Stromal Cell-derived Factor-1 (cid:1) Induces Tube-like Structure Formation of Endothelial Cells through Phosphoinositide 3-Kinase*

Stromal cell-derived factor-1 (cid:1) (SDF-1 (cid:1) ) is a CXC che-mokine, which induces tube formation of endothelial cells. Although SDF-1 (cid:1) transduces signals via CXC receptor 4 (CXCR4), resulting in activating a panel of downstream signaling molecules, such as phosphoinositide 3-kinase (PI3-kinase), little is known about the SDF-1 (cid:1) -mediated signaling pathways leading to tube formation. Here we examined the signal transduction pathway involved in SDF-1 (cid:1) -mediated tube formation by primary human umbilical endothelial cells and murine brain capillary endothelial cell line (IBE (im-mortalized murine brain capillary endothelial) cells). SDF-1 (cid:1) stimulated tube formation by IBE cells, which was blocked by LY294002 and pertussis toxin, suggesting that PI3-kinase and G i protein were involved in this process. SDF-1 also stimulated tube formation of human umbilical endothelial cells, and the response was LY294002-sensitive. SDF-1 (cid:1) activated PI3-kinase in IBE cells. In stable IBE cell lines expressing either the mutant p85 subunit of PI3-kinase (denoted (cid:2) Immune Complex PI3-Kinase Assay— The method used for determi- nation of PI3-kinase immunoprecipitates anti-phosphoty-rosine was described previously (25). In brief, serum-starved cells were lysed in Nonidet P-40 lysis buffer and incubated with anti-phosphoty-rosine antibody followed by absorption with protein A-agarose beads. After extensive washing, immunoprecipitates were incubated with phosphatidylinositol and [ (cid:2) - 32 P]ATP, and reaction products were sepa- rated by thin layer chromatography on silica gel 60 plates. Incorpora-tion of [ (cid:2) - 32 P]ATP into phosphatidylinositol was measured by Image Analyzer BAS 5000 (Fuji) followed by exposure on x-ray films (Amer-sham Biosciences). Immunoprecipitation and Immunoblotting— Serum- and growth fac-tor-starved IBE cells were either stimulated or left unstimulated with 500 ng/ml SDF-1 (cid:1) in the presence of orthovanadate (50 (cid:3) M ) for 10 min. c-Akt was immunoprecipitated with anti-Akt antibody followed by im- munoblotting with either anti-phospho-Akt or anti-Akt antibodies.

In the present study, we examined the signal transduction pathways leading to SDF-1␣-mediated tube-like structure formation of IBE cells and human umbilical cord vein endothelial cells (HUVECs). SDF-1␣ stimulated tube formation of both cells, and PI3-kinase inhibitor LY294002 blocked tube formation, suggesting that PI3-kinase might be involved in this process by these cells. PI3-kinase was activated by SDF-1␣-treatment in IBE cells, and the activation was dependent on c-Fes kinase activity. These results suggest that PI3-kinase is an important signaling molecule of SDF-1␣-induced tube-like structure formation of endothelial cells.

EXPERIMENTAL PROCEDURES
Reagents-Anti-phosphotyrosine (PY99) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rat monoclonal antibody against mouse vascular endothelial cadherin (VE-cadherin) was from Pharmingen. Mouse recombinant SDF-1␣ was obtained from R&D Systems (Minneapolis, MN). LY294002, PP2, PD98059, and pertussis toxin were from Calbiochem-Novabiochem and were dissolved in dimethyl sulfoxide (Me 2 SO) as a stock solution, except pertussis toxin, * This study was supported by a Japan Society for the Promotion of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Cell Culture-HUVECs and their culture medium were purchased from BioWhittaker, Inc. (Walkersville, MD) and cultured in endothelial cell basal medium supplemented with endothelial cell growth supplement, dexamethasone, fetal bovine serum, insulin, and epidermal growth factor as described in the protocol provided by the manufacturer. Parental IBE cells obtained from temperature-sensitive mutant SV40 large T transgenic mouse brain capillaries were cultured in Ham's F-12 medium containing fetal bovine serum, endothelial cell growth supplement, insulin, interferon-␥, and epidermal growth factor as has been described previously (17). Stable IBE cell lines expressing kinaseinactive (dominant negative) c-Fes (denoted KEFes 5-15 cells) were described elsewhere (20). A stable cell line expressing deleted mutant p85 PI3-kinase subunit, which does not interact with p110 catalytic subunit (22) (denoted ⌬p85-8 cells), was established, which demonstrated the dominant negative effect on epidermal growth factor-mediated PI3-kinase activation (23). Experiments using IBE cell lines were performed at 33°C rather than at 39°C because at the latter temperature, cells became senescent and lost responsiveness to extracellular stimuli (17).
Tube Formation Assay-For IBE cells, cells were cultured between two layers of type I collagen gels in Ham's F-12 medium containing 0.25% bovine serum albumin with or without indicated samples as described previously (17). For HUVECs, cells suspended in endothelial cell basal medium containing 0.5% fetal bovine serum were inoculated onto growth factor-reduced Matrigel (BD Biosciences) with or without indicated samples and cultured for 24 h as described previously (24). To quantify the length of newly formed tubes, three random phase-contrast photomicrographs (ϫ10 objectives) per well were taken, and the length of each tube was measured using NIH Image software (version 1.64). Tube length obtained from FGF-2-stimulated cells or SDF-1␣-FIG. 1. As shown in A, SDF-1␣ stimulates tube-like structure formation of IBE cells cultured between two layers of collagen gels. FGF-2 was used as a positive control. Tube length obtained from FGF-2-stimulated cells was set to 100. As shown in B, SDF-1␣-mediated tube formation is pertussis toxin-sensitive. IBE cells inoculated on collagen gels were incubated with 50 ng/ml of pertussis toxin for 1 h and then either stimulated or left unstimulated with 500 ng/ml SDF-1␣. Tube length obtained from SDF-1␣-stimulated cells was set to 100. As shown in C, SDF-1␣-mediated tube formation of IBE cells was PI3-kinase inhibitor LY294002-sensitive. Cells in the presence of PI3-kinase inhibitor LY294002 or vehicle (0.1% Me 2 SO (DMSO)) were seeded onto the first layer of collagen gels, and 1 h later, 500 ng/ml of SDF-1␣ was added. Tube length obtained from SDF-1␣-stimulated cells was set to 100. As shown in D, SDF-1␣ synergistically stimulates tube formation by IBE cells treated with either FGF-2 or Ang2. IBE cells were treated with FGF-2 or Ang2 in the presence (closed bars) or absence (open bars) of 500 ng/ml SDF-1␣ simultaneously. Tube length obtained from SDF-1␣-stimulated cells was set to 100. Data shown are reproduced from two to three experiments. Bar, 100 m. stimulated cells was set to 100 as described in the respective figure legends.
Immune Complex PI3-Kinase Assay-The method used for determination of PI3-kinase activity in immunoprecipitates of anti-phosphotyrosine was described previously (25). In brief, serum-starved cells were lysed in Nonidet P-40 lysis buffer and incubated with anti-phosphotyrosine antibody followed by absorption with protein A-agarose beads. After extensive washing, immunoprecipitates were incubated with phosphatidylinositol and [␥-32 P]ATP, and reaction products were separated by thin layer chromatography on silica gel 60 plates. Incorporation of [␥-32 P]ATP into phosphatidylinositol was measured by Image Analyzer BAS 5000 (Fuji) followed by exposure on x-ray films (Amersham Biosciences).
Immunoprecipitation and Immunoblotting-Serum-and growth factor-starved IBE cells were either stimulated or left unstimulated with 500 ng/ml SDF-1␣ in the presence of orthovanadate (50 M) for 10 min. c-Akt was immunoprecipitated with anti-Akt antibody followed by immunoblotting with either anti-phospho-Akt or anti-Akt antibodies.

RESULTS AND DISCUSSION
SDF-1␣ induces tube-like structure formation of primary human endothelial cells (10,11). To examine whether SDF-1␣ also stimulates tube formation of IBE cells, we tested the effect of SDF-1␣ on morphological changes of the cells. IBE cells respond to FGF-2 and angiopoietin 2 (Ang2) to form lumencontaining tube-like structures (18,19). As shown in Fig. 1A, FGF-2 induced multicellular aggregates of IBE cells followed by sprouting and fusion of aggregates, resulting in branching tube-like structures (26). SDF-1␣ induced the formation of the tube-like structures of IBE cells, which was similar to the effect of FGF-2. CXCR4 is a G protein-coupled receptor. We then examined the effect of pertussis toxin on SDF-1␣-induced tube formation. As shown in Fig. 1B, pertussis toxin at 50 ng/ml  HUVECs were serum-starved for 1 h and then stimulated with SDF-1␣ for 5 min. Particular proteins were immunoprecipitated with antiphosphotyrosine antibody. PI3-kinase activity in the immunoprecipitated proteins was measured as described under "Experimental Procedures." Relative phosphorylation was measured by the use of the BAS5000 BioImager (Fuji), and phosphorylation of phosphatidylinositol in untreated cells was set to 1.00. ori, start point; PIP, phosphatidylinositol 3-phosphate. As shown in B, SDF-1␣ mediated PI3-kinase activation depends on the c-Fes kinase activity and association with tyrosine phosphorylated proteins. KEFes 5-15 cells or ⌬p85-8 cells were serumstarved overnight. Cells were then either stimulated or left unstimulated with SDF-1␣, and PI3-kinase activity was measured. As shown in C, SDF-1␣ phosphorylates c-Akt at serine 473 in IBE cells but not in KEFes 5-15 cells. Cells were serum-starved for overnight and then either stimulated or left unstimulated with SDF-1␣ for 10 min. c-Akt was immunoprecipitated with anti-Akt antibody, and phosphorylated c-Akt was detected by anti-phospho-Akt antibody. Data shown are representative of two experiments. IB, immunoblots.

SDF-1␣-induced Tube Formation of Endothelial Cells
blocked SDF-1␣-induced tube formation, suggesting that G i protein was involved in this process. We then tested the effects of pharmacological inhibitors on SDF-1␣-induced tube formation. Neither Src family inhibitor PP2 nor mitogen-activated protein kinase/extracellular signal regulated kinase kinase inhibitor PD98059 inhibited SDF-1␣-induced tube formation of IBE cells (data not shown). Treatment of cells with PI3-kinase inhibitor LY294002 blocked SDF-1␣-induced tube formation (Fig. 1C). PI3-kinase inhibitor could not block FGF-2-and Ang2-mediated tube formation (19,25). FGF-2-and Ang2-mediated tube formation was dependent on c-Fyn kinase activity (19,21). Ang2 could not further stimulate FGF-2-induced tube formation (19), suggesting that Ang2 and FGF-2 utilized a common signaling pathway leading to tube formation, such as c-Fyn. To test whether SDF-1␣-induced tube formation requires different signaling pathways activated by FGF-2 and Ang2, we examined the additive effect of SDF-1␣ on FGF-2and Ang2-induced tube formation. As shown in Fig. 1D, SDF-1␣ further stimulated FGF-2-and Ang2-induced tube for-mation. These results suggest that SDF-1␣-induced tube formation of IBE cells may be dependent on PI3-kinase, which is not involved in FGF-2-and Ang2-mediated tube formation. We also examined the formation of tube-like structures by primary HUVECs on Matrigel. Fig. 2A shows that FGF-2 as well as SDF-1␣ induced the formation of tube-like structures. In addition, LY294002 markedly inhibited SDF-1␣-mediated formation of tube-like structures of HUVECs (Fig. 2B). LY294002 showed little effect on FGF-2-induced formation of tube-like structures of HUVECs. 2 Considered collectively, PI3-kinase may be involved commonly in SDF-1␣-mediated tube formation by endothelial cells from different origins.
We next examined the effect of SDF-1␣ on PI3-kinase activity. IBE cells were treated with either Me 2 SO (vehicle) or LY294002 for 30 min and then stimulated with SDF-1␣. Cells were lysed, and PI3-kinase activity in anti-phosphotyrosine immunoprecipitates was examined by thin layer chromatography. As shown in Fig. 3A, PI3-kinase activity in immunoprecipitates of anti-phosphotyrosine antibody from IBE cells was increased by SDF-1␣ treatment, suggesting that SDF-1␣ induced tyrosine phosphorylation of particular proteins followed 2 S. Kanda, unpublished observations. ⌬p85-8 cells (B). Indicated IBE cell lines were treated or untreated with 500 ng/ml of SDF-1␣ or FGF-2 (as a positive control) and cultured between two layers of collagen gels. Tube length obtained from FGF-2-stimulated cells was set to 100. Bar, 100 m. Data shown are representative of two experiments.

FIG. 5. As shown in
A, SDF-1␣ stimulates tight cell-cell contact (compaction) by IBE cells. Cells either treated with 500 ng/ml SDF-1␣ or left untreated were covered with the second layers of collagen gels. After 2 or 8 h, photographs were taken under a phase contrast microscopic observation. As shown in B, SDF-1␣-mediated tight cell-cell contact is not observed in cells treated with anti-VE-cadherin antibody. Cells were seeded on the first layer of collagen gels with either normal rat IgG or anti-VE cadherin antibody. One h later, cells were treated with SDF-1␣-at 500 ng/ml and cultured for 3 h. Culture medium was removed, and the cells were covered by the second layer of collagen gels. Photographs were taken after 8 h. As shown in C, SDF-1␣-induced tube formation is blocked by anti-VE-cadherin monoclonal antibody. IBE cells were seeded onto the first layer of collagen gels in the presence of either normal rat IgG or rat monoclonal antibody against mouse VE-cadherin at 100 g/ml. One 1 h later, SDF-1␣ was added, and after 3 h, cells were covered with the second layer of collagen gels. Tube length obtained from FGF-2-stimulated cells was set to 100.

SDF-1␣-induced Tube Formation of Endothelial Cells
by association with PI3-kinase. SDF-1␣ also increased the PI3kinase activity in anti-phosphotyrosine immunoprecipitates of HUVECs. c-Fes tyrosine kinase is exclusively expressed in endothelial cells and hematopoietic cells. Activation of c-Fes by extracellular stimuli requires oligomerization by its coiled-coil domain followed by autophosphorylation (27). c-Fes tyrosine kinase is expressed in both HUVECs and IBE cells (20). In a recent study, we showed that Ang2-mediated activation of PI3kinase was dependent on c-Fes (19). We then examined the effect of SDF-1␣ on PI3-kinase in KEFes 5-15 cells. As shown in Fig. 3B, SDF-1␣ could not elevate the PI3-kinase activity in anti-phosphotyrosine immunoprecipitates of KEFes 5-15 cells. This result suggests that SDF-1␣-mediated tyrosine phosphorylation of particular proteins, which subsequently associate with PI3-kinase, may be c-Fes-dependent. SDF-1␣ also failed to increase the PI3-kinase activity in ⌬p85-8 cells, suggesting that the association of the p110 catalytic subunit of PI3-kinase with tyrosine phosphorylated proteins was required in this reaction. c-Akt/protein kinase B is one of the important downstream targets of PI3-kinase and is involved in cell survival. We examined the activation of c-Akt by SDF-1␣. As shown in Fig. 3C, phosphorylation of c-Akt was increased by SDF-1␣treatment in IBE cells, and the increase was not observed in KEFes 5-15 cells. Considered collectively, these data suggest that SDF-1␣ utilized c-Fes, resulting in activation of PI3-kinase and c-Akt in IBE cells. We also examined the SDF-1␣mediated tube formation in KEFes 5-15 and ⌬p85-8 cells. As shown in Fig. 4, SDF-1␣ failed to stimulate tube formation in KEFes 5-15 cells as well as ⌬p85-8 cells. These results strongly suggest that SDF-1␣-mediated tube-like structure formation by endothelial cells requires PI3-kinase activation, possibly through c-Fes.
Although distinct methods are employed to assess the ability of cultured endothelial cells to form tube-like structures, common behavior involved in this process includes cell-cell contact. Endothelial cells contact each other to form cellular aggregates followed by lumen formation between aggregated cells (26, 28 -30). We then examined the effect of SDF-1␣ on endothelial cell-cell contact. When treated with SDF-1␣, IBE cells adhered to neighboring cells and formed compact aggregation at 8 h, which is characterized by the disappearance of borders between aggregated cells (Fig. 5A). This tight cell-cell contact, denoted cell compaction, is observed in cadherin-dependent cell-cell aggregation (31,32). Tight cell-cell contact was not observed in untreated IBE cells (Fig. 5A). As shown in Fig. 5B, cell compaction was inhibited by anti-VE-cadherin antibody, suggesting that VE-cadherin was required for cell compaction. Consequently, anti-mouse VE-cadherin antibody inhibited SDF-1␣-induced tube formation (Fig. 5C), suggesting that VEcadherin-mediated cell-cell contact may be involved in this process. As shown in Fig. 6, compaction was hardly observed in SDF-1␣-treated IBE cells in the presence of LY294002. Only loose cell-cell contact was observed in SDF-1␣-treated KEFes 5-15 cells and in ⌬p85-8 cells as well. These results suggest that SDF-1␣-induced tube formation may be regulated by PI3kinase-dependent tight cell-cell contact. Considered collectively, PI3-kinase and its downstream target molecules, such as c-Akt, seem to be required for SDF-1␣-induced tube formation. In fact, recent reports have demonstrated that c-Akt was involved in tube formation of endothelial cells (33)(34)(35). Although FGF-2 induced cell compaction of IBE cells as well, PI3-kinase inhibitor did not inhibit the cell compaction (data not shown). FGF-2 sufficiently promoted tube formation by KEFes 5-15 cells and ⌬p85-8 cells. FGF-2 activates PI3-kinase through activated Ras but not through binding to tyrosine phosphorylated proteins (23). Ang2 activated PI3-kinase through binding to tyrosine phosphorylated proteins, and the activation was dependent on c-Fes kinase activity (19). Ang2induced PI3-kinase activity was involved in chemotaxis of IBE cells. Treatment of cells with LY294002 did not inhibit Ang2induced tube formation (19), suggesting that PI3-kinase does not seem to be involved in this response. On the other hand, FGF-2 and Ang2 activated c-Fyn in IBE cells (19,21). Treatment of cells with PP2 or expression of kinase-inactive c-Fyn inhibited FGF-2-or Ang2-promoted tube formation of IBE cells, suggesting that signals through receptor tyrosine kinases leading to tube formation seem to require c-Fyn but not PI3-kinase (19,21). A previous study has shown that c-Fyn was activated by SDF-1␣ treatment in Jurkat T cells (15). However, we could not detect SDF-1␣-induced c-Fyn activation in IBE cells (data not shown). Additionally, PP2 failed to inhibit SDF-1␣-induced tube formation. Since induction of chemotaxis involves dissociation of cells, Ang2-mediated PI3-kinase activation would not be involved in cell compaction. Conversely, SDF-1␣-induced PI3-kinase activation was required for cell compaction. A number of protein kinases are activated by lipid products of PI3kinase (36,37). Depending on the culture condition (i.e. two dimensional culture for migration assay and three-dimensional culture for tube formation), qualitatively different signaling molecules may be accumulated into focal adhesion complexes or cell-cell contacts, where cross-talk between growth factor signaling and cell adhesion signaling exist. Therefore, PI3kinase activated by Ang2 or SDF-1␣ may regulate distinct sets of downstream signaling molecules. In conclusion, we have shown in the present study that SDF-1␣ induced tube formation of endothelial cells through c-Fes-dependent activation of PI3-kinase, and formation of cadherin-dependent tight cell-cell contact seemed to be involved in this process.