Kanda Shigeru, Mochizuki Yasushi, Nakamura Takao, Miyata Yasuyoshi, Matsuyama Toshifumi, Kanetake Hiroshi
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.
J Cell Sci. 2005 Mar 1;118(Pt 5):961-70. doi: 10.1242/jcs.01686. Epub 2005 Feb 15.
Pigment epithelium-derived factor (PEDF) exerts anti-angiogenic actions. However, the signal-transduction pathways regulated by PEDF remain to be elucidated. We show here that PEDF inhibited fibroblast growth factor 2 (FGF-2) induced capillary morphogenesis of a murine brain capillary endothelial cell line (IBE cells) and of human umbilical-vein endothelial cells (HUVECs) cultured on growth-factor-reduced Matrigel. We previously showed that FGF-2-mediated capillary morphogenesis was blocked by the Src-kinase inhibitor PP2 and that expression of dominant negative Fyn in IBE cells inhibited capillary morphogenesis. We examined the effect of PEDF on kinase activity of Fyn and found that PEDF downregulated FGF-2-promoted Fyn activity by tyrosine phosphorylation at the C-terminus in a Fes-dependent manner. In a stable IBE cell line expressing kinase-inactive Fes (KE5-15 Fes cells), PEDF failed to inhibit FGF-2-induced capillary morphogenesis or Fyn activity. PEDF induced the colocalization of Fyn and Fes in IBE cells expressing wild-type Fes, but not in KE5-15 Fes cells. In addition, wild-type Fes increased the tyrosine phosphorylation of Fyn in vitro, suggesting that Fes might directly phosphorylate Fyn. Expression of constitutively active Fyn (Y531F) in IBE cells exhibited capillary morphogenesis in the absence of FGF-2 and was resistant for PEDF treatment. Our results suggest that PEDF downregulates Fyn through Fes, resulting in inhibition of FGF-2-induced capillary morphogenesis of endothelial cells.
色素上皮衍生因子（PEDF）具有抗血管生成作用。然而，PEDF所调控的信号转导途径仍有待阐明。我们在此表明，PEDF抑制了成纤维细胞生长因子2（FGF - 2）诱导的小鼠脑毛细血管内皮细胞系（IBE细胞）以及在生长因子降低的基质胶上培养的人脐静脉内皮细胞（HUVECs）的毛细血管形态发生。我们先前表明，FGF - 2介导的毛细血管形态发生被Src激酶抑制剂PP2所阻断，并且在IBE细胞中显性负性Fyn的表达抑制了毛细血管形态发生。我们研究了PEDF对Fyn激酶活性的影响，发现PEDF以Fes依赖的方式通过C末端的酪氨酸磷酸化下调FGF - 2促进的Fyn活性。在表达激酶失活Fes的稳定IBE细胞系（KE5 - 15 Fes细胞）中，PEDF未能抑制FGF - 2诱导的毛细血管形态发生或Fyn活性。PEDF在表达野生型Fes的IBE细胞中诱导了Fyn和Fes的共定位，但在KE5 - 15 Fes细胞中未诱导。此外，野生型Fes在体外增加了Fyn的酪氨酸磷酸化，表明Fes可能直接磷酸化Fyn。在IBE细胞中组成型活性Fyn（Y531F）的表达在没有FGF - 2的情况下表现出毛细血管形态发生，并且对PEDF处理具有抗性。我们的结果表明，PEDF通过Fes下调Fyn，从而抑制FGF - 2诱导的内皮细胞毛细血管形态发生。
