Xia P, Aiello L P, Ishii H, Jiang Z Y, Park D J, Robinson G S, Takagi H, Newsome W P, Jirousek M R, King G L
Research Division, Joslin Diabetes Center, Boston, Massachusetts 02215, USA.
J Clin Invest. 1996 Nov 1;98(9):2018-26. doi: 10.1172/JCI119006.
Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen which mediates its effects by binding to tyrosine kinase receptors. We have characterized the VEGF-activated intracellular signal transduction pathway in bovine aortic endothelial cells and correlated this to its mitogenic effects. VEGF induced concentration- and time-dependent increases in protein kinase C (PKC) activation with a maximum of 2.2-fold above the basal level at 5 x 10(-10) M within 10 min as measured both by in situ and translocation assays. Immunoblotting analysis of PKC isoforms in cytosolic and membrane fractions indicated that after VEGF stimulation the content of Ca(2+)-sensitive PKC isoforms (alpha and betaII) was increased in the membrane fractions, whereas no changes were observed for PKC isoforms delta and epsilon. The stimulation of PKC activity by VEGF was preceded by the activation of phospholipase Cgamma (PLCgamma). This was demonstrated by parallel increases in PLCgamma tyrosine phosphorylation, [3H]inositol phosphate production, and [3H]arachidonic acid-labeled diacylglycerol formation in bovine aortic endothelial cells. In addition, VEGF increased phosphatidylinositol 3-kinase activity 2.1-fold which was inhibited by wortmannin, a phosphatidylinositol 3-kinase inhibitor, without decreasing the VEGF-induced increase in PKC activity or endothelial cell growth. Interestingly, genistein, a tyrosine kinase inhibitor, and GFX or H-7, PKC inhibitors, abolished both VEGF-induced PKC activation and endothelial cell proliferation. VEGF's mitogenic effect was inhibited by a PKC isoform beta-selective inhibitor, LY333531, in a concentration-dependent manner. In contrast, antisense PKC-alpha oligonucleotides enhanced VEGF-stimulated cell growth with a simultaneous decrease of 70% in PKC-alpha protein content. Thus, VEGF appears to mediate its mitogenic effects partly through the activation of the PLCgamma and PKC pathway, involving predominately PKC-beta isoform activation in endothelial cells.
血管内皮生长因子（VEGF）是一种强效的内皮细胞有丝分裂原，它通过与酪氨酸激酶受体结合来介导其作用。我们已经对牛主动脉内皮细胞中VEGF激活的细胞内信号转导途径进行了表征，并将其与有丝分裂作用相关联。VEGF诱导蛋白激酶C（PKC）激活呈浓度和时间依赖性增加，通过原位和转位测定法测量，在5×10^(-10) M时，10分钟内最高比基础水平增加2.2倍。对胞质和膜组分中PKC同工型的免疫印迹分析表明，VEGF刺激后，膜组分中对Ca(2+)敏感的PKC同工型（α和βII）含量增加，而PKC同工型δ和ε未观察到变化。VEGF对PKC活性的刺激之前是磷脂酶Cγ（PLCγ）的激活。这通过牛主动脉内皮细胞中PLCγ酪氨酸磷酸化、[3H]肌醇磷酸产生和[3H]花生四烯酸标记的二酰基甘油形成的平行增加得到证明。此外，VEGF使磷脂酰肌醇3激酶活性增加2.1倍，该活性被磷脂酰肌醇3激酶抑制剂渥曼青霉素抑制，而不降低VEGF诱导的PKC活性增加或内皮细胞生长。有趣的是，酪氨酸激酶抑制剂染料木黄酮以及PKC抑制剂GFX或H-7消除了VEGF诱导的PKC激活和内皮细胞增殖。VEGF的有丝分裂作用被PKC同工型β选择性抑制剂LY333531以浓度依赖性方式抑制。相反，反义PKC-α寡核苷酸增强了VEGF刺激的细胞生长，同时PKC-α蛋白含量降低了70%。因此，VEGF似乎部分通过激活PLCγ和PKC途径来介导其有丝分裂作用，在内皮细胞中主要涉及PKC-β同工型的激活。
