eph b4 receptor signaling mediates endothelial cell migration and

Eph B4 receptor signaling mediates endothelial cell migration and proliferation

via the PI3K pathway

Jena J. Steinle1, Cynthia J. Meininger1, Reza Forough1, Guoyao Wu1,2, Mack H. Wu1 and Harris J. Granger1

1Cardiovascular Research Institute and Department of Medical Physiology, College of Medicine, The Texas A&M University System Health Science Center,

Temple, TX 76504and

2Department of Animal Science, Texas A&M University, College Station, TX 77843

Running Title: EphB4 activates endothelial cell proliferation and migration

Corresponding Author: Jena J. Steinle, PhD The Texas A&M University System HSC 702 SW HK Dodgen Loop Medical Research Building, Room 202A Temple, TX 76504 Phone: 254-742-7144 Fax: 254-742-7145

Email: jsteinle@tamu.edu

This work was supported by NIH grant NHLBI 446221 (H.J.G.) and JDRF grants 2000-437 and 2002-228 (C.J.M. and G.W.).

JBC Papers in Press. Published on September 13, 2002 as Manuscript M207221200 by guest on A

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Summary

The goals of this study were twofold: 1) to determine whether stimulation of Eph

B4 receptors promotes microvascular endothelial cell migration and/or

proliferation and 2) to elucidate signaling pathways involved in these responses.

The human endothelial cells used possessed abundant Eph B4 receptors with no

endogenous ephrin B2 expression. Stimulation of these receptors with ephrin

B2/Fc chimera resulted in dose- and time-dependent phosphorylation of Akt.

These responses were inhibited by LY294002 and ML-9, blockers of

phosphatidylinositol-3-kinase (PI3K) and Akt, respectively. Eph B4 receptor

activation increased proliferation by 38%, which was prevented by prior blockade

with LY294002, ML-9, and inhibitors of protein kinase G (KT5823) and MEK

(PD98059). Nitrite levels increased over 170% after Eph B4 stimulation,

indicating increased nitric oxide production. Signaling of endothelial cell

proliferation appears to be mediated by a PI3K/Akt/eNOS/PKG/MAP kinase

cascade. Stimulation with ephrin B2 also increased migration by 63% versus

controls. This effect was inhibited by blockade with PP2 (Src inhibitor),

LY294002 or ML-9, but was unaffected by the PKG and MEK blockers. Eph B4

receptor stimulation increased activation of both matrix metalloproteinase-2 and

9. The results from these studies indicate that Eph B4 stimulates migration and

proliferation and may play a role in angiogenesis.

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Introduction

Eph receptors are a family of fourteen receptor tyrosine kinases, first noted in

the nervous system1. They all possess an N-terminal globular domain, which

folds into a compact β-sandwich and is necessary for ligand binding1. The

ligands for the Eph receptors are arranged into two families based upon their

attachment to the plasma membrane. Class A ephrins are attached to the outer

leaflet of the plasma membrane via a glycosylphosphoinositide (GPI) anchor.

Class B ephrins are transmembrane proteins. While ephrin ligands are insoluble

in their natural state, soluble chimeric ephrin B2 ligands are commercially

available to use as a tool to probe downstream signaling pathways.

A role for ephrins in vascular growth and remodeling was first noted when

gene knockout studies of Eph B4 or its ligand ephrin B2 resulted in embryonic

lethality due to cardiovascular defects 2,3. These malformations involved

disrupted angiogenesis of the yolk sac manifested as an apparent block in

capillary plexus remodeling. There was limited ingrowth of capillaries into the

neural tube, as well as defective interactions between endothelial and supporting

cells in mutant mice3. Additionally, work in Xenopus laevis using dominant

negative Eph B4 receptors or missense expression of ephrin B1 or ephrin B2

demonstrated that interactions between Eph B4 and ephrin ligands are critical

regulators of embryonic angiogenesis 4. After the initial studies on knockout mice

and Xenopus, much work has been done to determine cellular interactions of

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ephrins with one another 2,5 and with other known signaling components 6,7.

Activation of some Eph receptors promotes tube formation in renal endothelial

cells but not in umbilical vein endothelial cells, suggesting that Eph receptor

activation is specific for different types of endothelial cells 5. While much work

has been done to determine phosphorylation sites for the Eph receptors 8,

downstream signaling events resulting from receptor activation have received

less attention.

In this study, we first determined whether Eph receptors were present on

human mesentery vascular endothelial cells. Once we concluded that these cells

possess abundant Eph B4 receptors, we hypothesized that stimulation of Eph B4

receptors with ephrin B2 would result in activation of either the migration or

proliferation phase of angiogenesis. Finally, we sought to examine the signaling

pathways regulating these angiogenic events.

Experimental Procedures

Cell Culture.

Human microvascular endothelial cells were isolated by enzymatic

digestion of blood vessels taken from the mesentery of the small bowel and

cloned by limiting dilution (designated MM1 cells). Endothelial cell identity was

verified by positive staining for factor VIII-related antigen. Cells are grown in

gelatin-coated dishes and maintained in Dulbecco’s modified Eagle’s Medium

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(DMEM) with high glucose (Gibco, Rockville, MD) supplemented with 20% fetal

bovine serum, 3.7g/L sodium bicarbonate, 1mM sodium pyruvate, 30mM HEPES

buffer, 25IU/mL heparin, 100IU/mL penicillin, 100µg/mL streptomycin, and

0.25µg/mL amphotericin B. After the cells reach 80-90% confluence, they are

passaged with the use of 0.25% trypsin and 0.02% EDTA. Starvation medium

contains all of the above ingredients except 0.1% bovine serum albumin instead

of fetal bovine serum.

Pharmacological Inhibitors

The following pharmacological inhibitors were used to complete these

studies: PP2, 1µM, Src inhibitor, Calbiochem; KT5823, 1µM protein kinase G

inhibitor, Calbiochem; LY294002, 3µM, PI3K inhibitor, Calbiochem; ML-9,

100µM Akt inhibitor, Biomol; PD98059, 10µM, MEK inhibitor, Calbiochem; MMP-

2/MMP-9 inhibitor, 1µM, Calbiochem; and FTI III, 1µM, Ras inhibitor,

Calbiochem. Calbiochem is located in San Diego, CA. Biomol is located in

Plymouth Meeting, PA.

Determination of Eph receptor expression in microvascular endothelial cells.

Western blotting was conducted to determine if MM1 cells (passage 12-

18) expressed either Eph B4 receptor or ephrin B2 ligand or both. Cells in 60-

mm dishes were lysed and 50µg of protein was separated on a 4-12% pre-cast

polyacrylamide gel (Invitrogen, Carlsbad, CA), blotted onto a nitrocellulose

membrane, and membranes were blocked with Super Block (Pierce, Rockford,

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IL) overnight at 4°C. The following day, membranes were probed with primary

antibodies (5µg/ml, Santa Cruz, Santa Cruz, CA) to either Eph B4 receptor or

ephrin B2 ligand for 2 hours at room temperature. Horseradish peroxidase-

conjugated anti-rabbit or anti-goat secondary antibodies, respectively, were

applied at a 1:10,000 dilution for 2 hours at room temperature. Immunoreative

bands were detected by enhanced chemiluminescence (LumniGlo, Cell

Signaling, Beverly, MA) using Kodak BioMax ML film and scanned into the

computer using reflectance scanning. Intensity of the bands was quantified using

NIH Image.

Immunoprecipitation and Western Blotting for phosphorylation of Akt.

To determine the optimal concentration and time course for Eph B4

receptor activation, immunoprecipitation and western blotting experiments were

performed. To conduct these experiments, 60-mm dishes (passage 12-16)

were used after exposure to 0, 1nM, 25nM, 50nM, or 100nM ephrin B2/Fc ligand

(R&D Systems, Minneapolis, MN) for 15 minutes. Cells were washed once with

phosphate buffered saline (PBS) and 150µL of cell lysis buffer (50mM Tris-HCl,

pH 7.4; 1% NP-40; 0.25% Na-deoxycholate; 150mM NaCl; 1mM EDTA; 1mM

PMSF; 1µg/ml each of aprotinin, leupeptin, pepstatin; 1mM Na3VO4; 1mM NaF;

0.1% SDS) was applied. Cells were scraped from dishes and placed into

Eppendorf tubes for sonication and centrifugation. Protein concentrations were

determined using the standard BCA protocol (BioRad, Hercules, CA). Protein

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(150µg), 20µL Protein A/G Plus (Santa Cruz) and an appropriate volume of a

solution containing 20mM Tris, pH 8.0, 1mM Na3VO4, 1µg/ml each of aprotinin,

leupeptin, pepstatin, were mixed to a final volume of 400µL. The tubes were

allowed to incubate with rocking for 1 hour at 25°C. The tubes were then spun

down for 5 minutes at 14,000g, the supernatant collected and 5µg of PKB/Akt

antibody was added and allowed to incubate for 2 hours at room temperature

with rocking. Another 20µL of Protein A/G Plus was then added and incubated

overnight at 4°C with rocking. The following day, beads were spun down and

washed three times with Tris buffered saline (TBS, 10mM Tris, pH 8.0, 140mM

NaCl). Beads were boiled in sample buffer and released protein was loaded and

separated on 4-12% pre-cast polyacrylamide gels (Invitrogen), blotted onto

nitrocellulose, and membranes were blocked with SuperBlock with 0.05%

Tween-20 (Pierce) overnight at 4°C with rocking.

Immunodetection of phosphorylated proteins was achieved using

phospho-PKB/Akt (Serine 473, 5µg/ml, Cell Signaling) antibody added to the

membrane and incubated for 2 hours at 25°C with rocking. After washing for 20

minutes with a wash solution (KPL Laboratories, Gaithersburg, MD)

supplemented with 0.05% Tween-20, a rabbit anti-goat horseradish-peroxide

conjugated secondary antibody (Santa Cruz, dilution of 1:10,000) was applied for

1 hour at 25°C with rocking, followed by washing for another 20 minutes.

Immunoreactive bands were observed by enhanced chemiluminescence

(LumniGlo, Cell Signaling). Images of the bands were scanned into the computer

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and intensity of the bands was quantified using NIH Image.

Once the optimal dose was determined to be 50nM ephrin B2/Fc chimera,

this dose was used to determine the time course of receptor activation.

Experiments were conducted at 0min, 5min, 15min, 30min, or 60min using the

same procedures as above.

Phosphorylation of MAP kinase and Src

Western blots to evaluate phosphorylation states of MAP Kinase (phospho

p42/44,1:1000, ERK1/2, Cell Signaling) or Src (phospho Src-416,1:1000, Cell

Signaling) were done as above with stimulation of 50nM ephrin B2 for 0, 3, 5, 15,

or 30 minutes. Some dishes also received KT5823 (protein kinase G inhibitor,

Calbiochem, San Diego, CA) for 30 minutes prior to stimulation with 50nM ephrin

B2. These blots were probed for phospho-p42/44. Other blots received PP2

(Src inhibitor, Calbiochem) for 15 minutes prior to 50nM ephrin B2 and probed for

phospho-Akt to determine if Src lies upstream of PI3K and Akt.

Measurement of changes in intracellular calcium in endothelial cells

Changes in intracellular calcium were measured using a fluorescence ratio

technique with the aid of a microscope photometry system (Photon Technology

International), which consists of a PowerFilter high-speed dual wavelength

illuminator, a high-grade quartz fiber optic bundle, a D-104B single channel

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microscope photometer, and a 710 photon-counting photomultiplier tube (PMT).

The cell permeable form of Fura 2 (Fura 2-AM, Molecular Probes) was used as

the calcium indicator.

MM1 cells were grown on 30mm uncoated culture dishes with a cover slip

on the bottom (MatTek Corporation, Ashland, MA). These dishes were exposed

to ultraviolet light for 12 hours before cells were seeded onto the dishes. Cells

were fed normal medium until reaching confluence. Once confluent, cells were

placed in starvation medium for 12-18 hours before experimentation. For the

experiments, 3µL of Fura 2-AM was loaded for 20-30 minutes. Cells were

observed on the optical axis of a Zeiss Axiovert microscope that was connected

to the PMT equipped with the photometry system. Emission fluorescence at

510nm during excitation at 340nm and 380nm was detected by the PMT photon-

counting system using FeliX software. In each experiment, the excitation

wavelength alternated between 340nm and 380nm at a rate of 650/s and the

fluorescence intensities were recorded for 10 minutes at 5-sec intervals.

Correction for background fluorescence was automatically performed by the

software program.

After loading the fura-2 and getting baseline values, 50nM ephrin B2 was

added and fluorescence intensities were recorded for 10 minutes. 1µM

bradykinin was used as a positive control to promote increased intracellular

calcium concentrations.

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Measurement of Nitrite Accumulation.

Nitrite, a stable end product of nitric oxide metabolism, was assessed in

the medium by reaction with 2,3-diaminonaphthalene (DAN) under acidic

conditions to yield 2,3 naphthotriazole (NAT), a highly fluorescent product 9.

Reversed-phase HPLC separates NAT from DAN [and other fluorescent

compounds present in biological samples] before fluorescence detection of NAT.

Two days prior to the experiments, the medium was replaced with DMEM

containing 0.4mM glutamine. Ephrin B2/Fc (50nM) was added to the MM1 cells

to stimulate Eph B4 receptors and downstream pathways. Medium was collected

after 48 hours and filtered through 10-KDa cutoff ultrafilters to remove large

molecular weight proteins. Filters were washed 4 times with deionized and

double-distilled water (DD-H2O) prior to use. Nitrate was converted to nitrite

using nitrate reductase as follows: 200 µl of diluted sample or nitrate standard

(0-2 µM), 10 µl of 1 U/ml nitrate reductase (Roche) and 10 µl of 120 µM NADPH

were mixed and incubated at room temperature for 1 hr. This solution was then

used directly for nitrite analysis. The conversion of nitrate to nitrite is 98%

complete 9 as determined with known amounts of both standards.

One hundred µl of sample [diluted with double-distilled water (DD-H20],

diluted blank medium OR sodium nitrite standard (0-2 µM) was mixed with 100 µl

of DD-H2O and 20 µl of 316 µM DAN (in 0.62 M HCl). These reaction mixtures

were incubated at room temperature for 10 min, followed by addition of 10 µl of

2.8 M NaOH. After mixing, 15 µl of the derivatized nitrite-DAN solution was

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injected into a 5-µm C8 column guarded by a 40-µm C18 column for

chromatographic separation of NAT. The mobile phase (1.3 ml/min) was 15 mM

sodium phosphate buffer (pH 7.5) containing 50% methanol (1 liter of 30 mM

Na2HPO4 and 125 ml of 30 mM NaH2PO4 mixed with 1.125 liter of 100%

methanol) (0.0-3.0 min), followed sequentially by 100% HPLC-grade water (3.1-

5.0 min), 100% methanol (5.1-8.0 min), 100% HPLC-grade water (8.1-10.0

min), and the initial 15 mM sodium phosphate buffer (pH 7.5)-50% methanol

solution (10.1-15.0 min). The use of 100% HPLC-grade water before and after

100% methanol is necessary to prevent abrupt marked increases in column

pressure, and is sufficient to regenerate the columns for automatic analysis of

multiple samples. All chromatographic procedures were carried out at room

temperature. Fluorescence was monitored with excitation at 375 nm and

emission at 415 nm. The retention time for NAT is 4.4 min.

Cell Proliferation Assay.

Endothelial cell proliferation was assessed using an assay based on the

cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial

dehydrogenases. Expansion in the number of viable cells results in an increase

in the overall activity of the mitochondrial dehydrogenases in the sample. The

augmentation in enzyme activity leads to an increase in the formazan dye

formed. The formazan dye produced by viable cells can be quantified by a

multiwell spectrophotometer by measuring the absorbance of the dye solution at

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440nm.

To perform the experiments, an aliquot of 50,000 MM1 cells was added to

each well of a 96-well tray in medium with 20% fetal bovine serum. After the

cells attached to the new plate, the cells were washed, and the high serum

medium was replaced with starvation medium overnight. The following morning,

all wells were rinsed with phosphate buffered saline. Negative control wells

received low serum only and positive control wells received 50nM ephrin B2/Fc

only. PP2, FTI III, LY294002, ML-9, KT5823, or PD98059 was added for 30

minutes before ephrin B2 to allow for complete blockade. Controls treated with

inhibitor alone were also included to determine their effect on proliferation. Cells

were allowed to incubate for 48 hours. At this time, the WST-1 reagent

dissolved in Electro Coupling Solution (Chemicon) was applied for 4 hours to

measure cell proliferation. The plates were read on a spectrophotometer and

data is presented as a percentage of negative control proliferation with P<0.05

being significant.

Migration Assay.

All migration assays were done using BD BioCoat Angiogenesis System-

Endothelial Cell Invasion plates according to supplied protocols with little

variation. Briefly, 100,000 MM1 cells were added to top chambers in 250µL of

medium. Starvation medium, ephrin B2 chimera (50nM), or ephrin B2 chimera

(50nM) with an inhibitor was added to each of the lower chambers. LY294002,

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ML-9, KT5823, PD98059, MMP-2/ MMP-9 inhibitor III, or PP2 were added for

30 minutes before ephrin B2/Fc to allow for complete pathway blockade. In some

assays, inhibitors were given without ephrin B2 to determine the role of inhibition

alone. No cells were added to the bottom chamber, so that background

measurements could be obtained. Plates were allowed to incubate for 26-29

hours at 37°C to allow for migration through the Matrigel-coated membrane.

Chambers were transferred to wells containing Calcein AM (Molecular Probes,

Eugene, OR) in Hank’s Balanced Salt Solution for 1.5 hours and then read on a

fluorescent plate reader (Bio-Tek, Winooski, VT, Model FL600, gain of 100) at

485/530nm. Migration for ephrin B2- and inhibitor-treated cells was expressed

as a percentage of controls (receiving only starvation medium), after background

fluorescence was subtracted. Data was analyzed using Prism software

(GraphPad, San Diego, CA) and is presented with significance at P<0.05.

MMP-2 and MMP-9 Activation Assay.

MM1 cells in 60-mm dishes (passage 12-16) were starved for 40 hours

prior to experiments. Treated dishes received 50nM ephrin B2/Fc chimera for 6

hours. At the end of 6 hours, the medium was collected and the cells counted

using a hemocytometer. Pre-cast zymogram gels (Invitrogen) were loaded with

15µL of the conditioned medium from the dish with the fewest cells and 4µL

sample buffer (same as used for polyacrylamide gels). All other samples were

compared to the medium with the fewest cells and volumes of medium

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corresponding to the same number of cells were loaded onto the gels with 4µL of

sample buffer. Gels were run at 120V for 1 hour. After electrophoresis, the gels

were washed with 2.5% Triton X-100 in water for 20 minutes. Gels were then

placed into a rotator at 45rpm overnight at 35°C in development buffer

(Invitrogen) containing calcium chloride. The gels were then placed in a staining

solution (10%ethanol/5%acetic acid, 1% Commassie Blue, water) for

approximately 20 minutes or until bands were observed. Gels were then

scanned using a reflectance scanner and bands measured using NIH image

software. Activated MMP-2 was detected at a molecular weight around 65kD,

and activated MMP-9 at a molecular weight of 85kD.

Results

Eph receptor expression in microvascular endothelial cell culture.

Western blot analysis of Eph B4 receptor and ephrin B2 ligand expression

revealed that these cloned human microvascular endothelial cells possess an

abundant amount of the Eph B4 receptor (Figure 1A), but no endogenous ephrin

B2 (Figure 1B), making this a suitable model to investigate Eph B4 signal

transduction in angiogenesis.

Cell Proliferation.

Stimulation of Eph B4 receptors with ephrin B2/Fc produced a 38%

increase in cell proliferation (P<0.01 vs. control, Figure 2). This increase in

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endothelial cell proliferation was not inhibited by administration of PP2 or

farnesyltransferase III (FTI III). The increased proliferation was, however,

significantly decreased using LY294002 (P<0.01 vs. B2, n.s. vs. control, Figure

2) and ML-9 (P<0.001 vs. B2, n.s. vs. control, Figure 2). Cell proliferation was

also blocked by giving KT5823 (P<0.01 vs. B2, n.s. vs. control, Figure 2) and

PD98059 (P<0.001 vs. B2, n.s. vs. control, Figure 2), indicating that nitric oxide

and ERK1/2 may be involved in ephrin B2-stimulated endothelial cell

proliferation. Administration of ML-9 alone did significantly attenuate endothelial

cell proliferation. It appears that ML-9 causes blockade of both myosin light

chain kinase and Akt, which is resulting in detachment of cells from the dish over

Dose-response and time course curves for phosphorylation of Akt.

Immunoprecipitation analysis indicated that stimulation of Eph B4

receptor with 50nM ephrin B2/Fc chimera provided the optimal activation of the

receptor and phosphorylation of Akt (P<0.05 vs. 0 nM and other doses except

5nM where P<0.01, Figures 3A, 3B). Using the 50nM dose, immunoprecipitation

experiments were conducted to determine the best time for activation of the

receptor and phosphorylation of Akt. Ephrin B2/Fc chimera (50nM) stimulation of

Eph B4 receptor for 15 minutes produced substantial phosphorylation of Akt

(P<0.05 vs. 0 min, Figures 3C, 3D).

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Phosphorylation of ERK1/2 and Src416 after ephrin B2 stimulation.

Stimulation of mesenteric microvascular endothelial cells with ephrin

B2/Fc resulted in a time-dependent phosphorylation of ERK1/2. This response

peaked at 5 minutes and tailed off by 30min (P<0.01 vs. 0 minutes, Figure 4).

This phosphorylation was blocked by administration of KT5823 prior to ephrin B2

stimulation (data not shown). The phosphorylation of Src was not as rapid as

that of ERK 1/2 and peaked at 15 minutes (P<0.01 vs. 0 minutes, Figure 5). Src

phosphorylation is likely upstream of PI3K and Akt, as blocking Src with PP2

prevented the phosphorylation of Akt (data not shown). These results indicate

that stimulation of Eph B4 receptors does produce phosphorylation of both the

mitogen activated protein kinase (MAPK) and Src pathways.

Eph B4 receptor activation does not alter intracellular calcium levels.

Administration of ephrin B2/Fc to human mesenteric endothelial cells did

not alter intracellular calcium levels (data not shown). These cells responded to

1µM bradykinin (positive control) with a large increase in intracellular calcium.

Ephrin B2 stimulation increases nitrite production.

Stimulation of MM1 cells with ephrin B2/Fc for 48 hours increased nitrite

production from 2.4 ± 0.1nmol/ml to 4.1 ± 0.5nmol/ml (P<0.01 vs. not stimulated,

Figure 6).

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Eph B4 receptor activation promotes endothelial cell migration.

Stimulation of Eph B4 receptors with 50nM ephrin B2/Fc chimera resulted

in a 63% increase in migration over low serum controls (P<0.01, Figure 7). This

response could be blocked by prior administration of LY294002 (P<0.01 vs. B2,

n.s. vs. control, Figure 7), ML-9 (P<0.001 vs. B2, P<0.01 vs. control, Figure 7) or

PP2 (P<0.05 vs. B2, n.s. vs. control, Figure 7) in the presence of ephrin B2. This

response was not blocked by KT5823 or PD98059 (Figure 7). LY294002, ML-9,

KT5823, PD98059, or PP2 did not affect migration when administered in the

absence of ephrin B2 (data not shown).

MMP-2 and MMP-9 are activated following stimulation of ephrin B2.

Using zymography, both MMP-2 and MMP-9 were activated in both non-

treated and treated cells. Ephrin B2/Fc stimulation increased the amount of

activation of both MMP-2 (P<0.01, Figure 8A) and MMP-9 (P<0.05, Figure 8B).

Discussion

Eph B4 receptor is expressed in human microvascular endothelial cells.

A possible role for Eph receptor tyrosine kinases in the cardiovascular

system was suggested when it was noted that knockouts of either ephrin B2 or

Eph B4 produced significant cardiovascular defects, including disruption of

angiogenesis of the yolk sac 10. These authors noted that Eph receptors have

the potential for modulating capillary sprouting and downstream signaling in

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vasculogenesis. Other authors noted that ephrin B2-Eph B4 interactions are

required in vascular endothelial cells and their interaction may regulate

angiogenesis 11. Subsequent to suggestions for a role of Eph receptors in

vasculogenesis, a number of investigations have focused on signaling partners,

such as PI3K, Grb2, Grb10, Nck, RasGAP, 1 and Src 12, for the various Eph

receptor types. Once angiogenic potential was established, the role of Eph

receptors needed to be characterized in adult tissues. Investigators determined

locations of Eph B4 receptors in cells and possible roles for Eph B4 in specific

cellular functions 8. Members of the Eph receptor family were noted to promote

tube formation in renal vascular endothelium but not in endothelial cells from the

umbilical vein 5. Therefore, it was not entirely unexpected to discover that

human mesenteric microvascular endothelial cells possessed abundant Eph B4

receptors. The other advantage, however, was that there was no endogenous

ephrin B2 present in the MM1 cells. Using the knowledge of Eph B4 expression

in human mesenteric microvascular endothelial cells, we sought to address

whether Eph B4 receptor activation promotes the angiogenic events of migration

and proliferation and the signaling pathways utilized to activate these processes.

PI3K and Akt play central roles in Eph B4 receptor signaling

Receptor tyrosine kinases generally activate one or more of four main

signaling pathways: Ras, PI3K, Src, and phospholipase Cγ. Therefore, we

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determined which of these pathways could be activated following stimulation with

ephrin B2. Prevention of Ras translocation, and subsequent activation, using

FTIII had no effect on ephrin B2/Fc-induced endothelial cell proliferation.

Moreover, we found no evidence of phospholipase C activation using cytosolic

calcium as an indicator. Although Src appears to play a role in phosporylation of

PI3K and migration, the Src inhibitor PP2 had no effect on endothelial cell

proliferation elicited by Eph B4 activation. By contrast, PI3K and Akt were both

required to initiate proliferation and migration, suggesting that signaling of these 2

separate events diverged below Akt.

Eph B4 receptor activation mediates endothelial cell proliferation

Endothelial cell proliferation induced by ephrin B2/Fc was inhibited by

blockers of PI3K, Akt, PKG, and MEK. Surprisingly inhibition of Src had no effect

on the proliferation response. PI3K is known to initiate a series of events that

leads to Akt phosphorylation and translocation to the internal surface of the cell

membrane, resulting in activation of Akt . Calcium-dependent stimulation of

eNOS plays no role in Eph B4 signaling since we were unable to demonstrate a

change in the fura-2 signal in mesenteric microvascular endothelial cells

exposed to ephrin B2/Fc. Similar results were obtained from retinal endothelial

cells whereby stimulation with Eph B4 produces proliferation via phosphorylation

of PI3K and Akt without changes in calcium signaling (Steinle et al, submitted).

Others had shown that Akt can phosphorylate endothelial nitric oxide synthase

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(eNOS) on serine 1177 13, causing an increase in the activity of the enzyme. In

the present study, nitrite production increased almost two-fold following ephrin

B2 stimulation of Eph B4 receptors (Figure 8). The resultant increase in

downstream production of cyclic GMP via soluble guanylate cyclase accounts for

the recruitment of PKG in the proliferation process. Activation of the MAP kinase

cascade is probably not through Ras since the farnesyltransferase inhibitor FTI III

did not reduce ephrin B2/Fc-induced endothelial cell proliferation. Nitric oxide

donors and cyclic GMP analogs are known to initiate the phosphorylation of c-raf

by PKG, leading to activation of the MAP kinase cascade in a ras-independent

manner 14.

Eph B4 receptor activation causes endothelial cell migration

We found that significantly more (63%) endothelial cells invaded the

Matrigel-coated membrane when 50nM ephrin B2 was in the lower chamber

than those in which only low serum was in the lower chamber. This indicates that

ephrin B2 promotes endothelial cell migration in these microvascular endothelial

cells. As with cell proliferation, both PI3K and Akt were required to support the

migration response. By contrast, blockade of PKG and MEK had no effect on the

ephrin B2/Fc-induced stimulation of transmembrane movement of endothelial

cells. Thus, signaling of migration downstream of Akt diverges from the

aforementioned PKG/MAP kinase connection apparently responsible for Eph B4

signaling of proliferation.

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The mechanism of the Akt-mediated cellular migration remains unknown.

Cell motility involves changes in cytoskeletal dynamics, surface adhesion

properties and activity of extracellular proteases15. MMP-2 and MMP-9

degrade collagen within the basement membrane. Therefore, activation of these

matrix metalloproteinases likely allows enhanced movement of the endothelial

cells through the pores of the migration membrane. Akt has been linked to

activation of MMP-2 16 and MMP-9 15. This is believed to occur as activated

Akt increases transcriptional activation of NFκB 15. MMP-9 has a NFκB binding

site in its promoter. Other transcription factors have also been shown to activate

MMP-2 and MMP-9. From our studies, it is clear that MMP-2 and MMP-9 are

activated and are functioning in MM1 cell migration induced by ephrin B2/Fc.

Work in human retinal endothelial cells indicates that MMP-2 and MMP-9 are

also important for migration of these vascular endothelial cells (Steinle et al,

submitted). Furthermore, inhibition of these specific matrix metalloproteinases

resulted in substantial declines in endothelial cell migration.

In conclusion, activation of the PI3K pathway produces both endothelial

cell migration and proliferation. The signaling pathways for each process are

different, however, the phosphorylation of PI3K and Akt are critical for both

functions. Mitogenesis appears to signal through the PI3K/Akt/nitric oxide/ PKG/

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MAP kinase pathway, whereas migration involves the PI3K/ Akt/ MMP pathway

that does not require PKG or the MAP kinase cascade. The results of these

studies indicate that Eph B4 receptors may play a role in adult angiogenesis

through modulation of endothelial cell proliferation and migration.

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ligands: embryogenesis to

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F. and Anderson, D. J. (1999)

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Symmetrical mutant phenotypes of

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Figure Legends.

Figure 1. Eph B4 expression in mesenteric endothelial cells. A. MM1 cell lysates

were separated and blotted onto nitrocellulose. The blot was probed with anti-

Eph B4 polyclonal antibodies. B. MM1 cell lysates were separated, blotted and

probed with anti-ephrin B2 polyclonal antibodies. N=4.

Figure 2. Effects of Eph B4 receptor activation on MM1 cell proliferation.

Endothelial cells were treated with starvation medium (control), 50nM ephrin B2/Fc

chimera (B2) or ephrin B2/Fc plus various inhibitors to determine their effects on

proliferation. N=7; *P<0.05 vs. control; #P<0.05 vs. B2.

Figure 3. Dose response and time course of phosphorylation of Akt following

Eph B4 receptor stimulation. A., Western blot showing dose-response for Akt

phosphorylation. Blots were probed with anti-Akt (Ser 473) antibodies. B.,

Densitometric measurements of three blots of dose-response data on the

phosphorylation of Akt. *P<0.05 vs. 0nM control. C., Western blot showing time

course of phosphorylation of Akt following Eph B4 receptor activation. Blots were

probed with anti-Akt (Ser 473) antibodies. D., Densitometric measurements

from the Western blots of 3 time course studies for the phosphorylation of Akt

after 50nM ephrin B2/Fc had been given. *P<0.05 vs. controls.

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Figure 4. Phosphorylation of ERK1/2 following Eph B4 receptor activation. A.,

Western blot indicating the time course for ERK1/2 phosphorylation after ephrin

B2/Fc stimulation of Eph B4 receptors. Blots were probed with anti-phospho-

tyrosine p42/44. B., Densitometric measurements of three experiments

investigating the phosphorylation of ERK1/2. *P<0.05 vs. 0 minutes; **P<0.01

vs. 0 minutes.

Figure 5. Phosphorylation of Src 416 following Eph B4 receptor stimulation. A.,

Western blot of time course of Src 416 phosphorylation following Eph B4 receptor

stimulation with ephrin B2/Fc in MM1 cells. Blots were probed with anti-Src 416

antibodies. B., Densitometric measurements of the time course of three

experiments of Src phosphorylation. **P<0.01 vs. 0 minutes.

Figure 6. Role of Eph B4 in nitrite accumulation. Endothelial cells were treated

with 50nM ephrin B2/Fc for 24 hours and the media collected for nitrite

measurement using HPLC. N=4; **P<0.01.

Figure 7. Effects of Eph B4 receptor activation on MM1 cell migration. Data on

the effects of starvation medium (control), ephrin B2/Fc (B2) or ephrin B2/Fc plus

various antagonists on MM1 cell migration through a Matrigel-coated membrane.

N=7, *P<0.05 vs. control, #P<0.05 vs. B2.

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Figure 8. Role of Eph B4 phosphorylation of MMP-2 and MMP-9 activation. A.,

Densitometric measurements of MMP-2 activation following Eph B4 receptor

activation by 50nM ephrin B2/Fc. N=4, **P<0.01. B., Densitometric

measurements of MMP-9 activation following ephrin B2 stimulation of Eph B4

receptors in MM1 cells. N=4, *P<0.05.

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J. GrangerJena J. Steinle, Cynthia J. Meininger, Reza Forough, Guoyao Wu, Mack H. Wu and Harris

PI3K pathwayEph B4 receptor signaling mediates endothelial cell migration and proliferation via the

published online September 13, 2002J. Biol. Chem.

10.1074/jbc.M207221200Access the most updated version of this article at doi:

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eph b4 receptor signaling mediates endothelial cell migration and

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