Regulation of vascular endothelial growth factor receptor 2 trafficking and angiogenesis by Golgi localized t-SNARE syntaxin 6

Abstract

Vascular endothelial growth factor receptor 2 (VEGFR2) plays a key role in physiologic and pathologic angiogenesis. Plasma membrane (PM) levels of VEGFR2 are regulated by endocytosis and secretory transport through the Golgi apparatus. To date, the mechanism whereby the VEGFR2 traffics through the Golgi apparatus remains incompletely characterized. We show in human endothelial cells that binding of VEGF to the cell surface localized VEGFR2 stimulates exit of intracellular VEGFR2 from the Golgi apparatus. Brefeldin A treatment reduced the level of surface VEGFR2, confirming that VEGFR2 traffics through the Golgi apparatus en route to the PM. Mechanistically, we show that inhibition of syntaxin 6, a Golgi-localized target membrane-soluble N-ethylmaleimide attachment protein receptor (t-SNARE) protein, interferes with VEGFR2 trafficking to the PM and facilitates lysosomal degradation of the VEGFR2. In cell culture, inhibition of syntaxin 6 also reduced VEGF-induced cell proliferation, cell migration, and vascular tube formation. Furthermore, in a mouse ear model of angiogenesis, an inhibitory form of syntaxin 6 reduced VEGF-induced neovascularization and permeability. Our data demonstrate the importance of syntaxin 6 in the maintenance of cellular VEGFR2 levels, and suggest that the inhibitory form of syntaxin 6 has good potential as an antiangiogenic agent.

Figure 1

VEGF stimulates exit of VEGFR2 from the trans-Golgi complex. (A) Serum-starved HUVECs were labeled with mAb against VEGFR2 (55B11) and TGN46 (Golgi marker). Total cell-associated and Golgi-localized fluorescence intensity of VEGFR2 was quantified by image analysis. Values are expressed as a fraction of the total VEGFR2 in the Golgi apparatus. (B) Homogenates prepared from serum-starved HUVECs were fractionated on a self-generated Optiprep gradient (10%, 20%, 30%) and immunoblotted with antibodies against proteins enriched in the PM (vascular endothelial-Cadherin); the trans-Golgi complex (TGN46); or endosomes (EEA1). (C) Percentage of total VEGFR2 and TGN46 in each fraction, based on quantification of density of bands in each fraction obtained by Optiprep gradient centrifugation. (D) Effects of VEGF-A treatment on VEGFR2 localization at the Golgi apparatus. Serum-starved HUVECs were treated with CHX (10 μg/mL), and immunofluorescence imaging was carried out for VEGFR2 and TGN46 localization. (E) Quantification of the Golgi-localized VEGFR2 (overlapping with TGN46) shown in panel D. Values are expressed as a percentage of change in intensity of Golgi-localized VEGFR2 signal (relative to initial intensity in at 0 minutes chase at 37°C, data not shown). Percentages in panels A and E represent mean (± SD) in n = 90 cells for each condition from 5 separate experiments. For panel E, P ≤ .05. (F-G) Effects of BFA treatment on VEGFR2 transport in HUVECs. (F) Representative images of immunofluorescence analysis of untreated and BFA-treated cells stained with VEGFR2 antibody are shown. (G) Biotinylation-based analysis of cell-surface VEGFR2. Surface proteins labeled with the biotinylation reagent sulfo-NHS-SS–biotin were pulled down with streptavidin-Sepharose, and 5% of the total cell lysate and biotinylated cell-surface protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Western blot analysis with antibody against VEGFR2. (H) Quantification of band density for the cell-surface VEGFR2. Percentage is expressed as the change in surface VEGFR2 after BFA treatment (relative to initial levels). The percentage represents mean (± SD) for n = 3 and P ≤ .05. Scale bar represents 5 μm.

Figure 2

Inhibition of syntaxin 6 function decreases the levels of VEGFR2 but not the levels of VEGFR1. (A,D) Uninfected (Control) and syntaxin 6-cyto or syntaxin 16-cyto expressing HUVECs (after 20 hours of infection) were stained with mAb against VEGFR2 (55B11), syntaxin 6 (in Control and syntaxin 6-cyto–treated cells) or syntaxin 16 (in syntaxin 16-cyto–treated cells). Samples were then fixed and observed under fluorescence microscope. (B) HUVECs were subjected to siRNA-mediated syntaxin 6 or syntaxin 16 knockdown, and immunostained for VEGFR2. Representative images showing staining for intracellular VEGFR2 in cells in which endogenous syntaxin 6 or syntaxin 16 was knocked-down over 90% after 72 hours of siRNA treatment. (C) Samples were fixed and observed as in panel A, but stained with goat pAb against VEGFR1 and antibodies against syntaxin 6 or syntaxin 16. (D) Quantification of intracellular VEGFR2 or VEGFR1 in syntaxin 6-cyto and syntaxin 16-cyto expressing cells, and in cells in which endogenous syntaxin was knocked down by siRNA treatment (as in panels A-C). Epifluorescence images were acquired and total cell-associated fluorescence was quantified by image analysis. Values represent relative change in the levels of VEGFR2 or VEGFR1 normalized to an arbitrary value of 100 for untreated controls. Percentage is expressed as mean (± SD) of n = 90 cells for each condition from 3 separate experiments; P ≤ .001. (E) Lysates were prepared from uninfected, syntaxin 6-cyto– or syntaxin16-cyto–infected HUVECs (after infection for various periods of time, as indicated) and samples were immunoblotted for VEGFR2 (55B11) and VEGFR1 (rabbit polyclonal). Relative level of endogenous syntaxin 6, syntaxin 16, or tubulin in cell lysate is shown. (F) VEGFR2 and VEGFR1 band density from panel E was quantified and results represent relative levels of VEGFR2 and VEGFR1 after normalization to an arbitrary value of 100 for 0 minutes after infection. Percentage is expressed as mean (± SD) for n = 3; P ≤ .005). Scale bar represents 5 μm.

Figure 3

Inhibition of syntaxin 6 function targets VEGFR2 to the lysosomes for degradation. (A,B) Uninfected (Control) and syntaxin 6-cyto– or syntaxin 16-cyto–infected HUVECs (20 hours of infection) were metabolically labeled with ³⁵S-methionine–³⁵S-cysteine for 20 minutes. After the indicated chase period in an excess of unlabeled methionine-cysteine, the cells were lysed and total cell-associated precipitated. Radioactive counts were normalized before immunoprecipitation with VEGFR2 antibody (55B11). Autoradiographic signals from films were measured using Image J software Version 1.38x (NIH) and are represented as a percentage of the time 0 values for uninfected controls. Values are mean (± SD) for n = 3; P ≤ .05. (C,E) After 18 hours of infection, uninfected (Control) and syntaxin 6-cyto– or syntaxin 16-cyto–treated HUVECs were incubated for an additional 6 hours in complete medium (with 0.05% dimethyl sulfoxide [control], 100μM CHQ, 100nM Baf, or 5μM Lac). Samples were then costained with antibodies against VEGFR2, EEA1 (data not shown), or Lamp2 and observed by fluorescence microscopy. Fluorescence images were used to quantitate total cell-associated VEGFR2 (D) or the colocalization of VEGFR2 with EEA1 or Lamp2 (E). Values in panels D and E represent mean (± SD) for n = 80 cells for each condition from 5 separate experiments; P ≤ .05. Scale bar represents 5 μm.

Figure 4

Syntaxin 6 inhibition does not target endocytic pool of VEGFR2 for degradation. (A-E) Serum-starved uninfected (Control) and syntaxin 6-cyto or syntaxin 16-cyto infected HUVECs (20 hours of infection). (A-B) Cells were surface biotinylated as described for Figure 1F. Samples were then incubated at 16°C for 30 minutes to allow biotinylated VEGFR2 to accumulate in endosomes. The remaining surface biotin was removed by treatment with sodium 2-mercaptoethanesulfonate (MesNa) and quenching of MesNa with iodoacetic acid. These samples (0-minute chase time point) were further incubated at 37°C for the indicated periods. To determine the levels of intracellular biotinylated VEGFR2 after different chase periods, cells were again treated with MesNa to remove any additional biotinylated VEGFR2 that had recycled to the cell surface. Intracellular biotinylated VEGFR2 was pulled down by streptavidin-Sepharose as described for Figure 1F. Levels of intracellular biotinylated VEGFR2 were quantitated by immunoblotting for VEGFR2. Total cell lysates were also immunoblotted with the indicated antibodies to assess relative protein abundance. (B) Densitometric analysis of immunoblots in panel A. Values are expressed relative to 0-minute chase time point (normalized to 100%). Values are presented as mean (± SD) for n = 3, P ≤ .05. (C,D) HUVECs were labeled with an E-tagged anti-VEGFR2 antibody (ScFv), at 10°C for 30 minutes, and then incubated with VEGF for an additional 30-minutes. Cells were washed and imaged to assess ScFv-binding at the cell surface. Samples in which ScFv was bound to the cell surface were further incubated at 16°C for 30 minutes. The remaining surface-bound antibody was removed by washing with low pH buffer. These samples (0-minute chase time point) were subjected to chase at 37°C in the presence of VEGF. Cell surface–localized and internalized ScFv-VEGFR2 complexes were detected using an FITC-conjugated antibody against the E-tag. (D,E) Graphs represent quantitative measurements of cell surface–localized and total internalized VEGFR2, as determined by image analysis. Values in panel E are expressed as the percentage of total internalized ScFv fluorescence at the 0-minute chase time point. Values in panels D and E represent mean (± SD) for n = 90 cells for each condition from 3 separate experiments, P ≤ .005. Scale bar represents 5μm.

Figure 5

Inhibition of syntaxin 6 blocks VEGF-induced cell proliferation, migration and, morphogenesis in Matrigel. (A,E) Serum-starved HUVECs were left uninfected (Control) or were infected with syntaxin 6-cyto or syntaxin 16-cyto. (A) Cell proliferation assays were carried out using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Values are expressed as fold change relative to that in controls. (B) Wound healing in the presence or absence (data not shown) of VEGF as monitored by time-lapse imaging for a period of 24 hours. The wound edges are marked by a solid black line. (C) Quantification of the number of cells migrating into the wound. (D) Directional migration of HUVECs toward VEGF (100 ng/mL) by Boyden chamber assay, with VEGF present in the lower well. The number of migrating cells was normalized to that in unstimulated control cells. (E-F) Matrigel-based tube-formation assay was carried out with HUVECs in the presence of VEGF (100 ng/mL). The number of tubes per field was quantified. Data in panels A, C, D, and F are mean (± SD) from 4 independent experiments. P ≤ .001 in panel A, P ≤ .005 in panel C, and P ≤ .05 in panels D and F.

Figure 6

Expression of syntaxin 6-cyto in mice blocks VEGF₁₆₄-induced angiogenesis and permeability. (A,G) Mice in each group (all of which were Nu/Nu mice) were given 2 injections under anesthesia. The first set of injections (1st) was PBS, syntaxin 6-cyto, or syntaxin 16-cyto. The second (2nd) was PBS or Ad-VEGF₁₆₄, and given 2 days later at the site of first injection as shown in panel A. At 7 days after the first set of injections, animals were euthanized and the ears were either photographed or removed for further processing. (B) Western blot analysis of syntaxin 6-cyto and syntaxin 16-cyto expression in mouse ear lysates prepared 5 days after Ad-VEGF₁₆₄ treatment as described in panel A. (C) Quantification of VEGF₁₆₄ levels in ear extracts using the Quantikine Mouse VEGF immunoassay kit (R&D Systems). Values represent mean (± SD) for n = 10 ears, P ≤ .005). (D) Representative images showing gross appearance of angiogenesis in mock, syntaxin 6-cyto– or syntaxin 16-cyto–injected mouse ears, 5 days before Ad-VEGF₁₆₄ administration. (E,G) Ears were collected and processed for detailed histologic assessment. (E) Representative images showing staining of 1-μm Epon spur sections stained with Toludine blue. Vertical line marks extent of edema developed after different treatments. (F-G) Immunohistochemical staining of mouse ear sections stained with antibodies against CD31 (F) or VEGFR2 (G). Red arrowheads mark blood vessels developed after different treatments. (H,I) In an experiment similar to that described in panel C, mice treated 5 days before with Ad-VEGF₁₆₄, were injected with Evans blue dye. (H) Mouse ears were photographed 30 minutes after injection, and representative images are shown. (I) Quantification of dye extravasation, carried out in another group of mice that were euthanized. Ears were removed and Evans blue dye was extracted and measured spectrophotometrically. Quantitated values represent mean (± SD) for n = 10 ears per data point, P ≤ .03.