Abstract
BackgroundReceptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) is a chondroitin sulphate (CS) transmembrane protein tyrosine phosphatase and is a receptor for pleiotrophin (PTN). RPTPβ/ζ interacts with ανβ3 on the cell surface and upon binding of PTN leads to c-Src dephosphorylation at Tyr530, β3 Tyr773 phosphorylation, cell surface nucleolin (NCL) localization and stimulation of cell migration. c-Src-mediated β3 Tyr773 phosphorylation is also observed after vascular endothelial growth factor 165 (VEGF165) stimulation of endothelial cells and is essential for VEGF receptor type 2 (VEGFR2) - ανβ3 integrin association and subsequent signaling. In the present work, we studied whether RPTPβ/ζ mediates angiogenic actions of VEGF.MethodsHuman umbilical vein endothelial, human glioma U87MG and stably transfected Chinese hamster ovary cells expressing different β3 subunits were used. Protein-protein interactions were studied by a combination of immunoprecipitation/Western blot, immunofluorescence and proximity ligation assays, properly quantified as needed. RPTPβ/ζ expression was down-regulated using small interference RNA technology. Migration assays were performed in 24-well microchemotaxis chambers, using uncoated polycarbonate membranes with 8 μm pores.ResultsRPTPβ/ζ mediates VEGF165-induced c-Src-dependent β3 Tyr773 phosphorylation, which is required for VEGFR2-ανβ3 interaction and the downstream activation of phosphatidylinositol 3-kinase (PI3K) and cell surface NCL localization. RPTPβ/ζ directly interacts with VEGF165, and this interaction is not affected by bevacizumab, while it is interrupted by both CS-E and PTN. Down-regulation of RPTPβ/ζ by siRNA or administration of exogenous CS-E abolishes VEGF165-induced endothelial cell migration, while PTN inhibits the migratory effect of VEGF165 to the levels of its own effect.ConclusionsThese data identify RPTPβ/ζ as a cell membrane binding partner for VEGF that regulates angiogenic functions of endothelial cells and suggest that it warrants further validation as a potential target for development of additive or alternative anti-VEGF therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0287-3) contains supplementary material, which is available to authorized users.
Highlights
Receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) is a chondroitin sulphate (CS) transmembrane protein tyrosine phosphatase and is a receptor for pleiotrophin (PTN)
In order to determine which of the two Tyr is responsible for vascular endothelial growth factor 165 (VEGF165)-induced cell migration, we used CHO cells that express VEGF receptor type 2 (VEGFR2) (Figure 1A), RPTPβ/ζ and αν [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]
In the same line and to what we have recently shown for PTN [11], VEGF165-induced cell surface NCL localization was only observed in CHO cells overexpressing wild type-β3 or β3Y785F, while in cells overexpressing 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)
Summary
Materials Human recombinant VEGF165 was prepared as previously described [39]. VEGF121 was purchased from RELIATech GmbH (Wolfenbüttel, Germany). Serum starved CHO cells or HUVEC were treated with 10 ng/ ml VEGF165 for 10 min (in the presence or absence of inhibitors of signaling molecules where appropriate), fixed and incubated with anti–phospho and anti-total p85 antibodies. Formation of PTN-RPTPβ/ζ complexes as evidenced by in situ PLA in HUVEC (A) and U87MG cells (B) in the absence or presence of CS-E II (100 ng/ml).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
