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First published online 15 February 2005
doi: 10.1242/jcs.01686

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Pigment epithelium-derived factor inhibits fibroblast-growth-factor-2-induced capillary morphogenesis of endothelial cells through Fyn

¹ Department of Molecular Microbiology and Immunology, Division of Endothelial Cell Biology, Nagasaki University Graduate School of Biomedical Science, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan
² Department of Urology, Nagasaki University Graduate School of Biomedical Science, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan
³ Department of Molecular Microbiology and Immunology, Division of Cytokine Signaling, Nagasaki University Graduate School of Biomedical Science, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan

View larger version (48K): [in a new window]   Fig. 1. (A) PEDF dose-dependently inhibits FGF-2-induced capillary morphogenesis of IBE cells. IBE cells were suspended in Ham's F-12 medium containing 0.25% BSA and seeded onto growth-factor-reduced Matrigel. Cells were cultured with or without 20 ng ml–1 FGF-2 in the presence or absence of PEDF at indicated concentrations for 24 hours. Photographs were taken under a phase-contrast microscope. Relative tube length was expressed as means±s.d. for three photographs. Bar, 100 µm. (B) PEDF inhibits FGF-2-induced capillary morphogenesis of HUVECs. HUVECs were suspended in EBM-2 containing 0.5% FBS and seeded onto Matrigel. Indicated samples were added to the cells and culture was continued for 24 hours. Photographs were taken under a phase-contrast microscope. Relative tube length was expressed as means±s.d. for three photographs. Bar, 100 µm. Reproducible results were obtained from two or three independent experiments.

View larger version (35K): [in a new window]   Fig. 2. PEDF fails to inhibit FGF-2-induced capillary morphogenesis of a stable IBE cell line expressing kinase-inactive Fes (KE5-15 cells). KE5-15 cells were suspended in Ham's F-12 medium containing 0.25% BSA and seeded onto Matrigel. Cells were culture with the indicated samples for 24 hours. Photographs were taken under a phase-contrast microscope. Relative tube length was expressed as means±s.d. for three photographs. Bar, 100 µm. Reproducible results were obtained from two independent experiments.

View larger version (44K): [in a new window]   Fig. 3. (A) PEDF increases the kinase activity of Fes in IBE cells. IBE cells expressing either FLAG-tagged wild-type (WT6-8 cells) or FLAG-tagged kinase-inactive (KE5-15 cells) Fes were suspended in Ham's F-12 medium containing 0.25% BSA and seeded onto Matrigel. Cells were cultured with or without 200 ng ml–1 PEDF for 2 hours and lysed, and FLAG-tagged Fes was immunoprecipitated from 90% of cell lysate, followed by an in-vitro kinase assay. Relative activity was estimated using an Image Analyzer BAS 5000 (Fuji). The remaining 10% of each lysate was assessed for the loaded amount of protein by immunoblotting. Reproducible results were obtained from two independent experiments. (B) PEDF inhibits FGF-2-induced activation of Fyn in IBE cells but not in KE5-15 cells. Cells were cultured on Matrigel in the presence or absence of the indicated samples for 2 hours. Cells were washed and lysed, and Fyn was immunoprecipitated from 90% of cell lysate, followed by an in-vitro kinase assay. Relative activity was estimated using an Image Analyzer BAS 5000. The remaining 10% of each lysate was assessed for the loaded amount of protein by immunoblotting. Reproducible results were obtained from two independent experiments. (C) PEDF inhibits Fyn activity in HUVECs. HUVECs were suspended in EBM-2 medium containing 0.1% BSA and seeded onto Matrigel in the presence or absence of the indicated samples. Fyn was immunoprecipitated from 90% of cell lysate and an in-vitro kinase assay was performed. The remaining 10% of each lysate was assessed for the loaded amount of protein by immunoblotting. (D) Anti-pY529-Src antibody recognizes the C-terminal tyrosine phosphorylated Fyn. Src immunoprecipitated from HUVECs or Fyn precipitated from IBE cells was incubated with or without recombinant CSK and proteins were separated by SDS-PAGE, followed by transfer onto PVDF membranes. Phosphorylation of inhibitory tyrosine residue at the C-terminus was examined by immunoblotting with anti-pY529 Src antibody. (E) PEDF phosphorylates the inhibitory tyrosine residue at the C-terminus of Fyn in IBE cells but not in KE5-15 cells. Cells were cultured on Matrigel in the presence or absence of 200 ng ml–1 PEDF for 2 hours. Cells were washed and lysed, and Fyn was immunoprecipitated. Tyrosine phosphorylation of Fyn was estimated by immunoblotting. Reproducible results were obtained from two independent experiments. (F) Wild-type Fes, but not kinase-inactive Fes, phosphorylates the C-terminal tyrosine residue of Fyn. FLAG-tagged Fes was immunoprecipitated from either WT6-8 cells or KE5-15 cells cultured on Matrigel in the presence or absence of 200 ng ml–1 PEDF and incubated with recombinant human Fyn (rFyn) in the presence of ATP. As a positive control, rFyn was incubated with CSK. Phosphorylation of terminal tyrosine residue of rFyn was examined by immunoblotting.

View larger version (41K): [in a new window]   Fig. 4. Intracellular localization of Fyn, FLAG-tagged Fes and CSK in IBE cells. (A) Localization of Fyn is modulated by PEDF treatment in cells expressing wild-type Fes (WT6-8 cells) but not in cells expressing kinase-inactive Fes (KE5-15 cells). WT6-8 cells or KE5-15 cells were cultured on Matrigel-coated cover slips in the presence or absence of PEDF. FLAG-tagged Fes and endogenous Fyn were double stained by indirect immunofluorescence. Arrowheads indicate the Fyn located at the cell periphery including the tip of protrusions and focal adhesions. (B) Expression of CSK is diffusely observed in the cytoplasm and is not modulated by PEDF treatment and CSK does not localize to focal adhesions in IBE cells. Cells were cultured on Matrigel-coated cover slips in the presence or absence of PEDF. FLAG-tagged Fes and endogenous CSK were double stained by indirect immunofluorescence.

View larger version (47K): [in a new window]   Fig. 5. Expression of constitutively active Fyn in IBE cells restores the PEDF-mediated inhibition of capillary morphogenesis. (A) Two stable clones, CAFyn-6 and CAFyn-12, ectopically express Myc-tagged constitutively active Fyn. Empty-vector-transfected cells (Mock cells) were used as a negative control. (B) CAFyn-6 and CAFyn-12 cells cause FGF-2-independent capillary morphogenesis and its morphology is resistant to PEDF treatment. Cells were seeded onto Matrigel and cultured for 24 hours in the presence or absence of the indicated samples. Photographs were taken under a phase-contrast microscope. Relative tube length was expressed as means±s.d. for three photographs. Bar, 100 µm. Reproducible results were obtained from two or three independent experiments.

View larger version (42K): [in a new window]   Fig. 6. FGF-2-induces tyrosine phosphorylation of p190RhoGAP, which is inhibited by PEDF. IBE cells or KDFyn-8 cells were cultured on Matrigel in the presence or absence of 20 ng ml–1 FGF-2 with or without 200 ng ml–1 PEDF for 2 hours. Cells were washed and lysed, and p190RhoGAP was immunoprecipitated. Tyrosine phosphorylation was examined by immunoblotting.