Milanini J, Viñals F, Pouysségur J, Pagès G
Centre de Biochimie, CNRS-UMR 6543, Université de Nice, Parc Valrose, 06108 Nice, France.
J Biol Chem. 1998 Jul 17;273(29):18165-72. doi: 10.1074/jbc.273.29.18165.
Vascular Endothelial Growth Factor (VEGF) is a potent mitogen for vascular endothelial cells that has been implicated in tumor neovascularization. We show that, in hamster fibroblasts (CCL39 cells), VEGF mRNAs are expressed at low levels in serum-deprived or exponentially growing cells, whereas it is rapidly induced after stimulation of quiescent cells with serum. CCL39 derivatives, transformed with Polyoma virus or with active members of the p42/p44 mitogen-activated protein (MAP) kinase pathway, Gly/Val point mutant of Ras at position 12 (Ras-Val12), MKK1 in which Ser218 and Ser222 were mutated to Asp (MKK1-SS/DD)), express very high levels of VEGF mRNA. To analyze the contribution of the p42/p44MAP kinase in this induction, we used the CCL39-derived cell line (Raf-1:ER) expressing an estradiol-activable Raf-1. We show a time and an estradiol dose-dependent up-regulation of VEGF mRNA clearly detectable after 2 h of stimulation. The induction of VEGF mRNA in response to conditioned activation of Raf-1 is reverted by an inhibitor of MKK1, PD 098059, highlighting a specific role for the p42/p44 MAP kinase pathway in VEGF expression. Interestingly, hypoxia has an additive effect on VEGF induction in CCL39 cells stimulated by serum or in Raf-1:ER cells stimulated by estradiol. In contrast to VEGF, the isoforms VEGF-B and VEGF-C are poorly regulated by growth and oncogenic factors. We have identified a GC-rich region of the VEGF promoter between -88 and -66 base pairs which contains all the elements responsible of its up-regulation by constitutive active Ras or MKK1-SS/DD. By mutation of the putative binding sites and electrophoretic mobility supershift experiments, we showed that the GC-rich region constitutively binds Sp1 and AP-2 transcription factors. Furthermore, following activation of the p42/p44 MAP kinase module, the binding of Sp1 and AP-2 is increased in the complexes formed in this region of the promoter. Altogether, these data suggest that hypoxia and p42/p44 MAP kinase independently play a key role in the regulation of the VEGF expression.
血管内皮生长因子（VEGF）是一种对血管内皮细胞有强大作用的促有丝分裂原，与肿瘤新生血管形成有关。我们发现，在仓鼠成纤维细胞（CCL39细胞）中，VEGF mRNA在血清饥饿或指数生长期的细胞中低水平表达，而在用血清刺激静止细胞后会迅速被诱导。用多瘤病毒或p42/p44丝裂原活化蛋白（MAP）激酶途径的活性成员、第12位为甘氨酸/缬氨酸点突变的Ras（Ras-Val12）、丝氨酸218和丝氨酸222突变为天冬氨酸的MKK1（MKK1-SS/DD）转化的CCL39衍生物，表达非常高水平的VEGF mRNA。为了分析p42/p44 MAP激酶在这种诱导中的作用，我们使用了表达雌二醇可激活的Raf-1的CCL39衍生细胞系（Raf-1:ER）。我们发现，在刺激2小时后，VEGF mRNA出现明显可检测到的时间和雌二醇剂量依赖性上调。用MKK1抑制剂PD 098059可逆转Raf-1条件性激活后VEGF mRNA的诱导，突出了p42/p44 MAP激酶途径在VEGF表达中的特定作用。有趣的是，缺氧对血清刺激的CCL39细胞或雌二醇刺激的Raf-1:ER细胞中的VEGF诱导有累加效应。与VEGF相反，VEGF-B和VEGF-C同工型受生长和致癌因子的调控较弱。我们在VEGF启动子-88至-66碱基对之间鉴定出一个富含GC的区域，该区域包含所有负责其被组成型活性Ras或MKK1-SS/DD上调的元件。通过对假定结合位点的突变和电泳迁移率超迁移实验，我们表明富含GC的区域组成型结合Sp1和AP-2转录因子。此外，在p42/p44 MAP激酶模块激活后，启动子该区域形成的复合物中Sp1和AP-2的结合增加。总之，这些数据表明缺氧和p42/p44 MAP激酶在VEGF表达调控中独立发挥关键作用。
