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
* Author for correspondence (e-mail: jin.chen{at}mcmail.vanderbilt.edu )
Accepted 16 May 2002
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Summary |
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Key words: Ephrin-B1, EphB1, Endothelial, Angiogenesis, Signal transduction
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Introduction |
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Transmembrane Eph receptor kinases are activated upon binding to
oligomerized ephrins (Davis et al.,
1994), and specific oligomerized forms signal distinct cellular
responses (Stein, 1998). EphB subclass receptors (EphB1-6) display overlapping
affinity for transmembrane ephrin-B subclass counter-receptors (ephrin-B1-3),
whereas EphA subclass receptors (EphA1-8) bind predominantly to the
glycerol-phosphatidylinositol-linked ephrin-A counter-receptors (ephrin-A1-6)
(Gale et al., 1996
;
Menzel et al., 2001
). Both
EphB receptors and ephrin-B counter-receptors have C-terminal sequences
capable of interacting with PDZ-domain-containing proteins, including PICK1,
syntenin, GRIP (Bruckner et al.,
1999
; Torres,
1998
) and PDZ-RGS (Lu,
2001
).
Evidence that ephrin-B subclass counter-receptors signal cell-autonomous
responses was first provided by the targeting behavior of axons in
EphB2-mutant mice (Henkemeyer et al.,
1996). Axonal projections that express ephrin-B2 were misdirected
in mice homozygous null for EphB2. However, these axonal projections targeted
correctly through migratory fields in animals that expressed EphB2 ectodomains
as a tyrosine-kinase-deficient ß-galactosidase fusion. Ephrin-B2
cytoplasmic domain sequences are c-src tyrosine kinase substrates that are
tyrosine phosphorylated in early embryos and show regulated tyrosine
phosphorylation sensitive to EphB2/Fc
(Holland et al., 1996
) and
PDGF (Bruckner et al., 1997
) in
transfected cell lines. This regulated tyrosine phosphorylation recruits
adaptor protein Grb4 and transduces signals to promote changes in the actin
cytoskeleton (Cowan and Henkemeyer,
2001
).
Ephrin-B2 expression is required for development of the embryonic vascular
system where its early expression is restricted to endothelial cells of
arterial, not venous, vascular structures
(Wang et al., 1998).
Homozygous mice null for ephrin-B2 show failure of embryonic vascular
development at a stage when extraembryonic yolk sac arterial plexus vessels
fail to interconnect with venous plexus vessels that express the EphB4
receptor. A similar failure of vascular embryonic development is displayed in
EphB4-null mice (Gerety et al.,
1999
) and in mice doubly homozygous for deletions in EphB2 and
EphB3 (Adams et al., 1999
).
More recently, the functional role of the ephrin cytoplasmic domain was
demonstrated in ephrin-B2
C/
C mice expressing a mutant
ephrin-B2 with a cytoplasmic deletion
(Adams et al., 2001
).
Ephrin-B2
C/
C mice exhibit vascular remodeling defects
that are reminiscent of a subset of phenotypes in ephrin-B2-null mice,
indicating that signaling through ephrin-B2 is required for vascular
development.
Cultured primary microvascular endothelial cells are useful systems for
defining EphB1 signaling pathways that impact upon responses relevant to
vascular development, including cell attachment, migration and capillary-like
assembly responses (Daniel et al.,
1996; Huynh-Do et al.,
1999
; Stein, 1998). Here we have asked whether ephrin-B1
transduces `outside-in' signals to alter cell attachment or migration in
microvascular endothelial cells that express endogenous ephrin-B1 (Stein,
1998). Our findings demonstrate that ephrin-B1 is coupled to integrin function
and assign a direct role to the C-terminal ephrin-B1 sequences. The relevance
of the in vitro culture results is supported by the in vivo effects of a
soluble EphB1/Fc fusion protein that promotes neovascularization.
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Materials and Methods |
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Antibodies and reagents
Polyclonal rabbit ephrin-B1 antibodies, P1, recognize the C-terminal 15
amino acids of both ephrin-B1 and ephrin-B2, whereas ephrin-B1 antibodies, P2,
recognize a non-conserved juxtamembrane spacer domain peptide unique to
ephrin-B1 (aa221-238) (Immunex, Seattle, WA).
Horseradish-peroxidase-conjugated antibody 4G10-HRP, anti-ERK1/2 and anti-JNK
polyclonal antibodies were from Upstate Biotechnology (Lake Placid, NY) and
streptavidin-HRP from Jackson (Westgrove, PA). Human IgG1 and
plasma fibrinogen were from Sigma (St Louis, MO), human fibronectin was from
Life Technologies. The GRGDTP and GRGESP peptides were from Calbiochem (La
Jolla, CA). Anti-integrin blocking mAbs from Chemicon (Temecula, CA) were
LM609 (vß3), P1F6
(
vß5) and JBS5
(
5ß1). Polyclonal antibodies against
phosphorylated forms of JNK, ERK1/2 and p38MAPK were from Promega (Madison,
WI). Other matrix proteins, peptides and antibodies were from previously cited
sources (Huynh-Do et al.,
1999
; Stein, 1998).
Plasmids, cell culture, transfection and FACS analysis
cDNA encoding ephrinB-1Cy (carrying a deletion of the C-terminal 50
amino acids) and ephrinB-1
PDZbd (carrying a deletion of four C-terminal
amino acids) were amplified by PCR, sequenced and subcloned into expression
vector pSR
. Human renal microvascular endothelial cells (HRMEC) were
cultured and passaged as described previously
(Martin et al., 1997
). CHO
cells were passaged in DMEM-F12 medium (Life Technologies) supplemented with
10% fetal bovine serum (Hyclone Laboratories, Logan, UT). CHO were transfected
using the Lipofectamine Plus method (Life Technologies) with vector alone
(sR
), plasmids expressing full-length human ephrin-B1, c-myc-tagged
ephrin-B1
Cy (lacking the C-terminal 50 amino acids) or
ephrin-B1
PDZbd (lacking the four C-terminal amino acids required for
interacting with the PDZ domain protein). Migration assays and FACS analysis
were performed 48 hours after transfection. Cell surface integrin expression
was analyzed 30 minutes after stimulation with IgG (Fc control) or EphB1/Fc (2
µg/ml) using anti
vß3 (LM609, 10
µg/ml). Analysis of ephrin B1 and mutant forms expressed on CHO cell
surfaces was conducted using EphB1/Fc (2 µg/ml) followed by FITC-conjugated
goat anti-human IgG-Fc (Jackson Labs, 1:200 dilution) on a FACSCaliber (Becton
Dickinson, San Jose, CA) instrument using an argon ion laser at 488 nm with
detection by a 530±30 nm band pass filter.
Surface biotinylation and ephrin immunoprecipitation
Cell surface proteins were covalently conjugated with biotin by incubation
of pre-washed cells for 30 minutes at 4°C with 0.5 mg/ml
sulfo-NHS-LC-Biotin in phosphate buffered saline (Pierce, Rockford, IL). Cells
were washed, quenched in 0.15 M glycine then lysed, and integrins or ephrin-B1
were immunoprecipitated as described previously
(Daniel et al., 1996).
Biotinylated proteins were detected following immunoblot transfer using
streptavidin-HRP with enhanced chemiluminescence (ECL Western Blotting
Detection, Amersham). Data are representative of three independent
experiments.
Ephrin-B1 tyrosine phosphorylation
Sixty mm (p60) tissue culture dishes were coated with fibronectin (0.5
µg/cm2) overnight at 4°C in bicarbonate buffer (Stein,
1998). Serum-starved HRMEC or CHO cells were replated
(15-20x105 cells/60 mm dish) for 60 minutes at 37°C.
Cells were stimulated with agonists for 15 minutes at 37°C then lysed in 1
ml RIPA buffer. Recovery and tyrosine phosphorylation of endogenous or
transfected Ephrin-B1 were assessed by immunoprecipitation with
anti-ephrin-B1, followed by anti-ephrin-B1 or anti-phosphotyrosine (4G10-HRP)
immunoblots, respectively.
Western blot analysis of MAP kinase phosphorylation
CHO cells stably expressing ephrin-B1 or transiently transfected with no
vector (MOCK), full-length human ephrin-B1, ephrin-B1Cy or
ephrin-B1
PDZbd were serum-starved for 24 hours in Opti-MEM, treated
with 0.5 mM suramin for 3 hours and then stimulated for the indicated times at
37°C with EphB1/Fc (2 µg/ml) or with varying concentrations of EphB1/Fc
for 20 minutes. For assessment of JNK, ERK1/2 and p38 MAPK activation, cells
were lysed in RIPA buffer, and 30 µg of proteins were loaded on a 10%
SDS-PAGE. After transfer to Immobilon/PVDF membranes (Millipore, Bedford, MA),
phosphorylated JNK, ERK1/2 or p38 was detected with antibodies against
phosphorylated forms of JNK, ERK1/2 or p38 MAPK. Membranes were then stripped
and reprobed with anti-JNK or anti-ERK1/2 to ensure equal loading of
proteins.
Cell attachment
Forty-eight-well plates (Falcon) were coated with fibrinogen (1
µg/cm2) overnight at 4°C in bicarbonate buffer. Two hours
prior to the assay, wells were washed twice then blocked at 37°C with 1%
BSA. Cells were starved for 48 hours in Opti-MEM, recovered by gentle
trypsinization, washed twice in serum-free medium containing 1% BSA, then
plated at a density of 0.5-0.8x105 cells per well. Controls
[no addition, NA or IgG (class-matched human Fc control)] or agonists
(EphB1/Fc) were added at the indicated concentrations at the time of plating.
After incubation at 37°C for 1 hour, unattached cells were dislodged by
brisk vertical contact of the plate with a horizontal surface until cells
plated on albumin-coated plates in the absence of matrix (no fibrinogen) were
fully detached (four to five slaps). Wells were washed with PBS, and adherent
cells were fixed with 2% glutaraldehyde, stained with 0.5% crystal violet (in
0.2 M boric acid) and quantified by OD reading at 570 nm. In some experiments,
cells were preincubated with the indicated peptides (100 µM) or
integrin-specific antibodies (5 µg/ml) for 15 minutes at room temperature
before plating. The data represent three independent experiments and are
expressed as means of values from four wells ± s.e.m.
Wound closure assay
Replicate circular `wounds', or defects (600-900 µm diameter), were
generated in confluent HRMEC or CHO cell monolayers using a silicon-tipped
drill press, as described previously
(Daniel et al., 1999).
Serum-free medium was supplemented with the indicated agonists at the time of
wounding. Residual fractional `wound' areas were measured at the indicated
times using a Bioquant (Nashville, TN) software package calibrated to a Nikon
Diaphot microscope. Mean fractional residual areas of three wounds, calculated
at each of the two or three time points were used to derive linear
regressions, reflecting migration rates (expressed as a percentage of
closure/hour, ±95% confidence intervals). Data are representative of
three independent experiments.
Mouse corneal angiogenesis assay
Hydron pellets incorporating sucralfate with vehicle alone, basic FGF (3
pmol/pellet; a gift from Scios, Inc), control IgG1 or EphB1/Fc (5.6
pmol/pellet) were made as described previously
(Kenyon et al., 1996). Pellets
were surgically implanted into corneal stromal micropockets that were created
1 mm medial to the lateral corneal limbus of C57L male mice (7-9 weeks old).
At day 5, corneas were photographed at an incipient angle of 35-50° from
the polar axis in the meridian containing the pellet using a Zeiss split lamp.
Images were digitalized and processed by subtractive color filters (Adobe
Photoshop 4.0): the fraction of the total corneal image that was vascularized,
the ratio of pixels marking neovascular capillaries, both within the
vascularized region (R) and within the total corneal image (T) were calculated
using the Bioquant software (Nashville, TN). Each summary value is the
mean±s.e.m. of nine corneas for each condition.
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Results |
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To address whether ephrin-B1 transduces `outside-in' signals to alter
endothelial cell function, we evaluated whether EphB1/Fc stimulated
endothelial cell migration. Fig.
2 shows that the rate of endothelial migration to close a circular
wound in a confluent monolayer was increased in serum-free medium supplemented
with EphB1/Fc, or PMA, but was unaffected by the Fc control, IgG1.
EphB1/Fc-stimulated endothelial cell migration was seen at a concentration
(0.5 µg/ml) comparable to that stimulating ephrin-B1 tyrosine
phosphorylation (Fig. 1). Both
basal and stimulated migration were attenuated by the antibody against
vß3, making assignment of a specific role
for this integrin in EphB1-stimulated migration difficult (data not
shown).
|
To assess whether ephrin-B1 signaling can alter endothelial cell-matrix
attachment, we exposed endothelial cells to soluble EphB1/Fc and evaluated
their attachment to fibrinogen-coated surfaces. Experiments were first
performed in the primary HRMEC. A more extensive characterization of cell
attachment was subsequently carried out in an endothelial cell line, human
dermal microvascular endothelial cell (HMEC-1). EphB1/Fc stimulated cell
attachment in both HRMEC and HMEC-1 in a dose-dependent manner
(Fig. 3A). A class-matched
human IgG1 (Fc fusion control) was inactive at these
concentrations, and anti-Fc-preclustered EphB1/Fc was functionally inactive as
either a promoter or inhibitor of endothelial attachment to fibrinogen (data
not shown). Thus, the dimeric form of EphB1 ectodomain (in the Fc fusion
protein) promotes endothelial attachment. This effect of EphB1/Fc to promote
endothelial attachment was also detected when EphB1/Fc is attached to solid
phase surfaces within narrow surface densities in an alternative attachment
assay (U.H.-D., unpublished) (Huynh-Do et
al., 1999).
|
Increases in cell attachment stimulated by EphB1/Fc were sensitive to
competition by a peptide containing RGD but not RGE sequences. Antibodies that
block RGD engagement by vß3 integrin (LM609)
or
5ß1 integrin (JBS5) attenuated
EphB1/Fc-induced attachment to varying degrees, whereas an
vß5 integrin blocking antibody was without
effect (Fig. 3B, bottom
panels). This is noteworthy as HRMEC express comparable levels of
vß3 and
vß5 integrins
(Huynh-Do et al., 1999
),
whereas HMEC-1 cells express more
vß5 than
vß3 (Fig.
3B, top panels). As shown in
Fig. 3., some of the basal
endothelial attachment in this assay was also dependent upon
vß3 integrin in HRMEC cells. The increase in
vß3-integrin-mediated attachment elicited by
EphB1/Fc occurs without changes in the abundance of surface-expressed
vß3 integrin, as demonstrated by FACS
analysis (Fig. 3B, top left
panel) and recovery of surface biotinylated
vß3 integrin by immunoprecipitation in HRMEC
cells (data not shown).
To assess the potential biological activity of EphB1/Fc in a relevant in
vivo assay, we implanted hydron pellets impregnated with vehicle control, bFGF
(3 pmol), control IgG1 (5.6 pmol) or EphB1/Fc (5.6 pmol) into mouse
corneal micropockets. Neovascularization responses were scored by vital
photography 5 days after implantation, and images were analyzed as described
in the Materials and Methods. EphB1/Fc promoted consistent neovascularization
responses that were not seen with the Fc fusion control IgG1. This
neovascularization response to EphB1/Fc was neutralized by coincident
implantation of a pellet impregnated with ephrin-B1/Fc to interrupt EphB1/Fc
interactions with endothelial ephrin-B1 (data not shown). Although
EphB1-induced neovascularization responses were not as brisk as those evoked
by bFGF, the fractional corneal area involved was 60%, and the microvessel
density within that area was also 60% of the bFGF response
(Fig. 4). Thus, the EphB1
ectodomain is active as an agonist to promote angiogenic responses when
presented in this context as a soluble Fc fusion protein in the corneal
stroma. Similar neovascularization responses were evoked by ephrin-B1 and
ephrin-B2 ectodomain Fc fusion proteins (H. Liu, unpublished), paralleling
responses to ephrin-A1 observed previously
(Pandey et al., 1995).
|
We next addressed whether cytoplasmic domain sequences of Ephrin-B1 mediate
EphB1/Fc-induced responses, as expected with `outside-in' signaling processes.
CHO cells do not express endogenous ephrin-Bs (1-3), as shown by FACS
analysis, using EphB1/Fc as a molecular probe for ephrin-B counter-receptors
(Fig. 5B, vector). Full-length
ephrin-B1 (Fig. 5B, ephrin-B1)
or cytoplasmic domain deletion versions lacking either the 50
(ephrin-B1Cy) or four C-terminal amino acids necessary for PDZ domain
binding (ephrin-B1
PDZbd) were transiently expressed in CHO cells at
comparable levels (Fig. 5B, FACS inserts). Migration responses for each CHO cell population to EphB1/Fc
were evaluated using the planar wound closure assay. Cells expressing intact
ephrin-B1 had increased rates of migration in response to EphB1/Fc at
concentrations that are active on endothelial cells (Figs
2 and
3) and that promote ephrin-B1
tyrosine phosphorylation in CHO cells (Fig.
5A). Neither of the cytoplasmic domain deletion forms of
ephrin-B1, ephrin-B1
Cy or ephrin-B1
PDZbd conferred EphB1/Fc
responsiveness. Thus, CHO migration responses were strictly dependent upon
integrity of the four most C-terminal amino acids, which are capable of
interacting with PDZ domain proteins
(Torres et al., 1998
) and
contain two tyrosine residues that are phosphorylated in vivo
(Kalo et al., 2001
).
|
Finally, we investigated signaling mechanisms that couple ephrin-B1
activation to cellular responses. As shown in
Fig. 6, stimulation of CHO
cells expressing wild-type ephrin-B1 with EphB1/Fc induced phosphorylation of
p46 JNK in a time and dose-dependent manner, with the highest phosphorylation
level at 10-45 minutes after stimulation. By contrast, activation of ephrin-B1
did not affect the phosphorylation status of ERK1/2 or p38 MAP kinases (data
not shown). Furthermore, cytoplasmic domain deletion forms of ephrin-B1,
ephrin-B1Cy or ephrin-B1
PDZbd failed to activate p46 JNK upon
EphB1/Fc stimulation (Fig. 6C),
suggesting that p46 JNK transduces signals in response to ephrin-B1 activation
through the four most C-terminal amino acids of the ephrin-B1 protein.
|
![]() |
Discussion |
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vß3 integrin is implicated in tumor
angiogenesis and in ocular neovascularization under conditions where its
expression in endothelial cells is induced and where it probably binds to
provisional extracellular matrix components, including fibrinogen and
vitronectin (Brooks et al.,
1994
). A recent description of
v knock-out mice
provides evidence for a role in embryonic developmental vascularization
(Bader et al., 1998
), yet the
vascular dysgenesis phenotype is less severe than that of the
ephrin-B2 gene deletion embryos
(Wang et al., 1998
). It seems
likely that integrin responses downstream of ephrin-Bs are not limited to
vß3, which is consistent with inhibition
evoked by antibody antagonism of
5ß1
integrin (Fig. 3C). De novo
ephrin-B1 expression in CHO cells conferred responsiveness to EphB1/Fc, as
scored by migration, yet blocking antibodies active against the dominant
integrin in these hamster cells,
5ß1, are
unavailable. All these data argue for a function for ephrin-B1 cytoplasmic
domain sequences in transducing a signal that is initiated by binding of EphB1
to the ephrin-B1 ectodomain and that is coupled to integrin-mediated
attachment and migration. Our results are consistent with previous findings
that engagement of class A ephrins, ephrinA5 and ephrinA2 by EphA-Fc reagents
increased adhesion to integrin ligands in culture
(Davy et al., 1999
; Davy et
al., 2000; Huai and Drescher,
2001
).
Cell migration is a multi-step process involving lamellipodium extension,
formation of new adhesions, cell body retraction and tail detachment. Cell
migration requires the precise regulation of integrin-mediated adhesion and
de-adhesion. Whether or not the cell migrates and the rate of the migration on
a given substratum depend on several variables related to integrin-ligand
interactions, including ligand levels, integrin levels and integrin-ligand
binding affinities. At low ligand concentration (e.g. 1-10 µg/ml
fibronectin for cells expressing 5 integrin), increased
adhesion leads to enhanced cell migration
(Palecek et al., 1997
).
However, at high ligand concentration increased adhesion blocks cell migration
(Palecek et al., 1997
). Since
EphB1/Fc did not affect the expression level of integrin
vß3 (Fig.
3B, left panel), EphB1/Fc-induced cell migration at low fibrinogen
concentration (1 µg/cm2) is probably caused by increased cell
adhesion, possibly through enhanced integrin-ligand binding affinities.
The in vivo angiogenic response evoked by EphB1/Fc (Fig. 4) suggests that unliganded ephrin-B1 (or ephrin-B2 or B3) counter-receptors exist in the endothelium of the corneal limbus adjacent the cornea. Moreover, they appear to be competent to promote endothelial activation and neovascularization. In principle, EphB1/Fc acts either as an agonist for B-ephrins or as a blocking agent that antagonizes B-ephrin activity, leading to new blood vessel formation. However, B-ephrins also induce corneal angiogenesis (H.L. and T.O.D., unpublished). If EphB1/Fc antagonizes B-ephrins, it would block angiogenesis. The fact that EphB1/Fc induced, rather than inhibited, corneal neovascularization suggests that it is likely to act as an agonist for B-ephrins.
The capacity for full-length ephrin-B1, but not cytoplasmic domain deletion
forms, to confer EphB1/Fc responsiveness in CHO cells provides strong evidence
that cytoplasmic domain sequences participate in transducing signaling
responses. The expressed ephrin-B1Cy lacks all of the five cytoplasmic
domain tyrosine residues implicated in regulated phosphorylation, which are
potential substrates for either c-src
(Holland et al., 1996
), FGF
receptors (Jones et al., 1998
)
or other candidate tyrosine kinases. Further, deletion of the C-terminal four
amino acids in ephrinB-1
PDZbd mutant removes PDZ domain
protein-interacting sequences, as well as two of the five conserved tyrosine
residues. This small deletion is sufficient to abrogate outside in signaling
responses in the reconstituted CHO cell system, suggesting either a
PDZ-domain-containing protein [such as PICK, syntenin, GRIP, or PDZ-RGS
(Bruckner et al., 1999
;
Lu, 2001
;
Torres, 1998
)] or other
adaptor proteins capable of binding phosphorylated tyrosine residues within
this region are critical to signaling process.
In summary, we provide evidence that ephrin-B1 transduces signals to
activate JNK and modulate integrin-mediated cell attachment, migration and
corneal angiogenesis. In the reciprocal direction, specifically oligomerized
forms of ephrin-B1/Fc also engage EphB1 receptor tyrosine kinase to promote
vß3 and
5ß1 activation
(Huynh-Do et al., 1999
).
Interestingly, in this more classic signaling scenario, JNK also appeared to
play a pivotal role linking EphB1 signaling to cytoskeletal responses
(Stein et al., 1998
). Such
reciprocity of signaling correlates with the developmental vascularization
defects shared between mice null for ephrin-B2
(Wang et al., 1998
) and its
binding partner, EphB4 (Gerety et al.,
1999
). Thus, both EphB receptors and ephrin-B counter receptors
are involved in bidirectional signaling, which plays critical roles in
vascular development in embryogenesis and possibly in adult angiogenesis as
well.
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Acknowledgments |
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