Inhibition of Angiogenesis by a Mouse Sprouty Protein*

Sang Hoon LeeDagger , Derrick J. SchlossDagger , Lesley Jarvis§, Mark A. Krasnow§, and Judith L. SwainDagger

From the Dagger  Department of Medicine, § Howard Hughes Medical Institute, Department of Biochemistry, Stanford University School of Medicine, S-102, Stanford, California 94305-5109

Received for publication, August 1, 2000, and in revised form, October 25, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Sprouty negatively modulates branching morphogenesis in the Drosophila tracheal system. To address the role of mammalian Sprouty homologues in angiogenesis, another form of branching morphogenesis, a recombinant adenovirus engineered to express murine Sprouty-4 selectively in endothelial cells, was injected into the sinus venosus of embryonic day 9.0 cultured mouse embryos. Sprouty-4 expression inhibited branching and sprouting of small vessels, resulting in abnormal embryonic development. In vitro, Sprouty-4 inhibited fibroblast growth factor and vascular endothelial cell growth factor-mediated cell proliferation and migration and prevented basic fibroblast growth factor and vascular endothelial cell growth factor-induced MAPK phosphorylation in endothelial cells, indicating inhibition of tyrosine kinase-mediated signaling pathways. The ability of constitutively activated mutant RasL61 to rescue Sprouty-4 inhibition of MAPK phosphorylation suggests that Sprouty inhibits receptor tyrosine kinase signaling upstream of Ras. Thus, Sprouty may regulate angiogenesis in normal and disease processes by modulating signaling by endothelial tyrosine kinases.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Construction of Recombinant Adenovirus-- Replication-deficient adenovirus with the cytomegalovirus (CMV) enhancer/promoter driving expression of either the Escherichia coli beta -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 beta -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 beta -galactosidase, anti-PECAM antibody (Pharmingen) and anti-beta -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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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, beta -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 beta -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).

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).

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 beta -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.


                              
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Table I
Phenotype of embryos injected with AdCMV/lacZ or AdCMV/mSpry4

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).

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 beta -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.

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 beta -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).

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 beta -actin (B).

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.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

We thank Dr. Mark Kay (Stanford University) for kindly providing the adenoviral and shuttle vectors as well as Dr. Joseph Nevins (Duke University) for the recombinant adenoviruses encoding the RasL61 and RasN17 mutants. We also thank Drs. Anjali Pathak and Frances Johnson for assisting with FACS analysis.


    FOOTNOTES

* This work was supported by National Institutes of Health (NIH) Grants HL 26831 (to J. L. S.) and T32 HL07708 (to S. H. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Department of Medicine, Stanford University, S-102, 300 Pasteur Dr., Stanford, CA 94305-5109. Tel.: 650-489-7778; Fax: 650-725-8381; E-mail: jlswain@stanford.edu.

Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M006922200


    ABBREVIATIONS

The abbreviations used are: FGF, fibroblast growth factor; bFGF, basic FGF; CMV, cytomegalovirus; EBM, endothelial cell basal medium; FACS, fluorescence-activated cell sorter; FBS, fetal bovine serum; HUVEC, human umbilical vein endothelial cell; MAPK, mitogen-activated protein kinase; PECAM, platelet endothelial cell adhesion molecule; TUNEL, TdT-UTP nick end labeling; VEGF, vascular endothelial cell growth factor; HA, hemagglutinin.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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