Receptor protein tyrosine phosphatase beta/zeta is a functional binding partner for vascular endothelial growth factor

It has been shown that phosphorylation of β₃ cytoplasmic Tyr 773 and 785 in response to VEGF₁₆₅ plays a role in endothelial cell migration [2]. In order to determine which of the two Tyr is responsible for VEGF₁₆₅-induced cell migration, we used CHO cells that express VEGFR2 (Figure 1A), RPTPβ/ζ and α_ν [8,11], but do not express β₃ and are mock-transfected or stably transfected to over-express wild-type β₃ or β₃ in which Tyr773 and/or Tyr785 are mutated to Phe [11]. VEGF₁₆₅ induced migration of CHO cells over-expressing wild type β₃ or β₃Y785F, but had no effect on CHO cells over-expressing β₃Y773F or β₃Y773F/Y785F (Figure 1B), suggesting that β₃ Tyr773 is important for VEGF₁₆₅-induced cell migration. In the same line and similarly to what we have recently shown for PTN [11], VEGF₁₆₅-induced cell surface NCL localization was only observed in CHO cells over-expressing wild type-β₃ or β₃Y785F, while in cells over-expressing β₃Y773F, NCL remained restricted in the cell nucleus, suggesting that β₃ Tyr773 but not Tyr785 phosphorylation is important for VEGF₁₆₅-induced cell surface NCL localization (Figure 1C). Since RPTPβ/ζ is involved in PTN-induced β₃ Tyr773 phosphorylation and cell surface NCL localization [8,11], these data lead to the hypothesis that RPTPβ/ζ may also be involved in VEGF₁₆₅-induced signaling that leads to endothelial cell migration.

Since RPTPβ/ζ is responsible for PTN-induced β₃ Tyr773 phosphorylation through dephosphorylation and activation of c-Src in human umbilical vein endothelial cells (HUVEC) [8], we examined whether RPTPβ/ζ also affects VEGF₁₆₅-induced c-Src activation and β₃ Tyr773 phosphorylation. Down-regulation of RPTPβ/ζ expression by two different siRNAs abolished VEGF₁₆₅-induced dephosphorylation of c-Src at Tyr530 (Figure 2A), suggesting that RPTPβ/ζ may be the missing link for c-Src activation upon stimulation of endothelial cells with VEGF₁₆₅. Indeed, the increase in dephosphorylated c-Src Tyr530 was mirrored by higher phosphorylation of c-Src at Tyr419 (Figure 2A), verifying activation of c-Src. Interestingly, inhibition of VEGFR2 tyrosine kinase activity by the selective inhibitor SU1498 or inhibition of VEGF-VEGFR2 interaction by bevacizumab, did not affect VEGF₁₆₅-induced c-Src Tyr530 dephosphorylation or c-Src Tyr419 phosphorylation (Additional file 1), suggesting that c-Src activation may be independent of VEGFR2 in these assays. RPTPβ/ζ directly interacts with c-Src [6], an observation that is in line with a role for RPTPβ/ζ in c-Src activation. Down-regulation of RPTPβ/ζ expression also abolished VEGF₁₆₅-induced β₃ Tyr773 phosphorylation (Figure 2B), suggesting that RPTPβ/ζ may be involved in VEGF₁₆₅-induced signaling related to cell surface NCL localization, as has been previously described for PTN [11]. Indeed, down-regulation of RPTPβ/ζ expression abolished VEGF₁₆₅-induced cell surface NCL localization (Figure 2C). Although it has been known for several years that VEGF₁₆₅ induces cell surface NCL localization [13], which is required for VEGF₁₆₅-induced cell migration [14], the receptor/pathway involved was unknown up to date. The observation that VEGF₁₆₅-induced cell surface NCL localization depends on RPTPβ/ζ suggests a role for RPTPβ/ζ in the angiogenic effects of VEGF₁₆₅.

To further investigate the involvement of RPTPβ/ζ in VEGF actions, we studied the role of several signaling molecules known to be activated by both VEGF₁₆₅ and RPTPβ/ζ on cell surface NCL localization. Inhibition of c-Src and phosphatidylinositol 3-kinase (PI3K) abolished VEGF₁₆₅-induced cell surface NCL localization, while inhibition of ERK1/2 had no effect (Figure 2D). In order to investigate whether PI3K lays up- or downstream of α_νβ_3, the effect of PI3K inhibition on VEGF₁₆₅-induced β₃ Tyr773 phosphorylation was studied. The c-Src inhibitor PP1 was used as a positive control, since c-Src is known to lay upstream of β₃ Tyr773 phosphorylation [2,8,11]. The p38 inhibitor (SB203580 10 μΜ, BioSource Europe, Nivelles, Belgium) was used as a negative control, since it has been previously shown not to affect RPTPβ/ζ-mediated β₃ Tyr773 phosphorylation and PI3K activation [11]. PI3K inhibition did not affect VEGF₁₆₅-induced β₃ Tyr773 phosphorylation (Figure 2E), suggesting that it lays downstream of α_νβ₃. By using an ELISA for activated PI3K in HUVEC, it was found that inhibition of c-Src abolished VEGF₁₆₅-induced PI3K activation (Figure 2F). Using the same assay in CHO cells over-expressing wild-type β₃, β₃Y773F or β₃Y785F, it was found that VEGF₁₆₅ significantly induced PI3K activation in CHO cells over-expressing wild-type β₃ or β₃Y785F, while it had no effect in cells over-expressing β₃Y773F (Figure 2G), suggesting that PI3K lays downstream of α_νβ₃ and requires β₃ Tyr773 phosphorylation. This pathway resembles the one we have recently shown for PTN-induced cell surface NCL localization [11], further supporting the notion that RPTPβ/ζ mediates this effect of VEGF₁₆₅. Similarly to what has been previously discussed for PTN, it remains unclear how PI3K affects cell surface NCL localization. Co-immunoprecipitation of NCL with PI3K [15,16] favors a direct regulation of NCL by PI3K and one possibility is by regulating the trafficking of exocytotic vesicles. NCL has been detected in cytoplasmic vesicles fused with the plasma membrane [17], while inhibition of PI3K interferes with the trafficking of such exocytotic vesicles, affecting the number of several receptors on the plasma membrane, such as the transferrin receptor [18], the glucose transporter GLUT4 [19] or β integrin [20]. Alternatively, PI3K may indirectly regulate cell surface NCL localization, by regulating recruitment of proteins containing pleckstrin homology domains onto the cell membrane [21]. NCL does not possess such sequences but may act as a ligand for proteins containing pleckstrin homology domains through its acidic motifs [22].

Based on the literature that c-Src–mediated phosphorylation of β₃ essentially regulates VEGF₁₆₅-induced interaction of α_νβ₃ with VEGFR2 in endothelial cells [2] and our observation that RPTPβ/ζ is required for VEGF₁₆₅-induced c-Src activation and β₃ Tyr773 phosphorylation (Figure 2), we tested the hypothesis that RPTPβ/ζ may have a role in the interaction of α_νβ₃ with VEGFR2. Down-regulation of RPTPβ/ζ expression by siRNA abolished the increased interaction of α_νβ₃ with VEGFR2 induced by VEGF₁₆₅, as evidenced by both immunoprecipitation/Western blot (Figure 3A) and proximity ligation assays (PLA) (Figure 3B). These data also show involvement of RPTPβ/ζ in VEGF-induced endothelial cell migration.

Since RPTPβ/ζ affects VEGF₁₆₅-induced signaling related to cell migration, we tested whether it also affects VEGF₁₆₅-induced endothelial cell migration. Down-regulation of RPTPβ/ζ expression by siRNA abolished VEGF₁₆₅-induced HUVEC migration (Figure 4), highlighting a role for RPTPβ/ζ in the angiogenic effects of VEGF₁₆₅.

It has been previously shown that RPTPβ/ζ interacts with both α_νβ₃ and NCL in HUVEC [8,11], both of which are involved in VEGF₁₆₅-induced endothelial cell migration [2,3,13,14]. Since VEGFR2 is also required for VEGF₁₆₅-induced endothelial cell migration [1], we tested whether RPTPβ/ζ interacts with VEGFR2. By performing immunoprecipitation/Western blot and PLA assays, we found that VEGFR2 does not associate with RPTPβ/ζ (Figure 5A). We then tested the possibility that VEGF directly associates with RPTPβ/ζ by performing PLA assays in HUVEC, which express endogenous levels of VEGF (Additional file 2). As shown in Figure 5B, endogenous VEGF formed complexes with RPTPβ/ζ, suggesting a direct interaction between the two molecules. Interestingly, addition of exogenous VEGF₁₆₅ to HUVEC, which led to increased VEGF immunostaining (Additional file 2), increased VEGF-RPTPβ/ζ PLA signals (Figure 5B), verifying the specificity of the signal. In these assays, the interaction of VEGF with VEGFR2 was used as positive control. Interaction of VEGF with RPTPβ/ζ was also observed in human glioblastoma U87MG cells, by using a combination of immunoprecipitation/Western blot, double immunofluorescence and PLA assays. These cells express higher levels of endogenous VEGF compared with HUVEC and the PLA signals showing interaction with RPTPβ/ζ were also higher than those observed in HUVEC (Additional file 2). Interestingly, VEGF was not found to interact with α_νβ₃ (Additional file 3).

Bevacizumab used at a concentration that inhibits VEGF-induced cell migration and binding to both VEGFR1 and VEGFR2 [23], did not affect interaction of VEGF with RPTPβ/ζ (Figure 5C), suggesting that this interaction involves a different region on the VEGF molecule than the VEGFR binding site. Supporting this notion, bevacizumab did not inhibit VEGF₁₆₅-induced cell surface NCL localization (Figure 5D), which is also mediated by RPTPβ/ζ as discussed above. Bevacizumab at the same concentration significantly inhibited interaction of VEGF with VEGFR2 (Additional file 4). These data clearly indicate that although binding to VEGFR2 is essential for VEGF-induced endothelial cell migration [2], some of the angiogenic actions of VEGF, such as cell surface NCL localization, are not inhibited by bevacizumab. Taking into account that angiogenic factors, such as PTN, hepatocyte growth factor and even VEGF itself act through cell surface NCL [12], the lack of effect of bevacizumab on VEGF₁₆₅-induced cell surface NCL localization may explain at least some of the cases of resistance development to bevacizumab, e.g. in glioblastomas, where classical VEGF signaling through VEGFRs has been found to remain inhibited [24]. Moreover, our data provide a mechanistic support to the notion that RPTPβ/ζ is a valuable target for glioblastoma therapies [25,26].

There are two transmembrane isoforms of RPTPβ/ζ, the long isoform that has been described as a CS proteoglycan, and the short isoform considered a glycoprotein. Up to date, it is still unclear which of the two RPTPβ/ζ isoforms are responsible for each of its actions and it is not known whether glycosylation of these isoforms differs among different types of cells. Efforts to identify its CS glycanation suggest that it is regulated both developmentally and in different pathophysiological situations [27] and our unpublished observations based on Western blot analyses of RPTPβ/ζ expressed in different cell types support the notion of a cell-type specific RPTPβ/ζ glycosylation. PTN binding to RPTPβ/ζ involves both low (K _d  = 3 nM) and high (K _d  = 0.25 nM) affinity binding sites [28], which represent more than one sites of interaction that involve both the protein core and the CS chains of the receptor [5]. Oversulfation of CS is essential for PTN affinity and PTN-mediated functions [29-32], which is in line with our observation that CS-E inhibits interaction of PTN with RPTPβ/ζ in both HUVEC and U87MG cells (Additional file 5). It should be noted, however, that although inhibition in U87MG cells was at the level of 70% (total signals per cell in control: 23 ± 7 and in CS-E treated cells: 8 ± 1), in HUVEC it was smaller, at the level of 50% (total signals per cell in control: 6 ± 0.4 and in CS-E treated cells: 3 ± 0.3).

Since VEGF has been shown to be able to bind to CS-E similarly to heparan sulphate, but does not bind CS-C or CS-A [33], we investigated whether CS-Ε also inhibits VEGF interaction with RPTPβ/ζ. CS-E decreased interaction of VEGF with RPTPβ/ζ in U87MG cells (Additional file 6), but had no effect on HUVEC (Figure 6A), in line with the observation that it did not affect VEGF₁₆₅-induced cell surface NCL localization (Figure 6B). This difference could be explained by the hypothesis that cell surface NCL localization depends on the interaction of VEGF with the short, non-proteoglycan isoform of RPTPβ/ζ. Interestingly and in favor of such a possibility, in U87MG cells that express higher amounts of endogenous VEGF than HUVEC, we performed IP/Western blot assays and observed that CS-E inhibits interaction of both PTN and VEGF with the long, but not the short RPTPβ/ζ isoform (Additional file 6). These data suggest that interaction of VEGF (and PTN) with the short RPTPβ/ζ isoform may not involve CS-E chains. On the other hand, CS-E abolished VEGF₁₆₅-induced HUVEC migration (Figure 6C), an effect that may involve a different cell surface molecule or parallel signaling pathways that are important for cell migration. This point is under further investigation.

The idea that the interaction of VEGF with RPTPβ/ζ does not involve its heparin-binding properties is further supported by the observation that VEGF₁₂₁, which induces HUVEC migration but does not contain the heparin-binding site of VEGF [34], also induces cell surface NCL localization (Figure 6D). Collectively, our data suggest that interaction of VEGF with RPTPβ/ζ may involve a part of the molecule that is distinct from those involved in VEGFR or glycosaminoglycan binding. This notion is similar to the recent finding that the high affinity neuropilin-1 binding of VEGF-A involves the exon 8-encoded C-terminal Arg [35]. Tuftsin, a naturally occurring TKPR peptide with sequence similarity to the sequence coded by exon 8 of VEGF, blocks VEGF₁₆₅-induced autophosphorylation of VEGFR2, without inhibiting VEGF binding to VEGFR2 [36]. Taking into account that PTN binding to RPTPβ/ζ involves PTN's carboxy terminal region that is rich in basic amino acids [37], and that PTN inhibits VEGF binding to RPTPβ/ζ (see below), one can speculate that binding of VEGF to RPTPβ/ζ might be mediated by its exon 8-encoded sequence.

Finally, we found that the interaction of VEGF with RPTPβ/ζ was decreased in the presence of PTN in both HUVEC (Figure 7A) and U87MG cells (Additional file 7), suggesting that these two growth factors share a common binding site on RPTPβ/ζ. This is further supported by the observation that the PTN-RPTPβ/ζ interaction was also decreased in the presence of exogenous VEGF₁₆₅ (Figure 7B). It has been previously suggested that besides the CS chains, the core RPTPβ/ζ protein is also involved in binding to PTN [28,38], which may also be the site of interaction of the short RPTPβ/ζ transmembrane isoform with VEGF, as discussed above.

PTN stimulates human endothelial cell migration by approximately 40% [6,8,11], an effect that is smaller than that of other growth factors, such as VEGF. Interestingly, PTN decreased VEGF₁₆₅-induced HUVEC migration to the levels of its own, smaller stimulatory effect (Figure 7C), in agreement with a previous study showing that PTN did not abolish but decreased VEGF₁₆₅-induced endothelial cell infiltration of matrigel in vivo to the levels of its own stimulation [39]. These data favor the notion that PTN and VEGF compete for binding to RPTPβ/ζ, as discussed above, and highlight a possible role for PTN as a regulator of angiogenesis, by limiting the aberrant effect of VEGF, while it still induces a smaller, significant stimulatory effect.

It is important to note that although RPTPβ/ζ is required for cell migration induced by PTN [6] and VEGF₁₆₅ (present study), it is not sufficient by itself to induce cell migration, based on our previous data showing that midkine [8] or PTN_112–136 [40], which lead to c-Src Tyr530 dephosphorylation and β₃ Τyr773 phosphorylation through RPTPβ/ζ, do not induce endothelial cell migration. It seems that other receptors/signaling pathways are activated in parallel with the RPTPβ/ζ/c-Src/α_νβ₃ pathway to complementarily induce cell migration. Such a pathway for VEGF₁₆₅ involves at least VEGFR2 and the cross-talk of signaling molecules activated by VEGF₁₆₅ through RPTPβ/ζ, α_νβ₃ and VEGFR2 or other cell surface binding molecules in different cell types is under further investigation.