells were pooled and fixed in 1% paraformaldehyde in PBS for 15 min at 4°C. Cells were then washed once in PBS and permeabilized in pure methanol for at least 20 min at –20°C. DNA fragmentation, as a result of apoptosis, was analyzed by labeling DNA strand breaks with TdT, catalyzing polymerization of fluoresceinlabeled dUTP nucleotides (TUNEL reaction) [24 ], using the in situ cell death detection kit, Fluorescein (Roche Applied Science, Mannheim, Germany). Permeabilized cells were washed twice in PBS before being incubated for 1 h at 37°C in the TUNEL reaction mixture. Cells were stained with Hoechst 33258 (2 µg/ml; Invitrogen) for 15 min before analysis on the LSR II (Becton Dickinson) equipped with the FACSDiVa software (Becton Dickinson).

Signaling in Jurkat TAg cells
Jurkat TAg cells were starved in RPMI medium without serum overnight before signaling experiments were performed. Prior to signaling, cells were washed twice in warm PBS (37°C) before resuspension in PBS and incubation at 37°C for 30 min.

The cells were incubated with cross-linked EphA2-Fc- and/or antibody-coated beads at 37°C for the indicated durations before being spun down and lysed in lysis buffer [0.5% Nonidet P-40, 150 mM NaCl in 10 mM Tris-Cl, pH 7.5, 0.2 mM PMSF, 2.8 µg/ml aprotinin, 1 mM sodium pervanadate, and 10 µl phosphatase inhibitor cocktail (Sigma-Aldrich)] on ice for 30 min. Lysates were cleared by high-speed sentrifugation (1.8x104 g for 5 min at 4°C).

Immunoprecipitation and Western blotting
Immunoprecipitations were performed with antibodies preadsorbed to Protein G beads. Antibodies (0.7 µg/sample) were added to washed Protein G Dynabeads (Dynal; 10 µl/sample) in PBS and rotated for 1 h at room temperature. Unoccupied Protein G-binding sites were blocked with control-Fc for 30 min at room temperature before washing to remove excess antibody and fusion protein. Beads with antibody were added to lysates and rotated overnight at 4°C to acquire immunoprecipitates. Protein G beads were washed twice in lysis buffer after lysate incubation.

To isolate the ephrin-A4 signaling complex in Jurkat TAg cells, precipitations were performed by adding Protein G beads directly to the lysates to precipitate EphA2-Fc-associated proteins. Incubations were performed overnight at 4°C. EphA2-Fc (0.3 µg) was added to unstimulated samples. Protein G beads were washed twice in lysis buffer after lysate incubation.

Total cell lysates and immunoprecipitations were separated in SDS-polyacrylamide gels cast using RapidGel-40%-acrylamide/bis-acrylamide (19:1; GE Healthcare Bio-Sciences, Uppsala, Sweden) or ready-cast gels (Pierce, Rockford, IL, USA). Protein membranes were treated for 1 h at room temperature or overnight at 4°C with primary antibody before treatment with the appropriate HRP-linked secondary antibody. Signals after antibody incubation were detected using the ECL Plus Western blotting detection system (GE Healthcare Bio-Sciences).

Band intensity measurements were done in Quantity One, Version 4.6.3 (Bio-Rad Laboratories, Hercules, CA, USA), using local background subtraction.


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RESULTS
 
Ephrin-A expression in T cells
Expression of cell surface-bound, functional ephrin-A ligands was tested by incubating T cells and Jurkat TAg cell with soluble EphA-Fc fusion proteins. EphA2 has been shown to bind all members of the ephrin-A family [25 ], and EphA4 has a somewhat different binding pattern [2 ]. Soluble EphA2-Fc and EphA4-Fc fusion protein was used to detect functional receptor-binding ligands on the cell surface of CD4+ T cells and Jurkat TAg cells. Binding of the receptor fusion protein to cells was detected by flow cytometry. Our results show no or only weak EphA2-Fc binding to freshly isolated CD4+ T cells from blood (Fig. 1A ). Five days of incubation in medium with 10% human serum showed induced EphA2-Fc binding to the CD4+CD45RA+ population, consisting mostly of naïve T cells (Fig. 1A) . No EphA2-Fc binding was seen in the CD4+ CD45RA– population, identified as CD45RO+ (data not shown). We could not detect EphA4-Fc binding to CD4+ T cells (Fig. 1A) . Incubation of cells in medium with 10% FCS also led to increased EphA2-Fc binding but lower than with human serum (data not shown). Incubation in serum-free medium (X-VIVO 15) led to weakly induced EphA2-Fc binding (data not shown). Different CD4+ T cell isolation protocols, positive or negative isolation, did not affect the EphA2-Fc binding after serum stimulation (data not shown). In addition, depletion of dendritic cells by MHC class II antigen depletion before serum stimulation did not affect the EphA2-Fc binding (data not shown). After screening of a number of Jurkat-TAg cell lines, one line was found to bind EphA2-Fc and EphA4-Fc but not ephrin-A1-Fc (Fig. 1B) , indicating that this cell line expresses ephrin-A members but not EphA receptor members.


Figure 1
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  		Figure 1. Expression of ephrin-A ligands. (A) Costaining of CD4+ cells with anti-CD45RA and control-Fc, EphA2-Fc, or EphA4-Fc. Days of incubation in medium with human serum are indicated above the panels. (B) Left panel: Binding of control-Fc (dotted line), ephrin-A1-Fc (solid line), and EphA2-Fc (dashed line) to Jurkat TAg cells. Only EphA2-Fc binding is detected. Right panel: Binding of control-Fc (dotted line) and EphA4-Fc (dashed line) to Jurkat TAg cells. FL2, Fluorescence 2. (C) Costaining of single cell suspensions of lymph node tissue with anti-CD4 and control-Fc or EphA2-Fc. Three cases, 1–3, show significant binding of EphA2-Fc. Case 1 is composed of a monomorphic tumor population, whereas the remaining two cases contain a prominent background of inflammatory cells. (D) Quantitative RT-PCR of ephrin-A mRNA in CD4+ cells (upper panel) and Jurkat TAg (lower panel). The specific ephrin-A member is shown below the bars. Black bar, day 0; gray bar, day 5. A representative experiment of three is shown. (E) Effects of ephrin-A1- and -A4-directed siRNA on CD4+ T cells. Costaining of CD4+ cells with anti-CD45RA and EphA2-Fc. Cells were transfected with the indicated siRNA (above the panels) and incubated for 5 days. (F) Effects of ephrin-A1- and -A4-directed siRNA on Jurkat TAg cells. Binding of control-Fc (dotted line) and EphA2-Fc or EphA4 (dashed line) to Jurkat TAg cells, which were transfected with the indicated siRNA (above the panels) and incubated for 2 days in medium. (G) Detection of ephrin-A4 protein expression by immunoprecipitation. Ephrin-A4 antibody precipitations were performed in lysates prepared from 50 x 106 Jurkat TAg cells or 0.2 x 106-transfected 293T cells (upper panel). E4 is an immunoprecipitate from ephrin-A4-transfected HEK293T cells. Mock is an immunoprecipitate from HEK293T lysate transfected with an empty expression vector. Actin antibody incubation serves as a loading control for input lysate. The lower panel shows ephrin-A4 immunoprecipitation from CD4+ T cells. Numbers above this panel indicate days of incubation in medium with human serum. (H) Detection of ephrin-A4 after EphA2-Fc precipitation from Jurkat TAg or transfected HEK293T cell lysates. Lane 1, Control-Fc precipitation from 30 x 106 Jurkat TAg cells. Lane 2, EphA2-Fc precipitation from 30 x 106 Jurkat TAg cells. Lane 3, Jurkat TAg cell lysate (~1.5x106 cells). Lane 4, Mock-transfected HEK293T cell lysate (~1x105 cells). Lane 5, Ephrin-A1-transfected HEK293T cell lysate (~1x105 cells). Lane 6, EphA2-Fc precipitation from 3 x 105 ephrin-A1-transfected HEK293T cells. Lane 7, Ephrin-A4-transfected HEK293T cell lysate (~1x105 cells). Lane 8, EphA2-Fc precipitation from 3 x 105 ephrin-A4-transfected HEK293T cells.

Seventeen cases of peripheral T cell lymphomas and T cell leukemias were also analyzed for the expression of functional cell surface-bound ephrin-A members (see Supplementary File 1). The samples were obtained from patients who presented with T cell lymphoma or leukemias. The samples are taken at primary diagnosis of untreated patients. Blood samples or single cell suspensions of lymph node tissue were prepared and stored as described (see Materials and Methods) before phenotypic analysis. A four-color flow cytometry assay was used to identify the CD4 T cells and binding of EphA2-FC. Three cases of CD4+ T cell lymphomas showed significant binding of EphA2-Fc to CD4+ T cells (Fig. 1C) . One of these (Case 1) was composed of a monomorphic tumor population, whereas the remaining two cases contained a prominent background of inflammatory cells. It is therefore likely that in the latter cases, mostly reactive T cells showed EphA2-Fc binding and not the tumor cells.

Expression of ephrin-A ligands was investigated at the mRNA level by real-time PCR analysis. The results from fresh isolated CD4+ T cells show ephrin-A1 and -A4 expression but not -A2, -A3, or -A5 (Fig. 1D) . The data for ephrin-A2, -A3, and -A5 are not shown in Figure 1D . The expression of ephrin-A1 was higher than ephrin-A4. A primer efficacy test was performed on the different ephrin primers showing minor differences (data not shown). Expression of ephrin-A1 and -A4 was induced after culturing CD4+ T cells in medium containing 10% human serum (Fig. 1D) . Jurkat-TAg cells express ephrin-A1, -A2, -A3, and -A4, as shown by real-time PCR. Here, ephrin-A4 expression was higher than the others (Fig. 1D) .

The ephrin mRNA expression data led us to investigate further which ephrin members were responsible for binding EphA2 and EphA4. We performed transfection of normal T cells and Jurkat TAg cells with siRNA specific for ephrin-A1 or ephrin-A4. Normal T cells were then incubated for 5 days in medium with 10% human serum, and Jurkat TAg cells were incubated for 1 day in normal growth medium. The cells were then analyzed for binding of EphA2-Fc or EphA4-Fc to the cell surface by flow cytometry. The results show that ephrin-A1 is the major ephrin responsible for binding EphA2-Fc in normal CD4+ T cells (Fig. 1E) . In Jurkat TAg cells, only ephrin-A4 siRNA had an effect on EphA2-Fc or EphA4-Fc binding (Fig. 1F) .

Expression of ephrin-A4 protein on the protein level was also investigated in Jurkat TAg cells and in CD4+ T cells. Difficulties in evaluation and detection of endogenously expressed ephrin-A4 from total cell lysates by Western blot analysis led us to perform immunoprecipitation for detection. Ephrin-A4 protein expression was clearly detected in Jurkat TAg cells (Fig. 1G) . Here, ephrin-A4-transfected HEK293T cells served as our positive control. In addition, we could detect ephrin-A4 after precipitation with EphA2-Fc (Fig. 1H) . We could also detect ephrin-A4 protein after immunoprecipitation in T cells stimulated for 7 days with serum compared with freshly isolated cells (Fig. 1G) . We could not detect endogenously expressed ephrin-A1 protein as a result of weak antibody reactivity.

Signaling through ephrin-A ligands on T cells affects cell growth and survival
As described above, the T cell line Jurkat TAg used in this study expresses mainly ephrin-A4 but not EphA receptors. These features make the cell line suitable for the study of separate ligand function and signaling. Previously, signaling through overexpressed ephrin-A5 or ephrin-A2 has been described to be involved in integrin-mediated adhesion mechanisms [18 19 20 ]. The effect on adhesion after signaling through ephrin-A4 in Jurkat TAg cells was tested. The experiments were performed by comparing binding of Jurkat TAg cells with fibronectin in the presence of EphA2-Fc or control-Fc in a procedure similar to the one described [19 ]. We were not able to draw conclusions from these experiments. We then tested the effect on cell growth by incubating cells in fusion protein-coated wells followed by [3H]thymidine incorporation. The cells were stimulated with anti-CD3-coated beads to mimic antigen receptor stimulation. We observed a clear inhibition of cell growth when cells were incubated with beads (Fig. 2A ). Five or 7.5 anti-CD3 beads/cell were used in the experiments. Increasing numbers of anti-CD3-coated beads per cell led to increased growth inhibition. This negative effect on growth was partially abrogated when cells were grown in EphA2-Fc-coated wells, observed as an increase in thymidine incorporation. No significant difference in incorporation could be observed in cells grown in bead-free control-Fc- or EphA2-Fc-coated wells (data not shown).


Figure 2
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  		Figure 2. Effect of ephrin-A signaling on cell survival. (A) Jurkat TAg cells were cultured in wells coated with control-Fc (Fc) and EphA2-Fc (A2-Fc). Experiments were performed in triplicates. Relative DNA synthesis is given for cells cultured with or without 5 or 7.5 anti-CD3 beads (b) per cell. [3H]Thymidine was added for 24 h after 3 days incubation before thymidine incorporation was determined. Data from three experiments are given as relative proliferation obtained by normalizing the mean counts for each separate sample to the mean count for control-Fc-stimulated cells in medium ± SEM. (B) TUNEL experiments showing percentage of apoptotic cells. Columns are marked as explained in A. The mean percentage of apoptotic cells from two (7.5 beads), four (five beads), or three (no beads) experiments ± SEM is shown. (C) A representative TUNEL experiment showing apoptotic cells in the upper gates, indicating dUTP-FITC-labeled cells, which were cultured in control-Fc- or EphA2-Fc-coated wells containing five anti-CD3 beads per cell. FSC, Forward scatter.

It is well known that anti-CD3 stimulation of Jurkat cells can lead to AICD [26 ]. We therefore investigated if the growth-promoting effect of EphA2-Fc were a result of inhibition of apoptosis. Initial observations using propidium iodide staining to detect dead cells by flow cytometry indicated this (data not shown). To directly investigate the effect on apoptosis, we performed the TUNEL assay on cells stimulated with anti-CD3 in control-Fc- or EphA2-Fc-coated wells. As shown in Figure 2 B and C , anti-CD3 stimulation of Jurkat TAg cells led to induction of apoptosis. This effect could partially be inhibited by EphA2-Fc, indicating that signaling through ephrin-A4 favors cell survival (Fig. 2 B and C) .

Identification of molecules involved in ephrin-A4 signaling
We sought to identify the molecules involved in the ephrin-A4 signaling cascade to further understand the mechanism behind the observed inhibition of apoptosis. Previous studies have shown induced tyrosine phosphorylation on several proteins after ephrin-A5 and ephrin-A2 signaling [18 , 20 ]. The effect of signaling through ephrin-A4 at the phosphotyrosine level was therefore investigated. Cross-linked soluble EphA2-Fc was used to induce ephrin-A4 clustering and signaling. As shown in Figure 3A , induced tyrosine phosphorylation could be observed on a number of proteins already after 3 min of stimulation with EphA2-Fc. In particular, a prominent signal could be observed 75–80 kDa. These data show that signaling can be induced in Jurkat TAg cells after engagement of the cell-bound, GPI-linked ephrin-A4 ligand with the EphA2 receptor.


Figure 3
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  		Figure 3. Ephrin-A signaling induces tyrosine phosphorylation. (A) Proteins are tyrosine-phosphorylated after EphA2-Fc-induced ephrin-A4 signaling. The duration of signaling is shown in minutes. A total lysate Western blot was incubated with the general antiphosphotyrosine antibody (pY) and an antiphospho (p)-Src antibody. Actin antibody serves as loading control. (B) Lck and Fyn were immunoprecipitated (IP) from Jurkat TAg cell lysates. The duration of EphA2-Fc-induced ephrin-A4 signaling is shown in minutes. Phosphorylation of Src-family kinases was detected by incubating Western blots with an antiphospho-Src family antibody. Incubation with Lck or Fyn antibodies serves as loading control. (C) Syk was immunoprecipitated from lysates as described in B. Phosphorylation of Syk was detected with an antiphospho-Syk antibody. Incubation with a Syk-specific antibody serves as loading control.

The Src-kinase family member Fyn has been shown previously to be involved in ephrin-A5 signaling [18 ]. We therefore investigated the effect of ephrin-A4 signaling on Src family kinases in Jurkat TAg cells. An antiphospho-Src antibody was used to detect activated Src-family kinases. This antibody cross-reacts with most Src-family kinase members and recognizes a phosphorylated tyrosine residue in the activation loop of the kinase domain. Detection in total cell lysates showed induced phosphorylation of at least two proteins with a slightly different molecular mass after EphA2-Fc binding, indicating that more than one Src-family kinase member is activated (Fig. 3A) . Immunoprecipitation of different Src-family kinases was performed to identify the individual members followed by detection with the antiphospho-Src antibody. The Src-family members Lck and Fyn showed induced phosphorylation after EphA2-Fc binding (Fig. 3B) , indicating that these two kinases were detected by the antiphospho-Src antibody. Syk and Zap70 belong to a small family of kinases that is phosphorylated by Src kinases [27 ]. Phosphorylation of Syk was tested using phospho-specific antibodies to the tyrosine residues Y323, Y352, and Y525/526. Our results show a clear induction of phosphorylation on Y352 (Fig. 3C) but not on Y323 or Y525/526 (data not shown). No or low induced phosphorylation of Zap70 was observed using the general antiphosphotyrosine antibody 4G10 or a phospho-specific Y319 antibody (data not shown).

The Akt serine/threonine kinases have been shown to be involved in several cellular mechanisms, including the cell survival pathways [28 ]. We therefore investigated if Akt kinases were affected after EphA2-Fc binding to Jurkat TAg cells. The results presented in Figure 4A show induced phosphorylation of serine 473 on Akt after EphA2-Fc stimulation, observable already after 3 min. Phosphorylation of threonine 308 on Akt was also observed at this time-point (data not shown). Akt kinases are activated by the products of PI-3Ks [29 ]. To investigate the involvement of Src kinases in Akt activation and thus, in PI-3K activation, different Src kinase inhibitors were tested for their effect on Akt phosphorylation. The results show that the two Src kinase inhibitors tested (SU6656 and Damnacanthal) inhibited phosphorylation of Akt, indicating the role of Src kinases in PI-3K activation through ephrin-A4 (Fig. 4B) .


Figure 4
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  		Figure 4. Ephrin-A4 signaling leads to Akt phosphorylation (A) after EphA2-Fc-induced ephrin-A4 signaling detected in protein lysates with an antiphospho-Akt antibody. Akt serves as loading control. (B) Jurkat TAg cells were incubated in PBS or with the Src family kinase inhibitors Damnacanthal (DC) or SU6656 before stimulation with EphA2-Fc for the indicated time in minutes. Phosphorylation of Akt and Src family kinases was detected with antiphospho-specific antibodies as indicated. Detection with an Akt-specific antibody serves as loading control. (C) Relative intensity measurements of bands presented in Figure 4B . The measured signal intensity obtained by the phospho-sensitive antibodies was divided by the signal intensity of total Akt.

Identification of a complex involved in ephrin-A4 signaling
We sought out to isolate the signaling complex formed after EphA2-Fc binding to Jurkat TAg cells to understand the initial signaling events induced through GPI-linked ephrin-A4. Our approach took advantage of the cross-linked Fc-tag of EphA2-Fc to isolate the protein complex associated with ephrin-A4. Jurkat TAg cells were incubated for selected time-points with cross-linked EphA2-Fc, the cells were lysed, and the cell lysates were incubated overnight with Protein G beads (see Materials and Methods). The cross-linked EphA2-Fc bound to ephrin-A4 adsorbed to the Protein G beads. Unbound EphA2-Fc had been washed away before cell lysis. EphA2-Fc protein was added to the control lysate (0 min) to be able to immunoprecipitate ephrin-A4 from this lysate, also using this approach. The proteins were separated by SDS-PAGE before Western blot analysis. By this approach, a protein complex could be precipitated consisting of tyrosine-phosphorylated proteins with molecular masses of ~120, 75–80, and 60 kDa (Fig. 5A ). We then investigated if the Src-family kinases, already shown to be activated after EphA2-Fc stimulation (Fig. 3B) , could be a part of this complex. A phospho-Src antibody clearly showed induced phosphorylation on Src-family kinases in this complex (Fig. 5A) . Two representative experiments are shown here. In addition, specific signals could be obtained with a Lck and a Fyn antibody (Fig. 5A) . Thus, activated Fyn and Lck could be part of the ephrin-A4 signaling complex. The validity of this approach was shown by the identification of ephrin-A4 in the complex (Fig. 5B) . A stronger ephrin-A4 signal was observed here in the control sample as a result of the addition of EphA2-Fc after cell lysis, precipitating all ephrin-A4 available in the lysate rather than only on the cell surface.


Figure 5
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  		Figure 5. Identification of an ephrin-A4 signaling complex. (A) Jurkat TAg cells were stimulated with EphA2-Fc for the indicated time in minutes. Protein-G precipitations were performed directly in the lysates as described (see Materials and Methods). Detection of phosphorylated proteins is performed with a general antiphosphotyrosine and an antiphospho-Src antibody. Lck and Fyn were detected using specific antibodies. (B) Protein-G precipitations were performed in Jurkat TAg lysates, and the presence of ephrin-A4 was confirmed with an antiephrin-A4 antibody. E4 contains lysate of 293T ephrin-A4 transfectants. Actin is used as a loading control for input lysate.


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DISCUSSION
 
In this study, we show expression of ligands for EphA receptors on normal T cells and on the Jurkat TAg T cell line. In addition, we present data about lymphocytes isolated from patients with T cell lymphomas and leukemias, which in some cases also express ligands for EphA receptors. We show here that freshly isolated blood T cells do not, or only weakly, bind the soluble EphA2 receptor but do so after 4–7 days of incubation in medium with human serum. Thus, a combination of removing the CD4+ T cells from its natural environment and adding serum factors led to the observed, induced ephrin-A4 expression. Our data also show that the binding of EphA2 is restricted to the CD4+CD45RA+ T cell population, consisting mainly of naïve T cells. EphA2 binds to all ephrin-A members [25 ] and is an indicator for the presence of cell surface-bound ephrin-A members.

Our mRNA expression results reveal that EphA2-Fc most likely binds to ephrin-A1 expressed on CD4+ T cells. Real-time PCR showed an induced expression of ephrin-A1 and ephrin-A4 after serum stimulation, and ephrin-A1 expression was the highest. In addition, siRNA directed to ephrin-A1 almost completely abrogated the binding of EphA2-Fc, and no effect was seen with ephrin-A4-directed siRNA. In Jurkat TAg cells, ephrin-A4 mRNA expression was the highest, leading to binding of EphA2-Fc and EphA4-Fc. siRNA directed to ephrin-A4 showed some effect on EphA2-Fc and EphA4-Fc binding in Jurkat TAg cells, and no effect could be seen after ephrin-A1 siRNA transfection, indicating that ephrin-A4 is the major EphA-binding ligand in these cells.

An expression study about T cell lymphomas and leukemias was conducted using EphA2-Fc to detect ephrin-A ligands. Out of seventeen cases tested, only one showed expression of cell surface-bound ephrin-A ligand on the tumor cells. Ephrin-A ligand was also detected on the majority of T cells from two other cases, but here, the tumor cell population was evaluated to be a minor population on tissue sections. Thus, most likely, the CD4+ T cells expressing ephrin-A ligands here represent reactive normal T cells. As such, these data may represent examples of a physiological condition where normal T cells express ephrin-A ligand. We have not identified here the particular ephrin-A member expressed on these cells as a result of the limited material.

The leukemia T cell line Jurkat TAg used in our study did not bind ephrin-A ligands but expressed ephrin-A4, as shown by expression analysis and EphA2-Fc binding. This cell line was therefore chosen for further studies. Previous studies with overexpressed ephrin-A5 and ephrin-A2 have shown a role for ephrin-A signaling in integrin-dependent adhesion of cells to extracellular matrix proteins [18 , 19 ]. We could not conclude from our studies if this happened in Jurkat TAg cells. We observed that growth of these cells was affected when cells were incubated on plastic-bound EphA2-Fc in combination with antigen receptor stimulation (anti-CD3). In vivo, preactivated and expanded T lymphocytes that receive a restimulation signal via their TCR undergo AICD involving death receptors, such as CD95 (Apo-1/Fas) [26 ]. Stimulation of the TCR in Jurkat cells by anti-CD3 also leads to AICD [26 ]. This process was clearly inhibited or dampened when the cells were also stimulated with EphA2-Fc. This effect needed the presence of serum during the experiments. Induced AICD was also observed in cells grown without serum (50% death), but no effect of ephrin signaling was seen here (data not shown). Low induction of AICD (10% death) was observed when cells were grown in the serum-free medium X-VIVO 15, and no effect of ephrin signaling was observed here (data not shown). The serum factors cooperating with ephrin-A signaling in Jurkat TAg cells have not been discovered. So far, an antiapoptotic effect has not been reported previously after signaling through ephrins. Such effects have been reported through Eph receptor signaling in different cell systems, for example, through the EphB6 receptor in Jurkat cells [30 ], EphB and EphA receptors in the thymus [31 , 32 ], and EphB4 in breast cancer cells [33 ].

Signaling through ephrin-A4 in Jurkat TAg cells involved induced phosphorylation on several proteins. In particular, phosphorylation of the Src-kinases Fyn and Lck and the Ser/Thr kinase Akt was observed. In addition, ephrin-A4 signaling led to tyrosine phosphorylation of Syk and other not-yet-identified proteins with molecular masses of ~80 and 110–120 kDa. Previous studies have shown that ephrin-A signaling can involve Fyn, Rac1, Erk1/2 and unidentified tyrosine-phosphorylated proteins with molecular masses of 80 and 120 kDa [8 , 18 19 20 ]. In Jurkat TAg cells, no activation of Erk1/2 could be observed after EphA2-Fc stimulation within (data not shown) 30 min of stimulation. Whether the 80- and 110–120-kDa proteins observed after ephrin-A5 and ephrin-A2 signaling are the same as the proteins observed after signaling through ephrin-A4 in Jurkat TAg cells awaits further identification [18 , 20 ].

The activation of the Akt kinase might lead to the observed, antiapoptotic effect. Akt kinases have been shown to be involved in numerous signaling pathways, including cell survival [28 ]. We observe induced phosphorylation of Akt after ephrin-A4 signaling, indicating activation of PI-3Ks. Src kinases have been shown previously to be involved in PI-3K activation [34 ]. Different Src kinase inhibitors affected the activation of Akt in Jurkat TAg cells, but it is not yet clear whether Fyn or Lck or both are involved in this. Both have been reported previously to be involved in PI-3K activation, leading to induced Akt phosphorylation [34 , 35 ]. The consequence of the observed Syk phosphorylation after ephrin-A4 signaling is unclear, but other studies have shown that Syk can participate in PI-3K activation [36 ].

A model for activation of ephrin-A ligands has been suggested, where ephrin-A is localized to discrete membrane microdomains [20 ]. Signaling is initiated after binding of the cross-linked EphA receptor and might be transferred from the cell surface via a putative, unknown adaptor protein that recruits Src kinases and maybe their target proteins. This model has, so far, not been proven. Identifying the members of such a complex of signaling proteins associated with ephrin-A4 would be an important step in understanding the consequences of ephrin-A signaling. Our strategy here was to identify a putative signaling complex generated after binding of cross-linked EphA2-Fc to the ephrin ligand on Jurkat TAg cells. The ligand-receptor complex, with associated proteins, was then isolated via the bound, cross-linked EphA receptor. In this complex, the Src kinases Fyn and Lck and unidentified proteins of 80 and 110–120 kDa could be observed. Induced phosphorylation could be observed on all of these proteins. The Src kinases are well-known transducers of signals from GPI-anchored proteins [37 ]. These kinases anchor themselves onto the inner leaflet of the lipid bilayer via N-terminal lipid modification [38 ]. One model for transferring signal from GPI-linked proteins is that cross-linking of these in lipid rafts leads to the formation of membrane patches enriched in Src-family kinase and tyrosine-phosphorylated proteins [39 ]. Transduction of signals might also be transferred by transmembrane adaptor proteins localized close to or in contact with the GPI-linked proteins [20 ]. The identification of the phosphorylated 80- and 120-kDa proteins might shed some light on this matter. Molecules with similar masses have also been associated with signaling through the GPI-linked proteins CD59 [40 ] and Thy-1 [41 ].

We have not identified the direct link between the effect of ephrin-A4 signaling and inhibition of cell death by apoptosis. The signaling data here are presented for ephrin-A4 signaling alone and not in combination with anti-CD3, and anti-CD3 stimulation also induces the phosphorylation of Lck, Fyn, and Akt. No significant differences were observed in the induced phosphor levels of Lck, Syk, and Akt, with anti-CD3 alone or in the combination of anti-CD3 and EphA2-Fc (data not shown). Still, the localization of these molecules might be changed when introducing ephrin-A4 signaling together with an antigen receptor, although we do not show any evidence for this. This could then ultimately affect the observed effect on apoptosis. We could not observe any effect of ephrin-A4 signaling when cells were incubated with an anti-Fas antibody (CH11) [42 ] to directly induce apoptosis in Jurkat TAg cells (data not shown). This indicates that the antiapoptotic effect lies upstream of Fas signaling. We have also investigated if ephrin signaling affected anti-CD3-induced Fas ligand (FasL) mRNA and protein expression by real-time PCR or ELISA (Calbiochem FasL ELISA kit), respectively, without observing any effect (data not shown). A peak level of FasL mRNA was observed after 3 h in anti-CD3-stimulated cells, regardless of EphA2-Fc stimulation.

This study has revealed expression of ephrin-A ligands in different T cell settings. The physiological role of ephrin-A expression in normal T cells and which serum components induce the expression remain open questions. Our previous studies indicated that ephrin-A4 mRNA expression could be observed in the T cell-rich area in tonsils [22 ], indicating a physiological role there. In addition, the expression of ephrin-A ligands on reactive T cells from patients with T cell lymphomas further indicates a role for ephrin-A ligands on T cells at a distinct activation or differentiation stage. Our study shows that signaling through ephrin ligands on T cells can have an antiapoptotic effect. Signaling through ephrin-A4 on Jurkat TAg cells involves phosphorylation of several proteins including the Src-family kinases Lck and Fyn, Syk, Akt, and several not-yet-identified proteins. Our results indicate that a signal complex is generated after receptor interaction with ephrin-A4 involving Src-family kinases and unknown phosphorylated proteins. Although we have identified the components involved in the initial signaling through ephrin-A4, the specific cause of the antiapoptotic effect has not yet been elucidated.


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ACKNOWLEDGEMENTS
 
This work was supported by the Norwegian Cancer Society.

Received December 13, 2007; revised April 29, 2008; accepted June 9, 2008.


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