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Ephrin-B1 transduces signals to activate integrin-mediated migration, attachment and angiogenesis

¹ Vanderbilt-Ingram Cancer Center, Departments of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA
² Division of Nephrology and Hypertension, Department of Medicine and Clinical Research, University of Bern, CH-3010 Bern, Switzerland
³ Immunex Corporation, Seattle, WA 98101, USA
⁴ Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 USA
⁵ Cell Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA

View larger version (40K): [in a new window]   Fig. 1. EphB1/Fc stimulates endothelial ephrin-B1 tyrosine phosphorylation. Upper panel, ephrin-B1 is expressed in human renal microvascular endothelial cells (HRMEC). HRMEC or CHP100 (Davis et al., 1994) were surface biotinylated as described in the Materials and Methods, and ephrins were immunoprecipitated using non-immune rabbit serum (NRS) or rabbit polyclonal anti-ephrin-B1 antibodies recognizing C-terminal sequences (P1) or anti-ephrin-B1 recognizing an ephrin-B1-specific juxtamembrane spacer domain peptide (P2). Immunoprecipitated complexes were separated on a 10% SDS-PAGE under non-reducing conditions, transferred to PVDF membranes and detected with streptavidin-HRP using enhanced chemiluminescence (Amersham). The ephrin-B1-specific antibody (P2) was used in all subsequent experiments. Lower panel, phorbol myristate acetate and EphB1/Fc stimulate ephrin-B1 tyrosine phosphorylation. Serum-depleted HRMEC were replated on fibronectin-coated p60 tissue culture dishes for 60 minutes, then stimulated for 15 minutes at 37°C with vehicle (NA), phorbol myristate acetate (PMA, 20 ng/ml), control IgG1 (2 µg/ml) or EphB1/Fc at the indicated concentrations. Cells were lysed in RIPA buffer, immunoprecipitated with rabbit anti-ephrin-B1, and the levels of ephrin-B1 tyrosine phosphorylation were determined using the monoclonal antibody 4G10 conjugated to HRP followed by ECL detection. The results are representative of five independent experiments.

View larger version (26K): [in a new window]   Fig. 2. EphB1/Fc promotes endothelial cell migration. Confluent HRMEC were serum depleted prior to mechanical `wounding' to create circular defects of 600-900 µm diameter. The medium was supplemented with vehicle (NA), phorbol myristate acetate (PMA, 20 ng/ml), control IgG1 or EphB1/Fc (0.5 µg/ml each). Fractions of the areas remaining in triplicate wounds were determined by analysis of serial digital images obtained at the times indicated. Migration rates are expressed as a percentage of `wounds' closed per hour.

View larger version (26K): [in a new window]   Fig. 3. EphB1/Fc stimulates vß3 and 5ß1 integrin-mediated endothelial cell attachment. (A) EphB1/Fc stimulates endothelial cell attachment to fibrinogen. 48-well plates were coated with fibrinogen (1 µg/cm2), and endothelial cell attachment was assayed as described in the Materials and Methods. EphB1/Fc stimulated endothelial cell attachment at an optimal concentration of 2 µg/ml and 4 µg/ml for HRMEC and HMEC-1, respectively. An Fc fusion protein control, IgG, is inactive at these concentrations. (B) Upper panel, cell surface expression of integrin proteins in HRMEC and HMEC-1. Eph-B1/Fc does not increase the surface expression of endothelial vß3 in HRMEC cells (left panel). vß3 is expressed at a low level in HMEC-1 cells, whereas vß5 and 5ß1 are expressed at much higher levels, as assayed by FACS analysis (right panel). Lower panel, EphB1/Fc-induced endothelial cell attachment is mediated through vß3 and 5ß1 integrins. Assays of endothelial cell attachment to fibrinogencoated 48-well plates were performed as described in the Materials and Methods. Where indicated, cells were pre-incubated for 15 minutes at 22°C with blocking peptides (100 µg/ml) or anti-integrin antibodies (5 µg/ml) before plating. The data represent means±s.e.m. of three independent experiments. Group comparisons were performed using the Student's t-test. *P<0.05 versus IgG control; **P<0.05 versus RGE control; ***P<0.05 versus no inhibitor control, RGE control or avß5 treated; ****P<0.05 versus no inhibitor control, RGE control or vß5 treated.

View larger version (59K): [in a new window]   Fig. 4. EphB1/Fc stimulates mouse corneal angiogenesis. Hydron pellets impregnated with vehicle (PBS), bFGF (3.0 pmol), control IgG1 or EphB1/Fc (5.6 pmol) were implanted as described in the Materials and Methods and photographed at 5 days post-implantation. R, regional; T, total. The data are representative of four independent experiments.

View larger version (17K): [in a new window]   Fig. 5. EphB1/Fc stimulates migration of CHO cells transfected with ephrin-B1. (A) EphB1/Fc stimulates tyrosine phosphorylation of transfected ephrin-B1. CHO cells were transfected with plasmid expressing full-length ephrin-B1 as described in the Materials and Methods. 48 hours after transfection, cells were stimulated with the control IgG or EphB1/Fc (2 µg/ml), and ephrin-B1 was immunoprecipitated as in Fig. 1. Ephrin-B1 tyrosine phosphorylation (upper panel) was assessed by 4G10 immunoblot and recovery by anti-ephrin-B1 immunoblot (lower panel). (B) 48 hours after transfection with the plasmids indicated, wound closure assays were performed as described above. The migration rate is expressed as the percentage of closure/hour. The medium was replaced with serum-free medium (vehicle) or EphB1/Fc constructs as indicated in the bottom panel. Surface expression of ephrin-B1 ectodomain was analyzed by FACS analysis as described in the Materials and Methods. The data represent means±s.e.m of three independent experiments.

View larger version (39K): [in a new window]   Fig. 6. EphB1/Fc stimulates phosphorylation of p46 JNK. (A) Kinetics of EphB1/Fc-induced JNK activation. CHO cells stably expressing ephrin-B1 were serum-starved overnight and stimulated with EphB1/Fc following a time course as indicated. Cells were lysed and 30 µg of protein was subjected to western blot analysis as described in the Materials and Methods. (B) Concentration dependence of EphB1/Fc-induced JNK activation. CHO cells stably expressing ephrin-B1 were treated with EphB1/Fc from 0 to 4 µg/ml for 20 minutes, and cell lysates were subjected to western blot analysis. (C) Ephrin-B1 C-terminal deletion mutants block EphB1/Fc-induced JNK activation. CHO cells transfected with vector (MOCK), full-length wild-type human ephrin-B1, ephrin-B1Cy or ephrin-B1PDZbd were serum starved and stimulated with EphB1/Fc, and cell lysates were subjected to western blot analysis. The data are representative of three independent experiments.