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INTRODUCTION |
The Drosophila tracheal (respiratory) system and the
mammalian lung are both formed by patterned branching morphogenesis
(1). In Drosophila, Branchless, a homologue of mammalian
FGFs,1 is required for normal
tracheal branch patterning (2). Branchless activates Breathless, an FGF
receptor homologue, inducing tracheal cell migration and branching
(1-4). Loss of function mutations in the Drosophila sprouty
gene, which encodes a 63-kDa protein containing a cysteine-rich domain
that is highly conserved in three human and four mouse homologues
(5-8), cause enhanced tracheal branching (5). Further, inhibition of
murine Sprouty-2 by antisense oligonucleotides enhances
terminal branching of mouse cultured lung (6). These data suggest that
Sprouty proteins negatively modulate branching morphogenesis in the
Drosophila and mouse respiratory systems.
Drosophila sprouty is expressed at the tips of growing
primary branches of the tracheal system and also in other tissues such as eye imaginal disc, embryonic chordotonal organ precursors, and
midline glia (5, 9, 10). Mouse homologues of sprouty (mSpry1, mSpry2, and mSpry4) are
expressed in embryonic and adult tissues such as brain, heart, kidney,
lung, limbs, and skeletal muscle (6-8). FGF induces expression of
sprouty in Drosophila and the chick embryo (5,
8), and Drosophila Sprouty is a membrane-associated protein
that functions as a feedback inhibitor of the FGF signaling pathway (5,
9). Drosophila Sprouty can also antagonize epidermal growth
factor signaling pathways (9-11), and overexpression of Sprouty
can cause phenotypes resembling those of loss, or reduction, of
function of FGF and epidermal growth factor signaling pathways
(5, 9-11). Because angiogenesis shows some morphological similarities
to branching of the Drosophila tracheal system and because
it too requires receptor tyrosine kinase signals, including those
mediated through the FGF, VEGF, platelet-derived growth factor, ephrin
B, and Tie-2 receptors (12-21), we investigated whether a Sprouty
protein can regulate the sprouting and branching of blood vessels.
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MATERIALS AND METHODS |
Construction of Recombinant
Adenovirus--
Replication-deficient adenovirus with the
cytomegalovirus (CMV) enhancer/promoter driving expression of either
the Escherichia coli
-galactosidase gene
(AdCMV/lacZ, provided by the Institute of Human Gene
Therapy, University of Pennsylvania), the Ras mutants RasN17 and RasL61 (gift of Dr. Joseph Nevins,
Duke University), or murine Spry4 (AdCMV/mSpry4)
were used. The AdCMV/mSpry4 virus was generated using
methods described previously (22, 23). Briefly, a cDNA encoding
mSpry4 tagged with an HA epitope at its N terminus was subcloned into an E1-, E3-deleted type 5 adenoviral vector. The recombinant adenovirus was identified by Western blot analysis using an
anti-HA antibody (Roche Molecular Biochemicals), and the identity of
the virus was confirmed by DNA sequencing of selected regions. The
recombinant adenovirus was transfected into 293 cells and purified as
described (22, 23).
Whole Mouse Embryo Culture and Microinjection--
Whole mouse
embryo culture was performed as described previously (24, 25). Briefly,
embryos (embryonic day 8.0 to 8.25; 1-5 somites) were cultured in
medium consisting of 70% heat inactivated rat serum and 30% sterile
Dulbecco's phosphate-buffered saline (Life Technologies, Inc.).
Cultures were equilibrated with a mixture of 5% O2, 5%
CO2, and 90% N2 and rotated during incubation
at 37 °C for 12-16 h. Adenovirus (AdCMV/lacZ and
AdCMV/mSpry4) was injected into the sinus venosus of the
embryo as described previously (24, 25). Following injection, embryos
were cultured in medium equilibrated with 20% O2,
5% CO2, and 75% N2 for an additional 24 h (AdCMV/lacZ, n = 38;
AdCMV/mSpry4, n = 45). The embryos were subgrouped with respect to the following properties: presence of
beating heart, extent of neural tube closure, development of functioning extra-embryonic circulation, and progression of embryonic development as assessed by degree of turning of the embryos.
Microangiography was performed in a subset of the embryos
(AdCMV/lacZ, n = 20; AdCMV/mSpry4, n = 37) by injection of phenol
red dye into the vitelline artery.
FACS Analysis--
Human umbilical vein endothelial cells
(HUVECs) were isolated as described (26), infected with 5.0 × 103 particles/cell of either AdCMV/lacZ or
AdCMV/mSpry4 for 12 h, and were then cultured an
additional 24 h before being fixed and subjected to RNase
treatment and staining with propidium iodide. Cells were subjected to
FACS analysis on a Becton Dickinson FACScan. Cells were gated to
AdCMV/lacZ-infected cells and analyzed using the FLOWJO
analysis program.
Western Blot Analysis--
HUVECs were infected with recombinant
adenovirus for 16 h and harvested using cold lysis buffer (1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in
phosphate-buffered saline) containing a protease inhibitor mixture
(Roche Molecular Biochemicals), and samples were separated by
SDS-polyacrylamide gel electrophoresis in 10% Tris-glycine gels
(Novex, San Diego, CA). Transgene expression was detected by incubating
the membrane with a monoclonal antibody to HA (Roche Molecular
Biochemicals). Antibodies against Rb (G3-245, Pharmingen) and p21
(Transduction Laboratories, Lexington, KY) were used to detect
phosphorylation of RB and to detect p21 expression. To determine the
level of active MAPK, a monoclonal antibody against diphosphorylated
p42/p44 MAPK (Sigma) was used. To control for loading and transfer
efficiency, blots were probed with a monoclonal antibody against
-actin (Sigma).
Migration Assay--
Modified Boyden chambers (Transwell; Costar
Corp, Cambridge, MA) were utilized for cell migration assays. The
underside of the membrane of the upper chamber was coated with 10 µg/ml of collagen type I (Vitrogen 100, Cohesion, Palo Alto, CA) and
placed into the lower chamber containing medium (EBM with 0.5%
bovine serum albumin) plus either 10% serum, 30 ng/ml bFGF, 30 ng/ml VEGF, 30 ng/ml phorbol 12-myristate 13-acetate, or no additional factor. HUVECs infected as described previously were cultured in
serum-free medium for 5-6 h, trypsinized, and resuspended in EBM with 0.5% bovine serum albumin at 5.0 × 105
cells/ml. Cells were plated in the upper chamber and allowed to migrate
for 4 h at 37 °C before counting cells on the membrane.
Immunohistochemistry--
Cultured embryos injected with either
AdCMV/lacZ or AdCMV/mSpry4 were collected and
embedded in OCT and fixed in cold acetone. The sections were
incubated with anti-HA monoclonal antibody to detect transgene
expression. To detect coexpression of PECAM and
-galactosidase,
anti-PECAM antibody (Pharmingen) and anti-
-galactosidase antibody
(Sigma) were used.
TUNEL Staining--
Primary HUVECs were cultured on glass
coverslips and subsequently infected with recombinant adenovirus for
16 h. Cells were fixed in 4% paraformaldehyde and permeabilized
with 0.1% Triton X-100. Positive controls were obtained by incubation
with DNase I (Roche Molecular Biochemicals). TUNEL staining was
performed in situ (Roche Molecular Biochemicals) followed by
counterstaining with hematoxylin (Vector Laboratories, Burlingame, CA).
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RESULTS |
Endothelial Cell-specific Expression of Spr4
Transgene--
To test the effect of a Sprouty protein on angiogenesis
in vivo, mSpry4 was expressed in the developing
endothelium of the 9.0-day mouse embryo using an adenoviral vector.
Previous studies show that expression of transgenes in this manner is
restricted to endothelium (24, 25). Indeed, in sections of embryos
injected with AdCMV/lacZ,
-galactosidase was expressed in
PECAM-expressing endothelial cells of the dorsal aorta (Fig.
1, A and B).
Furthermore, immunohistochemistry revealed that expression of the
HA-tagged Sprouty protein was restricted to the developing vascular
endothelium, endocardium, and extra-embryonic vascular endothelium
(Fig. 1C and data not shown). No HA immunoreactivity was
observed in sections of AdCMV/lacZ-injected control embryos
(Fig. 1D). These results confirm that the Spry4
transgene was expressed exclusively in the endothelium of the embryonic
and extra-embryonic vasculature.

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Fig. 1.
Endothelial cell-specific expression of a
Sprouty transgene. The endothelial cell-specific marker PECAM
(A) and -galactosidase (B) are coexpressed in
the vascular endothelium of dorsal aorta of a control
AdCMV/lacZ-injected embryo, as demonstrated in
adjacent sections. The endothelial cell-specific expression of
mSpry4 transgene tagged with an HA epitope is shown in an
AdCMV/mSpry4-injected embryo (C). A control
AdCMV/lacZ-injected embryo shows no staining with the HA
antibody (D).
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Expression of Spry4 Inhibits Angiogenesis in Vivo--
To examine
the effects of Spry4 expression on yolk sac vascular development,
microangiography was performed (Fig. 2,
A and B). Phenol red dye injected into the
vitelline artery of control (AdCMV/lacZ) embryos circulated
throughout the yolk sac and embryo, demonstrating that extensive
vascular branching and network formation occurred in the control
embryos. In contrast, in the AdCMV/Spry4 embryos, although
large vessels with circulating blood cells were observed, there was no
evidence of organized sprouting of smaller vessels from large vessels.
Microangiography demonstrated that dye was pooled in the yolk sac at
the site of injection. Thus, Spry4 expression inhibits
sprouting and branch formation of small yolk sac vessels.

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Fig. 2.
Spry4 inhibits angiogenesis in
vivo. Microangiography shows well organized yolk sac
vasculature in a control (AdCMV/lacZ-injected) embryo (A).
The yolk sac of the AdCMV/mSpry4 embryo shows poorly
developed vascular branching and sprouting (B). To examine
the role of spry4 expression on the embryonic vasculature,
whole mount immunohistochemistry using an anti-PECAM antibody was
performed. A control embryo, injected with AdCMV/lacZ,
exhibits highly organized vasculature in the head (C), heart
(E), and intersomatic vessels (G). In contrast,
an embryo injected with AdCMV/mSpry4 exhibits poorly
developed vascular branching and sprouting in the head (D),
heart (F), and intersomatic vessels (H).
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To examine the effect of Spry4 expression on the development
of the vasculature of the embryo proper, whole mount staining with an
antibody against PECAM was performed (Fig. 2, C-H). The control embryos injected with AdCMV/lacZ formed a highly
organized vascular network in head, heart, and somatic vessels (Fig. 2, C, E, and G). In contrast, embryos
injected with AdCMV/mSpry4 exhibited a primitive vasculature
with poor branching and minimal sprouting of vessels (Fig. 2,
D, F, and H). Vascular development was
arrested at the primary plexus stage, with no formation of the highly
branched vascular network normally observed in the brain. The
AdCMV/mSpry4-injected embryos exhibited a less extensive endocardium, intermittent disruption of the endothelial lining of the
heart, and decreased branching of the intersomatic vessels (Fig. 2,
F and H).
The injected embryos were cultured for an additional 24 h, and
their gross morphology was evaluated. At the time of examination, the
development of the head, eyes, heart, and limb buds of
AdCMV/lacZ-injected control embryos expressing
-galactosidase had progressed normally, and the embryos had
completed turning within the yolk sac. The circulation of blood cells
through the yolk sac was clearly visible. In the control embryos, 36 of
38 developed normally, with complete turning of the embryos observed
(Table I). In contrast, in 45 embryos
injected with AdCMV/mSpry4, 41 showed delayed embryonic development and did not complete turning within the yolk sac. The
hearts of the embryos were beating but were incompletely developed. The
neural folds were not fused, and the development of the limb bud was
incomplete. Additionally, the same 41 embryos demonstrated abnormal
formation of the yolk sac vasculature. Since expression of the
Spry4 transgene was restricted to the endothelium, the abnormal embryonic development observed was probably secondary to
abnormal vascular development in the embryo proper.
Spry4 Inhibits Endothelial Cell Proliferation--
To determine
the effect of Spry4 expression on endothelial cell function,
a series of studies utilizing HUVECs was performed. To determine
the effects of Spry4 on HUVEC proliferation, cells were
infected with either AdCMV/lacZ or AdCMV/mSpry4.
The number of Spry4-infected HUVECs was less than that of
the lacZ-infected (control) cells at all time points
following infection (Fig. 3A). To determine whether Spry4 inhibits endothelial cell
proliferation mediated by bFGF or VEGF, cells were cultured in EBM
containing 2% FBS and either bFGF or VEGF at 25 ng/ml and harvested at
various time points. Expression of Spry4 inhibited both
bFGF- and VEGF-induced cell proliferation (Fig. 3, B and
C).

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Fig. 3.
Inhibition of endothelial cell proliferation
by Spry4. HUVECs were infected with AdCMV/lacZ (open circles) or
AdCMV/ mspry-4 (open squares) for
12 h was synchronized overnight in basal medium containing
2% FBS and plated on six-well dishes in growth medium
(A) or in basal medium containing 25 ng/ml bFGF
(B) or 25 ng/ml VEGF (C) for up to 96 h, and
cells were counted at the indicated times after the addition of growth
factors. As a negative control, cells were cultured in EBM containing
2% FBS (solid circles,
AdCMV/lacZ-infected; solid squares,
AdCMV/mSpry4-infected). Assays were performed in triplicate,
and data are expressed as mean ± S.D. (n = 3).
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To determine whether the reduction in cell number in
Spry4-expressing HUVECs results from cell death, TdT-UTP
nick end labeling (TUNEL analysis; Roche Molecular Biochemicals) was
performed. No TUNEL-positive cells were observed in either
AdCMV/mSpry4- or AdCMV/lacZ-infected cells (data
not shown). In addition, microscopic observation of cells infected with
AdCMV/mSpry4 did not reveal cells that were shrunken or
detached from the culture dishes, even after infection for 48 h.
These data suggest that cell cycle arrest, and not cell death, is the
primary consequence of overexpression of mSpry4 in HUVECs.
Spr4 Arrests Cell Cycle Progression--
To examine the effects of
Spry4 expression on the cell cycle, flow cytometric analysis
was performed. In the AdCMV/mSpry4-infected cells, an
increased percentage of the analyzed populations was detected in
G1 and G2, with a corresponding decrease in the
proportion in S phase, when compared with both
AdCMV/lacZ-infected and uninfected control cells (Fig.
4A). These results demonstrate
an inhibitory effect of Spry4 on progression through the
cell cycle.

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Fig. 4.
Spry4 arrests cell cycle progression in
G1. Uninfected HUVECs or HUVECs infected with
AdCMV/lacZ or AdCMV/mSpry4 as indicated were
cultured in growth medium for 36 h and then fixed and processed
for cell cycle analysis by FACS. The proportion of cells at each cell
cycle stage is shown in A. B, cell lysates were
harvested at different time points after infection and subjected to
10% SDS-polyacrylamide gel electrophoresis and immunoblotting with
antibodies to phosphorylated Rb (ppRb) (a), the
cell cycle inhibitor p21 (b), or -actin (c).
Lanes 1-4, AdCMV/lacZ-infected
HUVECs; lanes 5-8,
AdCMV/mSpry4-infected HUVECs. Note the decrease in
phosphorylated Rb and increased p21 in cells infected with
AdCMV/mSpry4.
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Cell cycle progression from G1 to S phase is regulated by
cyclin-dependent kinases. Rb protein is phosphorylated in a
cell cycle-dependent manner during the normal cell cycle.
In addition, induction of the cell cycle inhibitor p21 can arrest
progression from G1 to S. The expression of p21 and the
phosphorylation of Rb were examined in Spry4-expressing
cells and control
-galactosidase-expressing cells (Fig.
4B). In the control cells, Rb phosphorylation was maximally
increased by 24 h and decreased thereafter. In contrast, in cells
overexpressing Spry4, decreased phosphorylation of Rb and
increased expression of the cell cycle inhibitor p21 was observed. These changes may be responsible, at least in part, for the
G1 to S arrest observed in endothelial cells expressing
Spry4.
Spry4 Inhibits Cell Migration--
To determine whether the
Sprouty protein inhibits endothelial cell migration, studies were
performed using a modified Boyden chamber. HUVECs were cultured in
mitogen-poor medium, with or without the addition of bFGF or
VEGF. Both bFGF and VEGF stimulated cell migration in control
(lacZ)-infected cells (Fig. 5,
open bars). In contrast, in HUVECs expressing
mSpry4, bFGF and VEGF stimulated significantly less
migration (Fig. 5, solid bars). Thus, Spry4 is
capable of inhibiting migration induced by bFGF and VEGF.

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Fig. 5.
Spry4 inhibits endothelial cell
migration. HUVECs were infected with AdCMV/lacZ
(open bars) or AdCMV/mSpry4
(solid bars) and placed in modified Boyden
chambers, where they were exposed to either 10% FBS, no additional
growth factor, or bFGF or VEGF at 30 ng/ml as indicated. Cell migration
was assessed by counting cells on the membrane after 4 h.
Endothelial cells expressing Spry4 exhibited decreased
migration in response to bFGF or VEGF, compared with the control cells.
Data are expressed as mean ± S.D. (n = 3).
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Inhibition of MAPK Activation by Spry4--
To determine whether
Spry4 interferes with signaling through receptor tyrosine kinase
pathways and Ras/MAPK, we analyzed the effect of mSpry4 on
phosphorylated (activated) p42/p44 MAPK (Fig. 6). Treatment of the control
AdCMV/lacZ-infected HUVECs with either bFGF or VEGF for 10 min increased the level of phosphorylated MAPK above the basal level.
In contrast, in AdCMV/mSpry4-infected HUVECs, there was a
substantial reduction in both basal and bFGF- or VEGF-induced MAPK
phosphorylation. Expression of Spry4 did not inhibit MAPK
phosphorylation induced by phorbol 12-myristate 13-acetate, indicating
that the block in MAPK activation by Sprouty is selective for
activation via receptor tyrosine kinase pathways.

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Fig. 6.
Activation of p44/p42 MAPK by bFGF and VEGF
is inhibited by Spry4. HUVECs were infected with
AdCMV/lacZ (control; lanes 1-4) or
AdCMV/mSpry4 (lanes 5-8) for 12 h in medium containing 2% FBS. Cells were trypsinized and then
replated in serum-free medium for 4 h prior to treatment with 25 ng/ml of bFGF (lanes 2 and 6), VEGF
(lanes 3 and 7), or the phorbol ester
phorbol 12-myristate 13-acetate (lanes 4 and
8) or no additional mitogen (control; lanes
1 and 5). Cell lysates were prepared for Western
blotting and probed with an antibody to activated (diphosphorylated)
p44/p42 MAPK (A). Note the decrease in phosphorylated
p44/p42 MAPK in FGF- and VEGF-treated cells infected with
AdCMV/mSpry4. The increase in phosphorylated MAPK induced by
phorbol ester was not inhibited by Spry4. Equal protein loading was
confirmed by an antibody to -actin (B).
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Spry4 Inhibits Receptor Tyrosine Kinase Pathways--
To determine
where in the receptor tyrosine kinase signaling pathway Spry4 protein
acts, HUVECs were infected with AdCMV/lacZ or
AdCMV/Spry4 with or without concomitant infection of an
adenovirus expressing constitutively active RasL61 or
dominant negative RasN17. RasL61 expression
resulted in constitutive activation of MAPK, regardless of whether or
not cells were expressing Spry4 (Fig.
7). In contrast, in cells infected with
dominant negative mutant RasN17, stimulation of MAPK
phosphorylation by FGF and VEGF was inhibited, and mSpry4
had no effect on MAPK phosphorylation (Fig. 7). These data suggest that
Spry4 interferes with the tyrosine kinase signaling pathway upstream of
Ras.

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Fig. 7.
Constitutive activation of the Ras pathway by
RasL61 rescues the inhibitory activity of Spr4.
HUVECs were infected with AdCMV/lacZ (lane
1) or AdCMVmSpry4 (lane 5)
at 5000 particles/cell or were coinfected with adenovirus encoding the
constitutively active mutant RasL61 (A)
(lanes 2-4, lacZ plus
RasL61, 5000 particles/cell; lanes
6-8, mSpry4 plus RasL61, 5000 particles/cell; lanes 9-11, mSpry4
plus RasL61, 10,000 particles/cell). Note that the
expression of constitutively active RasL61 mutant activates
MAPK phosphorylation in the absence of growth factors and rescues the
inhibition of MAPK phosphorylation produced by Spry4. B,
cells were infected with AdCMV/lacZ alone (lane
1), or AdCMV/mSpry4 alone (lane
5) or were coinfected with an adenovirus encoding the
dominant negative RasN17 mutant (lanes
2-4, lacZ plus RasN17, 5000 particles/cell; lanes 6 and 7,
mSpry4 plus RasN17, 5000 particles/cell;
lanes 9-11, mSpry4 plus
RasN17, 10,000 particles/cell). The results indicate that
the dominant negative RasN17 mutant antagonized the
activation of MAPK mediated by FGF and VEGF and augmented the
inhibition of MAPK produced by Spry4.
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DISCUSSION |
The Sprouty protein is a novel receptor tyrosine kinase pathway
antagonist that regulates the formation of tracheal branches during
Drosophila development. The tracheal system undergoes
extensive branching morphogenesis, which is similar to the formation of a mature vascular system through angiogenesis (1). Since
Drosophila Sprouty can antagonize signaling through at least
two receptor tyrosine kinases, we hypothesized that mammalian Sprouty
homologs might inhibit sprouting and branching of blood vessels during angiogenesis by a similar mechanism.
In this study, we examined the effects of overexpression of
Sprouty on angiogenesis in vivo. Previous studies
indicate that a replication-defective adenovirus carrying a CMV
promoter/enhancer can drive the expression of transgenes in the
developing endothelium (24, 25). Therefore, an adenovirus encoding
murine Spry4 was constructed and delivered in this manner,
and the phenotype was examined. In cultured embryos overexpressing
murine Spry4, formation of a network of small and large
vessels and sprouting from existing vessels are disrupted. Further, the
vasculature of the yolk sac in the Spry4-expressing embryos
contained fewer branches and more similarly sized vessels rather than
exhibiting a complex vascular network. In addition, whole mount PECAM
staining demonstrated a disorganized and primitive vascular network in
the head, heart, and intersomatic vessels. These data indicate
that Sprouty inhibits branching and sprouting of the vasculature during angiogenesis.
Receptor tyrosine kinase activation triggers numerous cellular
responses. The activation of MAPK throughout the G1 phase
of the cell cycle is required for cells to enter S phase (27, 28). Previous reports suggest that MAPK signaling can regulate important processes in angiogenesis such as proliferation, migration, and differentiation (29, 30). In this study, overexpression of Spry4 inhibited endothelial cell proliferation and migration
in vitro. Spry4 arrested endothelial cell cycle progression
without inducing apoptosis. In addition, overexpression of
Spry4 inhibited MAPK activation induced by bFGF or VEGF but
did not inhibit that induced by other pathways, such as the one
stimulated by phorbol esters.
Drosophila Sprouty has been shown to antagonize at least two
different receptor tyrosine kinase pathways, FGF and epidermal growth
factor pathways. Our work demonstrates that a vertebrate Sprouty
protein can inhibit FGF as well as VEGF pathways, a third receptor
tyrosine kinase pathway. Our data begin to address the location in the
signaling pathway where inhibition by the vertebrate Sprouty occurs.
The expression of a constitutively active RasL61 resulted
in constitutive activation of MAPK, a downstream effector of Ras
signaling, even in the presence of mSpry4 expression. These data suggest that Spry4 inhibits the signaling pathway upstream of Ras.
Since neither the FGF or VEGF ligands nor the extracellular domain of
their cognate receptors share any significant structural similarities,
the data suggest that Spry4 protein inhibits receptor tyrosine
kinase-mediated signaling pathways at or downstream of the
intracellular domain of the receptor, but upstream of or at Ras.
The ability of Spry4 to inhibit tyrosine kinase-stimulated MAP kinase
activation probably results in the cell cycle arrest observed with
overexpression of Spry4. In addition, overexpression of
Spry4 in the vasculature of the murine embryo inhibited
branching and sprouting during in vivo angiogenesis. These
data suggest that Spry4 negatively modulates the MAPK signaling pathway
stimulated by receptor tyrosine kinase activation and that activation
of this pathway is required for angiogenesis. Although the present study indicates that Spry4 protein is a potent regulator of receptor tyrosine kinase signaling and angiogenesis when expressed in
endothelial cells, elucidation of the roles of Sprouty proteins in
developmental and pathological angiogenesis awaits the generation of
further mouse Sprouty mutants.