BOTH V-HA-RAS AND V-RAF STIMULATE EXPRESSION OF THE VASCULAR ENDOTHELIAL GROWTH-FACTOR IN NIH 3T3 CELLS
BOTH V-HA-RAS AND V-RAF STIMULATE EXPRESSION OF THE VASCULAR ENDOTHELIAL GROWTH-FACTOR IN NIH 3T3 CELLS
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DOI：
10.1074/jbc.270.43.25915
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发表时间：
1995-10-27
影响因子：
4.8
通讯作者：
MARME, D
中科院分区：
文献类型：
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作者：
GRUGEL, S;FINKENZELLER, G;MARME, D
用血小板衍生生长因子(PDGF)-BB和12-O-十四酰佛波醇-13-醋酸酯(TPA)刺激NIH 3T3细胞，可增强血管内皮生长因子(VEGF)基因的表达。为了探讨Ras和Raf是否参与了PDGF和TPA对血管内皮生长因子基因表达的诱导，我们检测了这两种因素对稳定表达v-Ha-ras或v-RAF的NIH3T3细胞的影响。在血清饥饿的NIH3T3细胞中，只能检测到低水平的mRNA表达，而ras和RAF转化的细胞株都表达4.3kb的高水平的VEGF转录本。经PDGF或TPA刺激后，所有细胞株的VEGF基因表达均升高，转化细胞中表达水平最高。免疫荧光研究证实，血管内皮细胞生长因子mRNA的表达增加与蛋白质水平的增加有关。在v-Ha-ras和v-RAF转化的细胞系中可检测到阳性的免疫荧光信号，而在未刺激的NIH3T3细胞中未检测到阳性的免疫荧光信号。用肝素-琼脂糖柱层析从v-RAF转化的NIH3T3细胞的条件培养液中部分纯化出VEGF。通过与重组人脐静脉内皮细胞I-125-VEGF(165)的竞争结合和刺激这些细胞的增殖，证明了该蛋白的生物学活性。
Stimulation of NIH 3T3 cells with platelet-derived growth factor (PDGF)-BB and 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances vascular endothelial growth factor (VEGF) gene expression. To address the question of whether Ras and Raf are involved in the induction of VEGF gene expression by PDGF and TPA, we examined the effects of both factors on NIH 3T3 cells stably transfected with v-Ha-ras or v-raf. In serum-starved NIH 3T3 cells, only low levels of mRNA expression can be detected, whereas both ras and raf transformed cell lines express enhanced levels of a 4.3-kilobase VEGF transcript. Stimulation with PDGF or TPA resulted in increased VEGF mRNA in all cell lines, with highest levels found in the transformed cells. Immunofluorescence studies confirmed that the elevated VEGF mRNA expression correlated with enhanced protein levels. Positive immunofluorescence signals could be detected in v-Ha-ras or v-raf transformed cell lines but not in unstimulated NIH 3T3 cells. VEGF from conditioned medium of v-raf transformed NIH 3T3 cells was partially purified by chromatography on heparin-Sepharose. Biological activity of this VEGF protein was demonstrated by competition with binding of recombinant I-125-VEGF(165) human umbilical vein endothelial cells and by its ability to stimulate proliferation of these cells.