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EphB ligand, ephrinB2, suppresses the VEGF- and angiopoietin 1-induced Ras/mitogen-activated protein kinase pathway in venous endothelial cells¹

National Creative Research Initiatives Center for Endothelial Cells and Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790–784, Republic of Korea; and
^* Regeneron Pharmaceuticals Inc., Tarrytown, New York, USA

³Correspondence: National Creative Research Initiatives Center for Endothelial Cells, Department of Life Science, Pohang University of Science and Technology, San 31, Hyoja-Dong, Pohang, 790–784, Republic of Korea. E-mail: gykoh{at}postech.ac.kr

SPECIFIC AIM

Although interaction between ephrinB2 and EphB in endothelial cells at the arterial-venous capillary interface are critical for proper embryonic capillary morphogenesis, the intracellular downstream signaling of ephrinB2-EphB in vascular endothelial cells is unknown. This study examined the effect of ephrinB2-induced activation of EphB kinases on vascular endothelial growth factor (VEGF) and angiopoietin 1 (Ang1) -induced Ras/mitogen-activated protein kinase- (MAPK) signaling cascades in human umbilical vein endothelial cells (HUVECs).

PRINCIPAL FINDINGS

1. HUVECs express EphB2, EphB3, EphB4, and ephrinB2
EphrinB2 is a ligand of the EphB2, EphB3, and EphB4 kinases. Among these kinases, EphB4 has ephrinB2 as its only known ligand. Our RT-PCR results indicate that HUVECs express all four EphB kinases as well as ephrinB2. Therefore, our subsequent results with ephrinB2-Fc were due to activation of all three endogenous EphB kinases and those with EphB4-Fc resulted from activation of endogenous ephrinB2.

2. EphrinB2-Fc suppresses VEGF- and Ang1-induced ERK1/2 phosphorylation
To measure MAPK activity, we measured the phosphorylation of two MAPKs: extracellular signal-regulated kinase 1 (ERK1; p44^MAPK) and ERK2 (p42^MAPK). VEGF (10 ng/mL) increased ERK1/2 phosphorylation as early as 2.5 min and produced a maximal effect at 10 min (Fig. 1 A). These effects declined and returned to control levels at 60 min. The maximum mean increases in ERK1 and ERK2 phosphorylation were 10.9- and 14.2-fold. To investigate how activation of EphB kinases affects the MAPK signaling pathway, we added ephrinB2-Fc to HUVECs. EphrinB2-Fc (10–1000 ng/mL) did not produce any changes in basal ERK1/2 phosphorylation (data not shown). However, addition of ephrinB2-Fc (500 ng/mL) inhibited 50–55% of the VEGF-induced ERK1/2 phosphorylation but did not produce any changes in basal ERK1/2 phosphorylation (Fig. 1B ). Anti-IgG Ab (5 µg/mL used to cluster ephrinB2-Fc) alone did not produce any changes in basal or VEGF-induced ERK1/2 phosphorylation (Fig. 1B ). Addition of ephrinB2-Fc (500 ng/mL) inhibited 45–55% of the VEGF-induced ERK1/2 phosphorylation at three time points: 5, 10, and 15 min (Fig. 1C ). Ang1 (100 ng/mL) increased ERK1/2 phosphorylation as early as 5 min and produced a maximal effect at 15 min (Fig. 2 A), returning to control levels at 60 min. The maximum mean increases in ERK1 and ERK2 phosphorylation were 5.8- and 8.4-fold. Addition of ephrinB2-Fc (500 ng/mL) inhibited 60–65% of the Ang1-induced ERK1/2 phosphorylation, whereas anti-IgG Ab (5 µg/mL) alone had no effect on either basal or Ang1-induced phosphorylation (Fig. 2B ). Addition of ephrinB2-Fc (500 ng/mL) inhibited 40–70% of the Ang1-induced ERK1/2 phosphorylation at 7, 15, and 25 min (Fig. 2C ). In turn, activation of endogenous ephrinB2 with EphB4-Fc did not alter basal, VEGF-induced, or Ang1-induced ERK1/2 phosphorylation at any time point tested.

View larger version (61K): [in this window] [in a new window]   Figure 1. EphrinB2 suppresses VEGF-induced MAPK activity. A) HUVECs were incubated with VEGF165 (10 ng/mL). B) HUVECs were incubated with control buffer (CB), ephrinB2-Fc (500 ng/mL, E), clustering Ab (5 µg/mL, CA), VEGF (10 ng/mL, V), E plus V (EV), or CA plus V (CAV) for 10 min. C) HUVECs were incubated with control buffer (CB), ephrinB2-Fc (500 ng/mL, E), VEGF (10 ng/mL, V), or E plus V (EV). Cell lysates were harvested after treatment. Each lane contains 50 µg of total protein from the cell lysates. Western blots were probed with anti-phospho-ERK antibody (upper panels). The blot was reprobed with anti-ERK antibody or anti-actin antibody (lower panels) to verify equal loading of protein in each lane. Results were similar in 3 independent experiments. Fold: Densitometric analyses are presented as the relative ratio of phospho-ERK1 to ERK1 or actin. The relative ratio measured in at time 0, or the ratio relative to control buffer, is arbitrarily presented as 1. Numbers represent the mean ± SD from 3 experiments. *P < 0.05 vs. time 0 or CB. #P < 0.05 vs. V.

View larger version (61K): [in this window] [in a new window]   Figure 2. EphrinB2 suppresses Ang1-induced MAPK activity. A) HUVECs were incubated with Ang1 (100 ng/mL). B) HUVECs were incubated with control buffer (CB), ephrinB2-Fc (500 ng/mL, E), clustering Ab (5 µg/mL, CA), Ang1 (100 ng/mL, A), E plus A (EA), or CA plus A (CAA) for 10 min. C) HUVECs were incubated with control buffer (CB), ephrinB2-Fc (500 ng/mL, E), Ang1 (100 ng/mL, A), or E plus A (EA) for the times indicated. After treatment, cell lysates were harvested. Each lane contains 50 µg of total protein from the cell lysates. Western blots were probed with anti-phospho-ERK antibody (upper panels). The blot was reprobed with anti-ERK antibody or anti-actin antibody (lower panels) to verify equal loading of protein in each lane. Results were similar in 3 independent experiments. Fold: Densitometric analyses are presented as the relative ratio of phospho-ERK1 to ERK1 or actin. The relative ratio measured at time 0 or the ratio relative to control buffer is arbitrarily presented as 1. Numbers represent the mean ± SD from 3 experiments. *P < 0.05 vs. time 0 or CB. #P < 0.05 vs. A.

3. EphrinB2-Fc suppresses VEGF- and Ang1-induced Ras activity
Because the ephrinB2-induced suppression of ERK1/2 did not involve receptor phosphorylation, we next examined Ras, an intermediary signaling mediator between receptors and ERK1/2. VEGF (10 ng/mL) increased Ras activity as early as 2.5 min, produced a maximal effect at 5 min, and then declined. The maximum mean increase in Ras activity was 10.4-fold. Addition of ephrinB2-Fc (500 ng/mL) inhibited 35–45% of the VEGF-induced Ras activity but produced no effect on Ras activity. Ang1 (100 ng/mL) increased Ras activity as early as 5 min and produced a maximal effect at 7.5 min, then declined. The maximum mean increase in Ras activity was 8.4-fold. Addition of ephrinB2-Fc (500 ng/mL) inhibited 50–60% of the Ang1-induced Ras activity but produced no changes in basal Ras activity. These results indicate that activation of EphB kinases suppressed VEGF- and Ang1-induced Ras/MAPK signaling through direct inhibition of Ras in endothelial cells.

4. EphrinB2-Fc induces phosphorylation of EphB2 and EphB4 and recruitment of p120-RasGAP to EphB2 and EphB4
Because a recent report indicated that ephrinB1-induced EphB2 phosphorylation suppressed Ras/MAPK activity through recruitment of p120-RasGAP to EphB2 in neuronal cells, we examined the effect of ephrinB2-Fc on phosphorylation of EphB2 and EphB4 and on recruitment of p120-RasGAP to EphB2 and EphB4. Addition of ephrineB2-Fc (1.0 µg/mL) increased phosphorylation of EphB2 in a time-dependent manner and increased recruitment of p120-RasGAP to EphB2 in HUVECs. Because a proper anti-EphB4 antibody for immunoprecipitation was not available, we examined the effect of ephrinB2-Fc on HEK 293 cells transfected with pFLAG-EphB4 gene. Addition of ephrinB2-Fc (1.0 µg/mL) increased phosphorylation of EphB4 in a time-dependent manner and increased recruitment of p120-RasGAP to EphB4 in HEK 293 cells. These results indicate that ephrinB2 suppressed Ras/MAPK signaling through recruitment of p120-RasGAP to EphB2 and EphB4 after their phosphorylation.

5. EphrinB2-Fc suppresses VEGF-induced proliferation and migration and Ang1-induced migration
MAPK functions in a protein kinase cascade that plays a critical role in regulating cell growth and differentiation. Because VEGF-induced ERK1/2 activation is involved in proliferation and migration in endothelial cells, we examined the effect of ephrinB2 on VEGF-induced proliferation and migration. Indeed, pharmacological inhibition of ERK1/2 activation with PD98059 suppresses basal and VEGF-induced proliferation and migration. Addition of ephrinB2-Fc (500 ng/mL) inhibited 50–55% of the VEGF-induced proliferative and migratory activities, but alone had no effect on proliferation or migration. Ang1 produces a migratory effect on endothelial cells but does not affect proliferative activity. Our pharmacological inhibition of ERK1/2 activation with PD98059 suppressed Ang1-induced migration, suggesting that intracellular activation of MAPK is required for Ang1-induced migration. Indeed, addition of ephrinB2-Fc (500 ng/mL) inhibited 40–70% of the Ang1-induced migratory activity. Activation of endogenous ephrinB2 with EphB4-Fc did not alter basal, VEGF-induced, or Ang1-induced proliferation/migration. These results indicate that activation of EphB kinases, but not activation of ephrinB2, suppressed VEGF- and Ang1-induced proliferation and/or migration through direct inhibition of the Ras/MAPK signaling cascade in endothelial cells.

CONCLUSIONS AND SIGNIFICANCES

To our knowledge, these results are the first to demonstrate that activation of endogenous EphB kinases in endothelial cells suppresses VEGF- and Ang1-induced Ras/MAPK signaling cascades through direct inhibition of Ras in endothelial cells, whereas activation of ephrinB2 did not alter the Ras/MAPK signaling cascades. These suppressive activities are mediated by recruitment of a negative regulator of Ras/MAPK signaling, p120-RasGAP, to EphB2 and EphB4 on their phosphorylation by ephrinB2. Activation of endogenous EphB kinases suppresses VEGF-induced proliferation, migration, and Ang1-induced migration. Thus, our results identify EphB kinases as putative negative regulators of the Ras/MAPK pathway, which exerts anti-mitogenic and anti-migratory effects on endothelial cells.

EphrinB2 and EphB4 display a remarkably reciprocal pattern of distribution within the developing vasculature; ephrinB2 is present in the endothelium of primordial arterial vessels whereas EphB4 is present in the endothelium of primordial venous vessels. These reciprocal expression patterns not only provide the earliest known markers distinguishing arterial and venous endothelium, but also suggest that these cells transmit some sort of bidirectional signal. The reciprocal signaling between presumptive arteries and veins through ephrinB2/EphB is required for proper morphogenesis of the intervening capillary beds as well as for interdigitation and differential growth of the larger vessels themselves. Our results suggest that the suppression of VEGF- and Ang1-induced Ras/MAPK activation by ephrinB2 may contribute to the proper morphogenesis of the intervening capillary beds and to proper interdigitation through the arrest of venous (at least) endothelial cell proliferation and migration (Fig. 3 ).

View larger version (41K): [in this window] [in a new window]   Figure 3. Model of ephrinB2-EphB signaling in an interconnected area of arterial and venous capillary endothelium during development. In venous and arterial capillaries, endothelial cells proliferate and migrate, stimulated by VEGF and Ang1. When the two sides meet, ephrinB2 in arterial endothelial cells binds to EphB in venous endothelial cells and bidirectional ephrinB2-EphB signaling occurs. Our data suggest that proliferation and migration of venous (at least) endothelial cells is arrested through suppression of MAPK activity by activation of EphB.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0805fje; to cite this article, use FASEB J. (May 21, 2002) 10.1096/fj.01-0805fje.

² These two authors contributed equally to this work.

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