Transactivation of vascular endothelial growth factor (VEGF) receptor Flk-1/KDR is involved in sphingosine 1-phosphate-stimulated phosphorylation of Akt and endothelial nitric-oxide synthase (eNOS).

Sphingosine 1-phosphate (S1P) and vascular endothelial growth factor (VEGF) elicit numerous biological responses including cell survival, growth, migration, and differentiation in endothelial cells mediated by the endothelial differentiation gene, a family of G-protein-coupled receptors, and fetal liver kinase-1/kinase-insert domain-containing receptor (Flk-1/KDR), one of VEGF receptors, respectively. Recently, it was reported that S1P or VEGF treatment of endothelial cells leads to phosphorylation at Ser-1179 in bovine endothelial nitric oxide synthase (eNOS), and this phosphorylation is critical for eNOS activation. S1P stimulation of eNOS phosphorylation was shown to involve G(i) protein, phosphoinositide 3-kinase, and Akt. VEGF also activates eNOS through Flk-1/KDR, phosphoinositide 3-kinase, and Akt, which suggested that S1P and VEGF may share upstream signaling mediators. We now report that S1P treatment of bovine aortic endothelial cells acutely increases the tyrosine phosphorylation of Flk-1/KDR, similar to VEGF treatment. S1P-mediated phosphorylation of Flk-1/KDR, Akt, and eNOS were all inhibited by VEGF receptor tyrosine kinase inhibitors and by antisense Flk-1/KDR oligonucleotides. Our study suggests that S1P activation of eNOS involves G(i), calcium, and Src family kinase-dependent transactivation of Flk-1/KDR. These data are the first to establish a critical role of Flk-1/KDR in S1P-stimulated eNOS phosphorylation and activation.

Nitric oxide (NO) 1 produced by endothelial NO synthase (eNOS) has a crucial role in the regulation of vascular tone, vascular remodeling, and angiogenesis (1)(2)(3). Recent evidence has established the involvement of NO in vascular endothelial growth factor (VEGF)-induced angiogenesis (4,5). For example, eNOS inhibitors block VEGF-induced endothelial cells (EC) migration, proliferation, and tube formation in vitro as well as VEGF-induced angiogenesis in vivo. VEGF is known to stimulate phosphoinositide 3-kinase (PI3K) and Akt-dependent phosphorylation of eNOS, resulting in activation of eNOS and increased NO production (6 -8). In EC the predominant VEGF receptor that mediates eNOS phosphorylation is Flk-1/ KDR (9,10).
Recently, sphingosine 1-phosphate (S1P), a bioactive lipid released by activated platelets, has emerged as an important mediator of angiogenesis. S1P induces migration, proliferation, and cytoskeletal changes of EC by binding to the endothelial differentiation gene (EDG), a family of G-protein-coupled receptors (GPCR) (11)(12)(13)(14). In the EDG family, EDG-1, EDG-3, EDG-5, EDG-6, and EDG-8 work as S1P receptors (15,16). Among them, EDG-1 is the best studied receptor and plays a major role in EC and smooth muscle cell function. Activation of EDG-1 receptor triggers several signaling pathways via pertussis toxin (PTx)-sensitive G i protein. Although the signaling pathways activated by S1P have been extensively studied in several cell types, the precise signaling mechanism by which S1P elicits angiogenesis remains unclear. Recent studies revealed that S1P binding to EDG-1 receptor increases NO synthesis through the PI3K-Akt pathway in EC, similar to VEGF (17)(18)(19).
The similarity in S1P and VEGF activation of eNOS through PI3K and Akt suggested shared upstream signaling mediators. Transactivation of receptor tyrosine kinases such as epidermal growth factor (EGF) receptor, platelet derived growth factor (PDGF) receptor and insulin-like growth factor receptor in response to activation of many GPCR has been reported (20 -27). Transactivation of the EGF receptor by S1P has also been reported (28). We hypothesized that transactivation of a growth factor receptor may be involved in S1P-stimulated PI3K-Akt-eNOS phosphorylation and investigated the specific role of Flk-1/KDR.
Here we show that S1P signaling increases tyrosine phosphorylation of VEGF receptor Flk-1/KDR. Importantly, we demonstrate that inhibition of tyrosine kinase activity of Flk-1/KDR reduces S1P-stimulated Akt and eNOS phosphorylation. These results demonstrate a critical role of Flk-1/KDR in S1P-induced PI3K-Akt-eNOS activation.
Immunoprecipitation-Lysates containing equal amounts (300 g) of protein were incubated with antibodies overnight at 4°C. After incubation with protein A/G PLUS-agarose for 2 h, precipitates were washed four times with lysis buffer and then resuspended in SDS-PAGE sample buffer. After being heated at 100°C for 5 min, samples were separated by SDS-PAGE and transferred to nitrocellulose membrane.

Rapid Activation of the 230-kDa Protein (VEGF Receptor
Flk-1/KDR) by S1P-BAEC were treated with 1 M S1P for 0 -60 min, cell lysates prepared, SDS-PAGE performed, and Western blots analyzed for phosphotyrosine. Very rapid and transient phosphorylation of a 230-kDa protein band (peak at 2 min) was observed (Fig. 1A, upper panel, B). Based on its molecular weight and tyrosine phosphorylation we predicted that this band was the VEGF receptor Flk-1/KDR. We reprobed the membrane with anti-Flk-1/KDR antibody and detected the same molecular mass band as a highly glycosylated mature form of Flk-1/KDR. A 200-kDa protein was also detected by anti-Flk-1/KDR antibody (presumably a less glycosylated form of Flk-1/KDR), but this band was not phosphorylated. As reported previously by Igarashi et al. (18) and Morales-Ruiz et al. (19), both Akt and eNOS are time dependently phosphorylated in response to S1P (Fig. 1A, middle and lower panels, B). The peak of Akt phosphorylation was at 3 min, that of eNOS was set at 5 min, and later both phosphorylations returned to baseline by 60 min. As shown in Fig. 1C, the same molecular mass protein, Flk-1/KDR, was time-dependently tyrosine-phosphorylated in BAEC treated with VEGF. The 230-kDa protein, Akt, and eNOS are dose dependently phosphorylated in response to S1P (Fig. 1D). The EC 50 values for phosphorylation of the 230-kDa protein, Akt, and eNOS were ϳ30 nM.
Phosphorylation of Flk-1/KDR, Akt, and eNOS Were Inhibited by VEGF Receptor Tyrosine Kinase Inhibitors-Comparison of the time course for phosphorylation of eNOS, Akt, and Flk-1/KDR indicated that phosphorylation of Flk-1/KDR was faster than eNOS or Akt (Fig. 1B). This finding suggests that transactivation of Flk-1/KDR is "upstream" of the phosphorylation of Akt and eNOS. To access this mechanism, BAEC were pretreated with the VEGF receptor tyrosine kinase inhibitors, SU 1498 and VTKi. Both compounds inhibited phosphorylation of Flk-1/KDR, Akt, and eNOS in response to either S1P or VEGF (Fig. 3). Based on these results it seems likely that transactivation of Flk-1/KDR is required for phosphorylation of Akt and eNOS induced by S1P.
To determine if the transactivation of the EGF receptor family (ErbB family) tyrosine kinase contributes to S1P-mediated phosphorylation of Akt and eNOS, we used AG1478, an inhibitor of ErbB family kinases. AG1478 (1 M) did not inhibit phosphorylation of eNOS, Akt, or Flk-1/KDR in response to S1P or VEGF stimulation. S1P did not stimulate the release of VEGF since the VEGF neutralization antibody 577B11 inhibited VEGF-stimulated phosphorylation of Flk-1/KDR, Akt, and eNOS, but not S1P-stimulated phosphorylation of these molecules.
Suppression of the Flk-1/KDR Protein Expression by Antisense Oligonucleotides-To verify the results of inhibitor studies described above, BAEC were pretreated with antisense Flk-1/KDR oligonucleotides and stimulated with S1P or VEGF. Because of low transfection efficiency of the antisense oligonucleotides to BAEC, Flk-1/KDR protein expression was de-S1P/EDG-1 Transactivates Flk-1/KDR creased by about 50% similar to data as reported by Bernatchez et al. (30) (Fig. 4). The expression of Akt and eNOS did not change. After antisense oligonucleotides treatment, both S1Pand VEGF-stimulated phosphorylation of Flk-1/KDR, Akt, and eNOS were inhibited to a similar extent. There was no change in cells treated with scramble oligonucleotides. Thus, transactivation of Flk-1/KDR is required for phosphorylation of Akt and eNOS induced by S1P.

Transactivation of Flk-1/KDR is G i -, Ca 2ϩ -, and Src
Family Kinase-dependent-To characterize the signal transduction pathway from the EDG-1 receptor to Flk-1/KDR, we examined the effects of several inhibitors on the phosphorylation of Flk-1/KDR, Akt, and eNOS after stimulation by S1P or VEGF (Fig.  5). As reported previously (18,19), the G i inhibitor PTx blocked phosphorylation of Flk-1/KDR, Akt, and eNOS by S1P but had by arbitrarily setting the densitometry of control cells (time ϭ 0) to 1.0 (shown is the mean Ϯ S.E., n ϭ 3). C, time course of VEGF-stimulated phosphorylation of Flk-1/KDR. BAECs were stimulated by 10 ng/ml VEGF for indicated times and tyrosine phosphorylation of Flk-1/KDR was analyzed by Western blotting. D, dose dependence for S1P stimulation. BAEC were stimulated by indicated concentration of S1P for 3 min, and phosphorylation of the 230-kDa protein, Akt, and eNOS were analyzed by Western blotting. Blots are representative of three experiments. Results were normalized by arbitrarily setting the densitometry of control cells (S1P ϭ 0 nM) to 1.0 for each blot and the ratios of phosphotyrosine (pY) to Flk-1/KDR, phospho-Akt (p-Akt) to Akt and phospho-eNOS (p-eNOS) to eNOS were obtained. S1P/EDG-1 Transactivates Flk-1/KDR no effect on VEGF-stimulated phosphorylation. BAPTA/AM inhibited Flk-1/KDR phosphorylation by S1P but not by VEGF. This means that Ca 2ϩ is required for the transactivation from EDG-1 to Flk-1/KDR. The Src kinase inhibitor, PP2, also blocked S1P-stimulated phosphorylation of Flk-1/KDR, but as expected had no effect on VEGF-stimulated phosphorylation of Flk-1/KDR. Interestingly, BAPTA/AM and PP2 inhibited both S1P-and VEGF-stimulated phosphorylation of Akt and eNOS. The protein kinase C inhibitor, cherelythrine, had no effect on both S1P and VEGF-induced phosphorylation. Wortmannin, an inhibitor of PI3K, did not block the phosphorylation of Flk-1/KDR by S1P or VEGF, but inhibited Akt and eNOS phosphorylation.
Transactivation of Flk-1/KDR by S1P Does Not Depend on the Release of Endogenous Ligand for Flk-1/KDR-EGF receptor transactivation by several GPCR ligands involves activation of metalloproteases and release of heparin binding-EGF, an endogenous membrane binding ligand (32). Because VEGFneutralizing antibody did not block S1P-stimulated Flk-1/KDR transactivation (Fig. 3), release of an endogenous ligand appeared unlikely. As shown in Fig. 1C VEGF time dependently phosphorylated Flk-1/KDR, but the peak was 5 min, which is slower than S1P-stimulated phosphorylation (Fig. 1A). The rapid activation by S1P is also not consistent with release of an endogenous ligand. To rule out this mechanism, we pretreated BAEC with the metalloprotease inhibitors, o-phenanthroline and GM6001, and stimulated with S1P or VEGF. No effect was observed with these inhibitors (Fig. 6A).
Transactivation of Flk-1/KDR Is Not Reactive Oxygen Species (ROS)-dependent-EGF receptor transactivation by AngII was reported to be ROS-dependent (33). Therefore we examined the effects of various anti-oxidants NAC, Tiron, ebselen, and DPI on tyrosine phosphorylation of Flk-1/KDR by S1P (Fig. 6B). NAC, Tiron, and ebselen had no effect on S1P-stimulated phosphorylation of Flk-1/KDR, while DPI showed a partial inhibition. All four compounds showed no effect on VEGF-stimulated phosphorylation of Flk-1/KDR. Based on these findings, ROS appear not to be involved in S1P stimulated Flk-1/KDR transactivation. DISCUSSION The major finding of the present study is that S1P transactivates the VEGF receptor Flk-1/KDR leading to phosphorylation and stimulation of the PI3K-Akt-eNOS pathway in endothelial cells. Based on our experiments and previously reported data we summarize that S1P stimulated eNOS activation as follows. VEGF receptor Flk-1/KDR localizes to caveolae (34), while EDG-1 receptor exists in both non-caveolae and caveolae membranes. After stimulation EDG-1 translocates and concentrates in caveolae (35). Upon G i protein-mediated activation of phospholipase C, intracellular Ca 2ϩ levels increase and Ca 2ϩ  complexes with calmodulin (CaM). The Ca 2ϩ /CaM complex then activates eNOS (36). Simultaneously, Ca 2ϩ and Src family kinase-dependent transactivation of Flk-1/KDR occurs. Flk-1/ KDR then stimulates Src family kinase and PI3K causing Akt and eNOS (Ser-1179) to be phosphorylated and activated. We speculate that different molecules of Src family kinase transduce the signaling before and after Flk-1/KDR. Ca 2ϩ /CaM and Ser-1179 phosphorylation synergistically activate eNOS (9). In addition, heat shock protein 90 and other molecules are also likely to regulate eNOS activation (37,38). Further studies are needed to identify individual Src family kinases and other molecules involved in this pathway.
EGF receptor transactivation by GPCR ligands has been well studied. The best elucidated mechanism involves binding of ligand to GPCR, activation of matrix metalloproteases, cleavage of membrane binding heparin binding-EGF, and binding of heparin binding-EGF to the EGF receptor (26,27,32). However Flk-1/KDR transactivation by S1P involves a different pathway since matrix metalloproteases are not involved (see Figs. 3 and 5B). In fact, from comparison of Flk-1/KDR phosphorylation by S1P and VEGF, S1P-stimulated Flk-1/KDR-phosphorylation is faster than VEGF-stimulated (Fig. 1C). We found involvement of G i , Ca 2ϩ , and Src family kinase in this transactivation pathway, suggesting a requirement for intracellular signals and/or membrane trafficking. Of interest S1P-mediated transactivation appeared not to require ROS (Fig. 6B). In contrast, Ushio-Fukai et al. (33) reported that antioxidants completely inhibit AngII-stimulated transactivation of EGF receptor, and we found that transactivation of the PDGF receptor was also inhibited by antioxidants (24). Further studies are necessary to elucidate the nature of Flk-1/KDR transactivation mechanism.
Previously, Igarashi and Michel (39) reported S1P-stimulated phosphorylation of Akt and eNOS. They proposed that G␤␥ directly activated PI3K␤ and excluded tyrosine kinases in this pathway because genistein (10 M), a broad spectrum tyrosine kinase inhibitor, did not inhibit the S1P-stimulated PI3K␤ activity. We believe the differences in our data are due to the fact that genistein at 10 M cannot completely inhibit the tyrosine kinase activity of Flk-1/KDR and Src family kinases. Similar to our data, Rikitake et al. (40) showed that the Src family inhibitor, PP2, inhibited S1P-stimulated phosphorylation of Akt and eNOS. We are confident of the role of Flk-1/KDR since two specific Flk-1/KDR tyrosine kinase inhibitors, (VTKi and SU1498) with different chemical structures inhibited S1Pstimulated phosphorylation of Flk-1/KDR, Akt, and eNOS. In addition the antisense Flk-1/KDR oligonucleotides experiment confirms the requirement for Flk-1/KDR expression (Fig. 4).