p42/p44 MAP kinase module plays a key role in the transcriptional regulation of the vascular endothelial growth factor gene in fibroblasts
p42/p44 MAP kinase module plays a key role in the transcriptional regulation of the vascular endothelial growth factor gene in fibroblasts
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DOI：
10.1074/jbc.273.29.18165
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发表时间：
1998-07-17
影响因子：
4.8
通讯作者：
Pagès, G
中科院分区：
文献类型：
--
作者：
Milanini, J;Viñals, F;Pagès, G
血管内皮生长因子（VEGF）是一种有效的血管内皮细胞有丝分裂原，与肿瘤新生血管形成有关。我们发现，在仓鼠成纤维细胞（CCL 39细胞），VEGF的mRNA表达在低水平的血清剥夺或指数生长的细胞，而它是快速诱导后，刺激静止的细胞与血清。CCL 39衍生物，用多瘤病毒或用p42/p44促分裂原活化蛋白（MAP）激酶途径的活性成员转化，Ras的12位Gly/瓦尔点突变体（Ras-Val（12）），其中Ser（218）和Ser（222）突变为Asp的MKK 1为了分析p42/p44 MAP激酶在该诱导中的作用，我们使用表达雌二醇可激活的Raf-I的CCL 39衍生的细胞系（Raf-1：ER）。我们显示了一个时间和雌二醇剂量依赖性的VEGF mRNA的上调明显检测后2小时的刺激。响应于Raf-I的条件活化的VEGF mRNA的诱导被MKK 1的抑制剂PD 098059逆转，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CCL 39细胞或雌二醇刺激的Raf-I：ER细胞中的VEGF诱导具有累加效应。与VEGF相反，同种型VEGF-B和VEGF-C受生长和致癌因子的调节较差。我们已经鉴定了VEGF启动子在-88和-66碱基对之间的富含GC的区域，其包含负责其通过组成型活性Ras或MKK 1-SS/DD上调的所有元件。通过突变的假定结合位点和电泳迁移率超移实验，我们表明，GC丰富的区域组成性结合Spl和AP-2转录因子。此外，在激活p42/p44 MAP激酶模块后，在启动子的该区域中形成的复合物中Spl和AP-2的结合增加。总之，这些数据表明，缺氧和p42/p44 MAP激酶独立地在VEGF表达的调节中发挥关键作用。
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-Val(12)), MKK1 in which Ser(218) and Ser(222) 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-l. 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-l 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-l: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 Spl and AP-2 transcription factors. Furthermore, following activation of the p42/p44 MAP kinase module, the binding of Spl 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.