Angiopoietin 2 stimulates migration and tube-like structure formation of murine brain capillary endothelial cells through c-Fes and c-Fyn

Yasushi Mochizuki1, Takao Nakamura1, Hiroshi Kanetake1 and Shigeru Kanda1,2,*

1 Department of Urology and
2 Department of Molecular Microbiology and Immunology, Division of Endothelial Cell Biology, Nagasaki University Graduate School of Medicine, 1-7-1, Sakamoto, Nagasaki 852-8501, Japan

*Author for correspondence (e-mail: shigeruk{at}net.nagasaki-u.ac.jp)

Accepted September 20, 2001


    SUMMARY
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 SUMMARY
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
The angiopoietin (Ang)/Tie2 system is exclusively involved in vasculogenesis and angiogenesis. Ang2 is known to inhibit Ang1-mediated phosphorylation of Tie2 as well as cellular responses during embryonic development. Recent studies have demonstrated that Ang2 has angiogenic activities in adult tissues and cultured endothelial cells. In the present study, we examined the downstream signaling pathways involved in Ang2-mediated cellular responses by murine brain capillary cell line, IBE cells. Tie2 was tyrosine phoshorylated by Ang2. Ang2 showed no effect on proliferation, but stimulated chemotaxis and tube-like structure formation. Phosphoinositide 3-kinase (PI 3-kinase) was activated by Ang2 through c-Fes and was involved in chemotaxis toward Ang2. Ang2 also activated c-Fyn in IBE cells. Cells expressing kinase-inactive c-Fyn attenuated Ang2-induced tube formation, suggesting that c-Fyn was responsible for Ang-2-mediated tube formation. Collecting these data, Ang2 activates c-Fes and c-Fyn, leading to migration and tube formation by murine capillary endothelial cells.

Key words: Angiopoietin 2, Tie2, Fes, Fyn, PI 3-kinase, IBE cells, Chemotaxis, Tube formation


    Introduction
 Top
 SUMMARY
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
During embryonic development, vasculogenesis as well as angiogenesis are observed. Vasculogenesis involves the formation of heart and large vessels as well as primary capillary networks. Following vasculogenesis, endothelial cells proliferate and migrate into avascular tissues, thus remodeling initial capillary networks; this is so-called angiogenesis, which occurs during embryonic development. These sequential vascular developments are tightly regulated by vascular endothelial growth factors (VEGF) and angiopoietins (Folkman and D’Amore, 1996; Gale and Yancopoulos, 1999; Lauren et al., 1998).

The Angiopoietins (Ang) family is composed of four members, Ang1 to 4 (Yancopoulos et al., 2000). Ang1 and 4 stimulate their specific receptor tyrosine kinase, Tie2, whereas Ang2 and 3 inhibit Ang1-induced tyrosine phosphorylation of Tie2 (Yancopoulos et al., 2000). The expression of Tie2 is exclusively observed in vascular endothelial cells during development. Targeted disruption of Tie2 as well as Ang1 demonstrated similar defects of maturation of myocardium and endocardium as well as vascular complexity (Dumont et al., 1994; Suri et al., 1996). In addition, Ang1 was clearly involved in angiogeneis during embryonic development (Suri et al., 1998; Takakura et al., 2000). Using tetracycline-responsive transgenic mice, loss of Tie2 induced rapid endothelial cell apoptosis in vivo (Jones et al., 2001), suggesting that Tie2 signals are involved in the maintenance of vasculature. Ang2 was cloned by Ang1-based homology screening and it was found that Ang2 inhibited Ang1-mediated autophosphorylation of Tie2 in human umbilical vein endothelial cells (HUVECs) (Maisonpierre et al., 1997). In addition, overexpression of Ang2 in transgenic mice demonstrated similar vascular anomalies to mice lacking Ang1, indicating that Ang2 is a natural antagonist for Ang1 during embryonic development (Maisonpierre et al., 1997).

Angiogenesis in mature tissues is prerequisite for many physiological and pathological processes, such as ovulation, wound healing, malignant tumor growth, retinopathies and rheumatoid arthritis (Colville-Nash and Willoughby, 1997; Folkman, 1995). Angiogenesis in mature tissue is composed of a series of cellular responses. Pericytes leave from basement membranes of pre-existing capillaries, followed by activation of endothelial cells by angiogenic factors, such as fibroblast growth factors (FGFs), VEGFs, hepatocyte growth factor and Ang. Activated endothelial cells produce proteases, which digest the basement membranes of blood vessels. Endothelial cells migrate into interstitial tissue, proliferate, and form lumen-containing, tube-like structures. Finally, basement membranes of newly formed vessels are surrounded by pericytes (Darland and D’Amore, 1999; Hanahan, 1997; Yancopoulos et al., 2000).

Compared with the developmental angiogenesis, the roles of Ang/Tie2 system in pathological angiogenesis were less understood. However, recent works concerning the examination of Ang/Tie2 system in adult tissues have shown new insights into the important roles of Ang2 in angiogenesis. Expressions of Ang2 and Tie2, but not Ang1, were increased in malignant endothelial cells, such as angiosarcoma and aquired-immunodeficiency syndrome-associated Kaposi’s sarcoma (Brown et al., 2000), as well as rodent tumor tissues (Holash et al., 1999). Ectopic expression of Ang2 in tumour cells induced angiogenesis in association with increased tumor growth in transplanted mice (Tanaka et al., 1999; Ahmad et al., 2001; Etoh et al., 2001). In the previous report, activation of Tie2 by Ang1 was antagonized by Ang2 in endothelial cells. However, when Tie2 was ectopically expressed in fibroblasts or BaF3 cells, Ang2 could induce autophosphorylation of Tie2 (Maisonpierre et al., 1997; Sato et al., 1998). Furthermore, high concentrations of Ang2 (Kim et al., 2000) or Ang2-treatment in a particular condition (Teichert-Kuliszewska et al., 2001) was found to stimulate autophosphorylation of Tie2 in HUVECs, which was associated with increased survival and tube formation in fibrin matrix, respectively. These data strongly support the idea that Ang2 has stimulatory action on angiogenesis in mature tissues.

Signal transduction pathways via Tie2 have been extensively examined previously. Tie2 directly associates with the p85 regulatory subunit of phosphoinositide 3-kinase (PI 3-kinase) at an autophsohorylation site, Y1101 (Y1100) (Jones et al., 1999; Kontos et al., 1998), which in turn activate PI 3 kinase, leading to cell motility and survival (Jones et al., 1999; Papapertropoulos et al., 2000). Tie2 also binds to Dok-R, which is structurally homologous to p62dok and insulin-receptor substrate 3 (Jones and Dumont, 1998). Dok-R recruits adaptor protein Nck and Ras-GTPase activating protein (Ras-GAP). Upstream molecules of Ras/MAPK pathways, Grb2 and Shp2 were also identified as binding partners of Tie2 (Huang et al., 1995). More recently, it was demonstrated that Grb14 bound to Y814 and Y1106, Shp2 associated with Y814 and Y1111, and Grb2 as well as Grb7 could bind to Y1100 (Jones et al., 1999). Signal transducers and activators of transcription 3 (STAT3) and STAT5 were activated by Tie2, in association with the increased expression of p21 cell cycle inhibitor (Korpelainen et al., 1999). However, neither MAPK activation nor induction of proliferation were activated by Tie2 (Huang et al., 1995; Jones et al., 1999; Witzenbichler et al., 1998). In addition, no candidate signaling molecule involved in tube formation of endothelial cells was identified downstream of Tie2.

We have previously established a murine brain capillary endothelial cell line from ts-A58-H-2Kb transgenic mice, denoted IBE (Immortomouse brain endothelial) cells. IBE cells can proliferate, migrate and form lumen-containing tube-like structures in response to FGF-2 treatment (Kanda et al., 1996; Rahmanian et al., 1997). Using this culture model, we have previously shown that expression of dominant-negative c-Src in IBE cells perturbed chemotaxis towards FGF-2 with the lack of MAPK activation within focal contacts (Shono et al., 2001a). Expression of dominant-negative c-Fes caused impaired chemotaxis independently on c-Src and MAPK (Kanda et al., 2000; Shono et al., 2001a). Dominant-negative c-Fyn inhibited FGF-2-mediated tube formation without major effect on proliferation or motility (S. Tsuda et al., unpublished).

In the present study, we examined the signal transduction pathways leading to Ang2-mediated cellular responses of parental IBE cells as well as examination of downstream signaling pathways of Tie2 by using stable IBE cell lines expressing dominant-negative c-Src, c-Fyn and c-Fes. We found that: (1) 2000 ng/ml Ang2 added to IBE cells (which express Tie2) induced autophosphorylation, suggesting that Ang2 could activate Tie2 in IBE cells; (2) Ang2 stimulated motility and tube formation; (3) PI 3-kinase was activated and was dependent on c-Fes kinase activity; (4) PI 3-kinase was involved in chemotaxis towards Ang2, as has been demonstrated in the case of Ang1 (Witzenbichler et al., 1998); and (4) expression of kinase inactive c-Fyn attenuated Ang2-mediated tube formation, suggesting that c-Fyn is involved in both FGF-2- and Ang2-mediated tube formations of IBE cells.


    Materials and Methods
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 SUMMARY
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Antibodies
Anti-c-Fyn (FYN3), anti-c-Src (N-16), anti-Tie2 polyclonal antibodies and anti-phosphotyrosine monoclonal antibody (PY99) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-FLAG monoclonal antibody (M2) was purchased from Sigma (St Louis, MO). Anti-p85{alpha} PI 3-K polyclonal antibody, anti-phosphotyrosine monoclonal antibody (4G10) and anti-Erk 1/2 polyclonal antibody were purchased from Upstate Biotechnologies (Lake Placid, NY). Anti-phospho-MAPK (activated MAPK) rabbit polyclonal antibody was purchased from New England Biolabs (Beverly, MA).

Cell culture
IBE cells were routinely cultured in Ham’s F-12 medium supplemented with 20% heat-inactivated fetal bovine serum (FBS), 75 µg/ml endothelial cell growth supplement (Sigma), 5 µg/ml bovine pancreas insulin (Sigma) and 10 ng/ml human recombinant epidermal growth factor (Roche Diagnostics Inc. Tokyo, Japan), as described previously (Kanda et al., 1996). All experiments were performed at 33°C, because cells at 37 or 39°C did not proliferate, migrate or form tube-like structures following FGF-2-treatment (Kanda et al., 1996). Establishment of IBE cell lines stably expressing kinase-inactive c-Fyn (S. Tsuda et al., unpublished), wild-type or kinase inactive c-Fes (Kanda et al., 2000) and kinase-inactive c-Src (Shono et al., 2001a) are described elsewhere.

Cell proliferation assay
The cell proliferation assay was performed as described previously (Kanda et al., 2000). Briefly, cells suspended in Ham’s F-12 medium containing 0.25% (w/v) bovine serum albumin (BSA) were inoculated into wells of fibronectin-coated 24-well-culture plates and cultured in the presence or absence of recombinant human FGF-2 (Roche Diagnostics) and/or human recombinant Ang2 (Genzyme-Techne, Minneapolis, MN) for 3 days. Cell numbers were counted with a hemacytometer. The number of cells counted in untreated samples was set as 100%.

Chemotaxis assay
The chemotaxis assay was performed as described previously (Kanda et al., 2000). In brief, cells were suspended in Ham’s F-12 medium containing 0.25% BSA and seeded onto the upper surface of Transwell membrane filters (Corning Coster Japan, Tokyo), of which were pre-coated with fibronectin. In the lower wells, known concentrations of FGF-2 and/or Ang2 were added to the same medium. Four hours later, cells were fixed and stained, and cells attached onto the lower surface of membranes were counted microscopically. The number of cells counted in untreated samples was set as 100%.

Tube formation assay
The tube formation assay was performed as described previously (Kanda et al., 1996). Briefly, cells were suspended in Ham’s F-12 medium containing 0.25% in the presence or absence of FGF-2 and/or Ang2 at indicated concentrations and seeded onto the first layer of collagen gels. After 4 hours, medium was aspirated and cells were covered with the second layer of collagen gels. After gelation, Ham’s F-12 medium containing 0.25% BSA was added onto the second layer of collagen gels and cultured for 16 hours, and then photographed under a phase-contrast microscope.

Immunoprecipitation and immunoblotting
Methods for immunoprecipitation and immunoblotting were described elsewhere (Kanda et al., 2000) with some modifications. Cells were incubated with serum- and growth supplement-free medium for 16 hours. Medium was changed to the fresh serum- and growth supplement-free medium, then cells were further incubated for 2 hours. After 20 minutes treatment of cells with 0.1 mM orthovanadate for 20 minutes, cells were either stimulated or left unstimulated with Ang2 (1 µg/ml) for 15 minutes. Cells were rinsed with TBS containing vanadate and lysed in NP-40 lysis buffer (50 mM Hepes, pH 7.2, supplemented with 0.15 M NaCl, 10% glycerol, 10 mM pyrophosphate, 50 mM NaF, 1% NP-40, 100 U/ml aprotinin, 1 mM PMSF, 0.1 mM orthovanadate, 10 µM leupeptin and 10 µM pepstatin A). After centrifugation to remove insoluble materials, cell lysates were immunoprecipitated with the indicated antibodies, followed by adsorption to Protein A-agarose beads. For the specification of anti-Tie2 antibody, 1 µg of antibody was incubated with 50 µg of immunized peptide (Santa Cruz) at 4°C overnight, then used for the immunoprecipitation. After washing the beads, proteins were eluted by heating in SDS-sample buffer and then separated by SDS-polyacrylamide gel electrophoresis. After the transfer of proteins onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA), blots were probed with the indicated antibodies. Antibody incubation was followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG, and detection was via the enhanced chemiluminescence reaction (ECL, Amersham Life Science, UK). Between two probings, stripping of the membrane was performed as described before (Kanda et al., 2000).

In vitro kinase assay for receptor tyrosine kinase
The in vitro kinase assay for receptor tyrosine kinase was described previously (Kanda et al., 1996). IBE cells (15 cm dishes) were serum-starved overnight and either stimulated or left unstimulated with 2 µg/ml Ang2 for 15 minutes in the presence of 100 µM orthovanadate. Cells were washed, lysed in Triton X-100 lysis buffer, and centrifuged. Cleared lysates (90%) were then incubated with anti-Tie2 antibody and Tie2 was also immunoprecipitated from the remaining 10% of lysate to examine the amount of Tie2 by immunoblotting. Incubated proteins with antibodies were absorbed with Protein A agarose beads. Beads were washed thoroughly and the autophosphorylation assay was carried out in 20 mM Hepes, pH 7.4, supplemented with 10 mM MgCl2, 2 mM MnCl2, 0.05% Triton X-100, 1 mM DTT and 0.037 MBq/sample of [{gamma}-32P] ATP at 4°C for 10 minutes. The reaction was stopped by addition of 2x sample buffer, boiled, and eluted proteins were then separated by SDS-PAGE. Gels were incubated with destain for 30 minutes, fixed with 2.5% glutaraldehyde, rinsed with water, treated with 1 M KOH for 30 minutes at 55°C to remove phosphorylated serine residues, and then incubated with destain for 30 minutes at room temperature. Gels were dried and analyzed for radioactivity of particular proteins using a Bio Imager BAS 5000 (Fuji, Tokyo), followed by exposure on X-ray films.

In vitro kinase assay for Src family kinases
Serum-straved cells were either stimulated or not stimulated with Ang2 (1 µg/ml) for 15 minutes, then rinsed and lysed in NP-40 lysis buffer (Kanda et al., 2000); 10% of the lysate was mixed with SDS-sample buffer, then stored until required for immunoblotting to investigate the levels of proteins. Particular proteins were immunoprecipitated with the corresponding antibodies followed by incubation with Protein A-agarose beads with or without anti-mouse IgG rabbit IgG as a bridging antibody. Beads were washed four times with NP-40 lysis buffer, twice with TBS and twice with kinase buffer (25 mM Tris-HCl, pH 7.4, containing 10 mM MnCl2 and 2 mM MgCl2). Kinase buffer supplemented with 0.37 MBq/sample of [{gamma}-32P] ATP and 0.5 mg/sample of acid-denatured rabbit muscle enolase (Roche Diagnostics) was added to the beads and incubated for 10 minutes at room temperature. The reaction was stopped by addition of 2x sample buffer, boiled, and eluted proteins were then separated by SDS-PAGE. Gels were treated to remove phosphorylated serine with 1 M KOH as described above. Gels were dried and analyzed for radioactivity of particular proteins using a Bio Imager BAS 5000 (Fuji, Tokyo), followed by exposure on X-ray films.

In vitro PI 3-kinase assay
Details of the in vitro kinase assay for PI 3-K are described elsewhere (Kanda et al., 1997). In brief, serum-starved IBE cells were either stimulated or not stimulated with Ang2 (1 µg/ml) for 15 minutes, then rinsed and lysed in Nonidet P-40 (NP-40) lysis buffer. In indicated experiments, Ang2 (1 µg) was preincubated with extracellular domain of either Tie1/Fc chimera or Tie2/Fc chimera (25 µg) at room temperature for 10 minutes, then added to the cells. Clarified cell lysates were immunoprecipitated with indicated antibodies, followed by the absorption with Protain A-agarose beads. In the examination using anti-phosphotyrosine immunoprecipitates, cells were treated with 0.1 mM orthovanadate for 20 minutes, then either stimulated or left unstimulated with Ang2. Beads were extensively washed, suspended in kinase buffer containing phosphatidylinositol (Sigma) and 0.37 MBq/sample of [{gamma}-32P] ATP, and were incubated for 10 minutes. The reaction was stopped and reaction products were extracted with chloroform, and separated by thin layer chromatography on silica Gel-60 plates (Merck). Phosphorylated products were detected and incorporation of [{gamma}-32P] ATP was measured by Image Analyzer BAS 5000 (Fuji) and exposed on X-ray films (Amersham-Pharmacia BioTech).


    Results
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 SUMMARY
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Ang2 stimulates motility and tube-like structure formation by IBE cells.
Tie2 is exclusively expressed in endothelial cells. To examine whether Tie2 is expressed in IBE cells, immunoblot analysis was performed. Fig. 1A shows that single Tie2 band was visualized in immunoprecipitates with anti-Tie2 antibody and preincubation of the antibody with immunized peptide of Tie2 abolished the precipitation of the band. This result suggests that IBE cells expressed Tie2. The net molecular weight was about 135 kDa. A previous study demonstrated that there were at least four transcripts of Tie2 mRNA (Partanen et al., 1992). It is therefore possible that Tie2 in IBE cells was smaller than HUVECs. We then examined the tyrosine phsophorylation of Tie2 by Ang2 in IBE cells. Fig. 1B shows that Tie2 was autophosphorylated by Ang2, suggesting that Ang2 activated Tie2 tyrosine kinase in IBE cells.



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Fig. 1. Ang2 stimulates autophosphorylation of Tie2. (A) Expression of Tie2 on parental IBE cells was examined by immunoprecipitation with or without immunized peptide, followed by immunoblotting. About 135 kDa single protein was observed. (B) Tie2 was immunoprecipitated from cells (15 cm dishes) either stimulated or left unstimulated with 2 µg/ml Ang2 for 15 minutes and immune complex kinase assay was performed using 90% of cell extracts. Tie2 was immunoprecipitated followed by immunoblotting from the remaining 10% of cell extracts to examine whether a similar amount of Tie2 protein was present in the immune complex kinase assay.

 
The effects of Ang2 on proliferation, migration and formation of tube-like structures by IBE cells were examined. FGF-2 positively regulates these cellular responses of IBE cells (Kanda et al., 1996). Fig. 2A shows that FGF-2, but not Ang2, stimulated proliferation of IBE cells. Ang2 had no remarkable effect on FGF-2-mediated proliferation. By contrast, Ang2 stimulated chemotaxis (Fig. 2B). These results are consistent with the previous data obtained from Ang1-treated human endothelial cells (Witzenbichler et al., 1998). In addition, additive effect of Ang2 on chemotaxis toward FGF-2 was observed, suggesting that signal transduction pathways leading to chemotaxis of IBE cells toward FGF-2 and Ang2 might be different. The effect of Ang2 on formation of tube-like structure was also determined. Fig. 3 shows that Ang2 stimulated tube formation and no remarkable effect was observed in FGF-2-mediated tube-like structure formation. This result is able to suggest that certain signaling molecules may be commonly involved in the downstream of Tie2 and FGF receptor.



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Fig. 2. Ang2 induces chemotaxis, but not proliferation of IBE cells. (A) Cells were inoculated into fibronectin-coated wells and cultured in Ham’s F-12 medium containing 10% FBS. On the following day, medium was replaced with Ham’s F-12 medium containing 0.25% BSA with (closed bar) or without (open bar) 5 ng/ml FGF-2 in the presence or absence of Ang2 and the culture continued for 3 days. Data are expressed as means±s.d. for triplicated wells and similar results were obtained from two independent experiments. The number of cells counted in untreated samples was set as 100%. (B) Cells were suspended in Ham’s F-12 medium containing 0.25% BSA and seeded onto the upper surface of a fibronectin-coated Traswell membrane. In the lower wells, 50 ng/ml FGF-2 was added to the same medium (indicated as closed bars) in combination with Ang2. Four hours later, cells attached on the lower surface of the membranes were counted. Data are expressed as means±s.d. for quadruplicated wells and similar results were obtained from two independent experiments. The number of cells counted in untreated samples was set as 100%.

 


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Fig. 3. Ang2 induces tube-like structure formation by IBE cells. Cells with or without 10 ng/ml FGF-2 in the absence or presence of 1 µg/ml Ang2 were cultured between two layers of collagen gels. Representative data were obtained from two independent experiments. Magnification is 100x.

 
Ang2 activates a panel of intracellular protein tyrosine and lipid kinases
We then examined the downstream signaling molecules of Tie2, which were already determined in FGF-2-stimulated IBE cells. c-Src (Shono et al., 2001a) as well as c-Fes (Kanda et al., 2000) contributed to FGF-2-mediated motility by IBE cells. c-Fyn was involved in FGF-2-mediated tube formation (S. Tsuda et al., unpublished). The effects of Ang2 on these kinases were examined. c-Fyn and c-Src were activated by Ang2-treatment (Fig. 4A,B). Activation of c-Fes could be assessed by its autophosphorylation (Kanda et al., 2000). We then used the IBE cells expressing either C-terminally FLAG-tagged wild-type c-Fes (denoted FesWT6-8 cells) or kinase-inactive c-Fes (denoted FesKE5-8 cells), and examined Ang2-dependent autophosphorylation by immunoprecipitation followed by immunoblotting. Fig. 4C shows that Ang2 stimulated tyrosine phosphorylation of wild-type c-Fes, but not kinase-inactive c-Fes. These results indicate that c-Fyn, c-Src and c-Fes were possible downstream molecules of Tie2, regulating Ang2-mediated cellular responses.



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Fig. 4. Ang2 activates a panel of non-receptor protein tyrosine kinases. (A,B) IBE cells (6 cm dishes) were either stimulated or left unstimulated with 1 µg/ml Ang2 for 15 minutes and c-Fyn (A) or c-Src (B) was immunoprecipitated from 90% of lysate and used for in vitro kinase assay. Acid-denatured enolase was used as a substrate. The remaining 10% total lysate was examined by immunoblotting to determine the amount of loaded proteins. (C) IBE cells expressing FLAG-tagged wild-type c-Fes (FesWT6-8 cells; 10 cm dishes) were either stimulated or left unstimulated with 1 µg/ml Ang2 for 15 minutes and FLAG-tagged c-Fes was immunoprecipitated followed by imunoblotting. Between two probings, membranes were stripped. Representative data were obtained from two independent experiments.

 
PI 3-kinase is one of the important signaling molecules downstream of Tie2. FGF-2-mediated PI 3-kinase activation was extremely weak, because the activation was only active Ras-dependent (van Weering et al., 1998) and did not depend on the binding to tyrosine phosphorylated proteins, which further increased its catalytic activity (Kanda et al., 1997; Klippel et al., 1992; van Weering et al., 1998). By contrast, activation of PI 3-kinase via Tie2 seemed to involve the direct association of tyrosine phosphorylated Tie2 (Jones et al., 1999; Kontos et al., 1998). In IBE cells, Ang2 stimulated PI 3-kinase activity (Fig. 5A) and was increased in anti-phosphotyrosine immunoprecipitates (Fig. 5C). This result suggests that the association of p85 regulatory subunit with phosphotyrosine-containing proteins and/or tyrosine phosphorylation of p85 were stimulated by Ang2. Activation of PI 3-kinase in the series of these experiments was Ang2 specific, because preincubation of Ang2 with Tie2-Fc chimera, but not with Tie1-Fc chimera, remarkably reduced the PI 3-kinase activity (Fig. 5B). In contrast with the previous study (Jones et al., 1999), no increase in PI 3-kinase activity was observed in anti-Tie2 immunoprecipitates (Fig. 5D). In addition, we could not detect the coprecipitated Tie2 or p85 subunit of PI 3-kinase in immunoprecipitates of anti-p85 or anti-Tie2 (data not shown). These results suggest that Ang2-mediated PI 3-kinase activation in IBE cells was independent of the association of the p85 regulatory subunit of PI 3-kinase to Tie2. Endothelial cells exclusively express c-Fes (Smithgall et al., 1998), which is involved in interleukin-4-induced activation of PI 3-kinase (Izuhara et al., 1996), but not in FGF-2-induced activation (Kanda et al., 2000). Using FesKE5-8 and FesWT6-8 cells, PI 3-kinase activation by Ang2 was examined. Fig. 5E clearly shows the dominant-negative effect of kinase-inactive c-Fes on Ang2-mediated activation of PI 3-kinase. This result suggests that c-Fes regulates Ang2-mediated PI 3-kinase activation in IBE cells.



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Fig. 5. Ang2 activates PI 3-kinase in a manner depenedent on c-Fes. In vitro PI 3-kinase assay was performed in immunoprecipitates with anti-p85 subunit (A,B,E), anti-phosphotyrosine (PY99; C) or anti-Tie2 (D) antibodies. Phosphatidylinositol was separated by thin layer chromatography and phosphorylation of phosphatidylinositol was measured by the BioImager BAS 5000. The activity of phosphatidylinositol triphosphates (PIP) in untransfected cells was set as 1.00. (A,C,D) IBE cells (6 cm dishes) were either stimulated or left unstimulated with 1 µg/ml Ang2 for 15 minutes and PI 3-kinase activity was examined. (B) Ang2 (1 µg) was preincubated with extracellular domain of either Tie1/Fc chimera or Tie2/Fc chimera (25 µg) at room temperature for 10 minutes, then added to the cells. (E) IBE cells expressing either FLAG-tagged kinase-inactive c-Fes (FesKE5-8 cells) or FLAG-tagged wild-type c-Fes (FesWT6-8 cells) were either stimulated or left unstimulated with 1 µg/ml Ang2, and PI 3-kinase activity was examined. Representative data were obtained from two independent experiments.

 
Chemotaxis toward Ang2 involves PI 3-kinase
c-Src was involved in chemotaxis of IBE cells toward FGF-2 (Shono et al., 2001a) and c-Fyn showed no significant effect on FGF-2-induced motility (S. Tsuda et al., unpublished). In these studies, we established the stable cell lines expressing kinase-inactive c-Src and c-Fyn, respectively. Among these cell lines, cells expressing kinase-inactive c-Src or c-Fyn, denoted SrcKD-2 and FynKD-8 cells, respectively, were used to determine the specificity of the dominant-negative effect on each kinase activity by treatment of cells with PDGF-BB. PDGF-BB is a potential activator of Src family kinases and IBE cells express PDGFß receptor (Kanda et al., 1996). In SrcKD-2 cells, only c-Src activation was inhibited and c-Fyn was clearly activated. In FynKD-8 cells, c-Src activation was intact with the lack of c-Fyn activation (S. Tsuda et al., unpublished). These data clearly support the notion that the trans-dominant-negative effect of other Src kinases was negligible. We then examined the chemotaxis towards Ang2 by using these cell lines. However, these cell lines demonstrated no dominant-negative effect on Ang2-mediated motility (Fig. 6). PI 3-kinase is involved in Ang1-mediated migration of HUVECs. IBE cells were then treated with PI 3-kinase inhibitor LY294002 and the chemotaxis towards Ang2 was examined. Fig. 7A shows that LY294002 strongly inhibited Ang2-mediated motility. This result was consistent with the data obtained by the use of Ang1. FesKE5-8 cells, but not FesWT6-8 cells, showed impaired chemotaxis towards Ang2 (Fig. 7B). Together, these data show that the additive effect of FGF-2 and Ang2 on the motility of IBE cells is caused by the use of different signal transduction pathways, c-Src and PI 3-kinase, respectively.



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Fig. 6. Stable cell lines expressing either kinase-inactive c-Src (denoted SrcKD-2 cells) or c-Fyn (denoted FynKD-8 cells) migrate toward Ang2. Chemotaxis assay was performed as described for Fig. 2B. Data are expressed as means±s.d. for quadruplicated wells. Representative data were obtained from two independent experiments.

 


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Fig. 7. Ang2-mediated chemotaxis is PI 3-kinase-dependent. (A) IBE cells were preincubated with either 0.1% DMSO (vehicle) or 50 µM LY294002 for 30 minutes and assayed for chemotaxis as described for Fig. 2B. (B) FesKE5-8 cells and FesWT6-8 cells were examined for chemotaxis towards 1 µg/ml Ang2. Data are expressed as means±s.d. for quadruplicated wells. Representative data were obtained from two independent experiments.

 
Ang2-mediated tube formation was c-Fyn-dependent
The role of PI 3-kinase in Ang2-mediated tube formation was examined. However, neither the treatment with LY294002 nor expression of kinase-inactive c-Fes inhibited Ang2-mediated tube formation (Fig. 8). FGF-2-mediated tube formation was dependent on c-Fyn kinase activity, because treatment with Src family kinase inhibitor PP2 as well as expression of dominant-negative c-Fyn attenuated FGF-2-induced tube formation (S. Tsuda et al., unpublished). We then examined the role of c-Fyn in Ang2-mediated tube formation. Fig. 9A shows that kinase-inactive c-Fyn stably expressed in IBE cells (FynKD-8 cells) had a dominant-negative effect on Ang2-mediated activation of c-Fyn. We then examined tube formation by using this cell line. Fig. 9B shows that Ang2 could not induce tube formation by FynKD-8 cells. These results clearly show that c-Fyn was involved in Ang2-mediated tube formation and PI 3-kinase did not contribute to Ang2-mediated tube formation.



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Fig. 8. Ang2-mediated tube formation is not dependent on PI 3-kinase. Treatment of IBE cells with PI 3-kinase inhibitor LY294002 (A) or FesKE5-8 cells, which have a dominant-negative effect on PI 3-kinase activation by Ang2 (B) form tube-like structures in the presence of 1 µg/ml Ang2. Representative data were obtained from two independent experiments.

 


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Fig. 9. Ang2-mediated tube-like structure formation was c-Fyn dependent. IBE cells expressing kinase-inactive c-Fyn (K299M), denoted FynKD-8 cells, were examined by in vitro kinase assay to determine the dominant-negative effect on Ang2 (A). Phosphorylation of acid-denatured enolase was measured by the use of BAS5000 Image Analyzer. FynKD-8 cells were examined for their ability to form tube-like structures mediated by Ang2 as described for Fig. 3B. Representative data were obtained from two independent experiments.

 

    Discussion
 Top
 SUMMARY
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
In the present study we have demonstrated novel signaling molecules downstream of Tie2, c-Fes and c-Fyn. FGF-2-mediated motility of IBE cells depended on c-Fes and c-Src activity with a lack of attenuation by PI 3-kinase inhibitors (Kanda et al., 2000; Shono et al., 2001a). Ang2-mediated PI 3-kinase activation through c-Fes was similar to that observed downstream of the interleukin-4 receptor. Thus, Ang2-mediated PI 3-kinase activation gave an additive effect to increase the motility of FGF-2-treated cells. By contrast, c-Fyn was identified as a common molecule that regulates FGF-2- and Ang2-mediated formation of tube-like structures in capillary endothelial cells.

Tie2-mediated activation of PI 3-kinase in the previous reports have suggested the direct interaction of p85 subunit with phosphorylated Y1100 (Y1101) of Tie2 (Jones et al., 1999; Kontos et al., 1998). However, these reports demonstrated this interaction by the use of a yeast two-hybrid assay, peptide competition assay, pull down of particular proteins from unstimulated endothelial cell lysate with GST fusion proteins or coprecipitation of p85 with Tie2 in 293T cells cotransfected with Tie2 and p85. To date, the increase of PI 3-kinase activity has been demonstrated in anti-p85 immunoprecipitates (Kim et al., 2000), but not in anti-Tie2 immunoprecipitates from Ang-treated endothelial cells. It is therefore possible that Ang-treated endothelial cells may use yet unidentified pathways to activate PI 3-kinase. In the present study, no increase in association of PI 3-kinase activity with Tie2 by Ang2-treatment was detected. However, kinase-inactive c-Fes exhibited the dominant-negative effect on Ang2-mediated activation of PI 3-kinase (Fig. 5D). This distinct mechanism of activation of PI 3-kinase via Tie2 may be due to the difference of cell types used between the studies, because c-Fes is exclusively expressed in endothelial and hematopoietic cells. Downstream of interleukin 4 receptor, c-Fes was not fully, but partially, involved in PI 3-kinase activation (Izuhara et al., 1996). In addition, kinase-activity-dependent association of c-Fes with two SH2 domain-containing fragments of p85 regulatory subunit was observed in yeast two-hybrid system (T.N. and S.K, unpublished). c-Fes has a typical YXXM motif surrounding Y633, which is recognized by p85 regulatory subunit of PI 3-kinase (Smithgall et al., 1998). It is therefore possible that activated c-Fes might associate with the p85 subunit of PI 3-kinase in endothelial cells. However, expression of kinase-inactive c-Fes had a dominant-negative effect on FGF-2-mediated motility without any effects on PI 3-kinase activity (Kanda et al., 2000). In IBE cells, only activated Ras seemed to be involved in PI 3-kinase activation mediated by FGF-2 and binding of the p85 regulatory subunit to tyrosine phosphorylated proteins was not involved downstream of FGF-2-treated cells (Kanda et al., 1997) (Y.M. et al., unpublished). Taken together, these data show that Ang2 could activate PI 3-kinase through c-Fes, which in turn regulates the binding of PI 3-kinase to phosphotyrosine-containing proteins in endothelial cells in a manner different from FGF-2-treatment.

Recent studies of signal transduction pathways downstream of Tie2 and the effects of Ang1 as well as Ang2 on cellular responses were examined mainly by the use of HUVECs. Ang1 could not stimulate tube formation of HUVECs in 3D collagen gels (Papapetropoulos et al., 1999). However, Ang2 mediated tube formation of HUVECs in fibrin gels in association with Tie2 activation (Teichert-Kuliszewska et al., 2001). These studies clearly demonstrate that tube formation by HUVECs depends on the type of extracellular matrix protein. Modulation of particular integrin functions may be involved in this process. Figrin is one of the ligands for {alpha}vß3 integrin and inhibition of this integrin perturbed tube formation in fibrin gels (Bayless et al., 2000; Dallabrida et al., 2000). It is therefore possible that signals via Tie2 may modify the {alpha}vß3 integrin function. Interestingly, angiopoietin family molecules have a fibrinogen-like domain (Davis et al., 1996; Gale and Yancopoulos, 1999). It is also possible that angiopoietin may have an adhesive property, which can bridge cell-cell association involved in tube formation, especially in fibrin gels. By contrast, IBE cells can efficiently form lumen-containing tube-like structures in type I collagen gels; type I collagen is also a ligand of {alpha}vß3 integrin (Kanda et al., 1996). However, the inhibition of {alpha}vß3 integrin by cyclic peptide did not block tube formation in collagen gels (Kanda et al., 1999), but attenuated FGF-2-mediated motility on fibronectin (Shono et al., 2001b). The different cellular responses might be caused by the different kinds of integrin required for tube-like structure formation.

c-Src was activated by Ang2-treatment in IBE cells. However, no dominant-negative effect on Ang2-mediated cellular responses was observed. Conversely, another member of the Src kinase family, c-Fyn, was involved in Ang2-mediated tube formation. In addition, c-Fyn was shown to play a part in FGF-2-mediated tube formation (S. Tsuda et al., unpublished). However, the morphology of tube-like structure was different in FGF-2-treated and Ang2-treated cells. FGF-2-mediated activation of c-Fyn was observed only transiently and in the later phase of stimulation. The dominant-negative effect of kinase-inactive c-Fyn on FGF-2-induced tube formation demonstrated cell aggregates, but no cord-like, continuous morphology. By contrast, inhibition of Ang2-mediated tube formation resulted in the defect of aggregate formation. This difference in the dominant-negative effect of c-Fyn on tube formation in FGF-2- and Ang2-treatment seems to suggest that c-Fyn might regulate tube formation by more than one mechanism.

Many molecules are commonly expressed in endothelial cells and hematopoietic cells, because their origin is the same during development. However, hematopoietic cells do not form tube-like structures. Identification of the endothelial cell-specific signaling molecule downstream of c-Fyn and its inhibition would thus help in the development of new anti-angiogenic therapies.


    ACKNOWLEDGMENTS
 
We are grateful to Marilyn D. Resh for the kind gift of the expression plasmid containing kinase-inactive c-Fyn, Carl-Henrik Heldin for PAE cells, and Wataru Ogawa and Masato Kasuga for cDNA encoding the bovine p85{alpha} subunit of PI 3-kinase. We also thank T. Shimogama, M. Yoshimoto and members of the Nagasaki University Radioisotope Center for their excellent help. This study was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan.


    REFERENCES
 Top
 SUMMARY
 Introduction
 Materials and Methods
 Results
 Discussion
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