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First published online 7 October 2008
doi: 10.1242/jcs.031898

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The protein tyrosine phosphatase TCPTP controls VEGFR2 signalling

¹ VTT Technical Research Centre of Finland, Medical Biotechnology, Turku FIN-20520, Finland
² Centre for Biotechnology, Turku University, Turku FIN-20520, Finland
³ MediCity Research Laboratory, Turku University, Turku FIN-20520, Finland

View larger version (57K): [in this window] [in a new window]   Fig. 1. Human endothelial cells express TCPTP in vitro and in vivo. (A) Immunoblotting of TCPTP, 1 and β1 integrins, and VEGFRs from HUVEC lysates. HeLa cell lysate is a positive control for expression of integrins and TCPTP, and tubulin blots serve as loading controls. Shown is a representative blot out of three, which each had similar results. (B) Immunoblotting of HUVECs and peripheral blood mononuclear cells (PBMCs, mainly consisting of T lymphocytes) isolated from different donors (numbers above the lanes) for TCPTP. (C) HUVECs grown on coverslips were stained for TCPTP in the absence (control) or presence of recombinant TCPTP (TC45). Note the specific TCPTP expression in the cytoplasm and membrane of the control HUVECs, which is competed out by pre-incubation of the antibody with the soluble target antigen. Scale bars: 10 µm. (D) Two-colour immunofluorescence stainings of human umbilical cords for TCPTP, 1 integrin and VEGFR2, and control IgGs. Individual channels and merged images with the indicated antibodies from an umbilical vein are shown. DAPI stainings reveal the nuclei of the endothelial lining. Scale bars: 20 µm. E, endothelial lining; L, lumen.

View larger version (18K): [in this window] [in a new window]   Fig. 2. TCPTP activity controls VEGF-dependent responses in endothelial cells. (A) Silencing of TCPTP enhances the proliferation of HUVECs. HUVECs were transfected with no oligo (`–' on the blot), siRNA against TCPTP or a control siRNA (Scr), and TCPTP expression was measured using immunoblotting (inset). Cells were treated with 80 ng/ml VEGF and proliferation of sparse cells that were plated on gelatin was assayed after 16 hours using a BrdU-based assay (mean ± s.d.; n=4-6; *P<0.05). (B) Activation of TCPTP decreases chemotaxis of HUVECs. Subconfluent HUVECs were transfected with the constitutively active TCPTP (TC37) or vector control (pCG) and chemotaxis towards VEGF was measured using a Transwell assay for 4 hours. Cells adhering to the bottom of the filter were fixed and stained with crystal violet (micrographs). The number of migrated cells was counted from four randomly selected fields of view in three parallel wells (mean ± s.e.m.; n=3; **P<0.01). The western blot shows expression of TC37 in the transfected cells (also detected is the 45-kDa endogenous TCPTP).

View larger version (38K): [in this window] [in a new window]   Fig. 3. TCPTP binds to VEGFR2. (A) HEK293 cells transiently transfected with VEGFR2 and a substrate-trapping mutant of TCPTP (TC45-D182A) were treated with VEGF (100 ng/ml, 5 minutes). Cell lysates were immunoprecipitated (IP) with anti-TCPTP or control (IgG) antibodies and blotted for VEGFR2 and TCPTP as indicated. Aliquots of the lysates were also analyzed to control for equal protein expression. (B) HEK293 cells transfected as in A, or an untransfected control, were treated with VEGF or left untreated. Immunoprecipitations were performed with anti-VEGFR2 or control (IgG) antibodies and blotted as indicated. The co-immunoprecipitations were done in the presence or absence of 100 µM sodium orthovanadate (Na3VO4) as indicated. Note the disappearance of the co-immunoprecipitated TCPTP when sodium orthovanadate is present.

View larger version (57K): [in this window] [in a new window]   Fig. 4. TCPTP dephosphorylates VEGFR2 in a phosphosite-specific manner and controls its activity. (A,B) HEK293 cells were transfected with VEGFR2, treated with VEGF and immunoprecipitated (IP) with anti-VEGFR2 antibody. Immunoprecipitates were incubated in the presence of recombinant TCPTP (TC45) or buffer control, and immunoblotted using total phosphotyrosine antibody (A) or phospho-specific VEGFR2 antibodies (B). Total VEGFR2 was blotted as a control. Representative blots out of three experiments with similar results are shown. The values under the blots represent VEGFR2 phosphorylation relative to the buffer controls (mean ± s.e.m.; n=3). (C,D) TCPTP activity controls the internalization of VEGFR2 in endothelial cells. (C) Subconfluent HUVECs were transfected with vector control (pCG) or constitutively active TCPTP (TC37), stimulated (+) or not (–) with VEGF, and stained for VEGFR2 (red) and nuclei (blue) after fixation and permeabilization. Arrows indicate representative VEGFR2 vesicles. (C, bottom) The number of VEGFR2-positive vesicles (mean ± s.e.m.; ***P<0.005) was quantitated using image analysis. The perinuclear bright Golgi-resembling staining was excluded in the analysis. Scale bar: 10 µm. (D) HUVECs were transfected with TC37, TC45 or vector (pCG), or left untransfected, and surface-labelled with cleavable biotin. VEGFR2 was allowed to internalize for 15 minutes in the presence or absence of VEGF (100 ng/ml). Biotin remaining on the cell surface was cleaved and VEGFR2 was immunoprecipitated using anti-VEGFR2 antibody. Internalized VEGFR2 was detected by blotting for biotin. Because biotin is cleavable by reducing reagents, a non-reducing gel was used, and this alters the mobility of the proteins compared to a reducing gel. The numbers below the blot show levels of internalized VEGFR2 (mean ± s.d., two individual experiments).

View larger version (53K): [in this window] [in a new window]   Fig. 5. Binding of HUVECs to collagen via integrin 1 activates TCPTP and attenuates VEGFR2 signalling in endothelial cells. (A) Confocal-microscopy images from three-colour immunofluorescence stainings show the localization of integrin 1β1, vinculin and TCPTP in membrane protrusions in HUVECs adhering to collagen IV but not to gelatin. Scale bars: 10 µm. (B) Phosphatase activities (mean ± s.d.; n=3) were measured in control (IgG), TCPTP and SHP2 immunoprecipitates from HUVECs grown on collagen I or gelatin. The immunoprecipitates were also resolved on SDS-PAGE gels and probed for SHP2 and TCPTP. The phosphatase activities of TCPTP and SHP2 were normalized to the amounts of immunoprecipitated (IP) molecules. A representative experiment out of three with similar results is shown (mean ± s.e.m.; n=9; *P<0.05; ***P<0.005; n.s., not significant). (C) Serum-starved subconfluent HUVECs were treated for 1 hour with the integrin-1 cytoplasmic-tail fusion peptide (1-TAT, 200 nM) followed by VEGF-induction (100 ng/ml, 15 minutes) as indicated. Cell lysates were resolved on SDS-PAGE and immunoblotted for phosphorylated VEGFR2 (or tubulin as a control). (D) HUVECs were treated as in C and VEGFR2 kinase activity (mean ± s.d.; n=3) was measured from the cell lysates. **P<0.01. (E) Recombinant, purified TCPTP was incubated with no peptide (–), 1 or 2 cytoplasmic tail peptides (L amino acids), or with 1 and scrambled TAT peptides (D amino acids) and analyzed for phosphatase activity (mean ± s.d., n=3). **P<0.01.

View larger version (31K): [in this window] [in a new window]   Fig. 6. Controlled TCPTP activation inhibits VEGF-driven morphological changes, chemokinesis and chemotaxis. (A) HUVECs were cultured in fibrin gels and treated with or without VEGF and TAT peptides (200 nM). After 24 hours, the changes in endothelial morphology and alignment were semi-quantitatively scored. Representative images show HUVECs at the beginning of the assay and untreated (control, ctrl) HUVECs without and with VEGF stimulation after 24 hours of incubation taken at 20x magnification. The graph shows the effects of the indicated treatments on the morphology index. **P<0.01. (B) HUVECs were plated on gelatin in the presence of the peptides (200 nM) or left untreated for 1 hour. The cells were stimulated as indicated and the migration of individual cells was tracked using time-lapse microscopy. Cumulative migration distance was plotted for randomly picked individual cells (B, top) (mean ± s.e.m.; n=40 cells; ***P<0.0005; B, bottom). (C) HUVECs were treated with the 200 nM peptides and the chemotaxis assay was performed as in Fig. 2B. *P<0.05. (D, top) HUVECs were transfected with no oligo, with siRNA against TCPTP or with a control siRNA (ScrsiRNA), and TCPTP silencing was analyzed by immunofluorescence. The same exposures of TCPTP (green) and DAPI (blue) stainings at 63x magnification are shown. Scale bars: 10 µm. (D, bottom) 48 hours post-transfection, cells were subjected to chemotaxis assay as in C.

View larger version (55K): [in this window] [in a new window]   Fig. 7. Controlled TCPTP activation inhibits VEGF-driven sprouting angiogenesis. (A,B) HUVEC spheroids embedded in three-dimensional collagen gel were treated with VEGF (20 ng/ml) and 400 nM TAT peptides as indicated. Spheroids were analyzed after 24 hours at 10x magnification (A) and the cumulative sprout length (A, bottom) and the number of sprouts per spheroid (B) were quantified using image analysis. Shown is a representative of two similar experiments (mean ± s.e.m., n=5 spheroids per treatment). (C) PAE spheroids embedded in three-dimensional collagen gel were treated with VEGF (20 ng/ml) and 400 nM TAT peptides as indicated. Spheroids were analyzed after 24 hours at 10x magnification and the cumulative sprout length was quantified using image analysis. Shown is a representative of two similar experiments (mean ± s.e.m.; n=5 spheroids per treatment).

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