BACKGROUND & AIMS:
:The EC rabbit endothelial cell line was transfected with the EJ-ras oncogene (EJ-ras EC). EJ-ras EC cells display over expression of the Ras oncogene, morphological changes and deregulation of the cell cycle, becoming more densely populated and serum-independent. In addition, EJ-ras-transfectant cells show higher levels of the syndecan-4 mRNA. In addition to the increase in the core protein, a parallel increase in the glycosylation of the syndecan-4 protein, a proteoglycan that bears heparan sulfate chains, also occurs. This increase is observed both for the heparan sulfate proteoglycan synthesized by the cells and for that secreted to the culture medium. This enhancement in heparan sulfate synthesis was observed through metabolic labeling of the cells, immunoprecipitation of syndecan-4 and heparitinases treatment. Furthermore, the EJ-ras-transfectant cells do not exhibit decreased synthesis of heparan sulfate during the G(1)-S phase transition, as observed for the parental cell line. Also, heparan sulfate synthesis is not stimulated by PMA as displayed by parental endothelial cells. Significant structural changes of heparan sulfate, such as decreased O-sulfation, were observed in the EJ-ras-transfected cells. Decreases in the mRNA levels of some enzymes (glucuronosyl C-5 epimerase, iduronosyl-2-O-sulfotransferase, glucosaminyl-6-O-sulfotransferase-1 and N-deacetylase/N-sulfotransferase-1), involved in the biosynthetic pathway of heparan sulfate, were also observed. The results suggest that overexpression of the EJ-ras oncogene alters the cell cycle, through signal transduction cascades, upregulates the expression of syndecan-4, and downregulates enzymes involved in the heparan sulfate biosynthesis related to chain modification, leading to the structural changes of the heparan sulfate syndecan-4 proteoglycan in endothelial cells.
背景与目标:
: 用EJ-ras癌基因 (EJ-ras EC) 转染EC兔内皮细胞系。Ej-ras EC细胞显示出ras癌基因的过度表达,形态变化和细胞周期的失调,变得更加密集和不依赖血清。此外,EJ-ras转染细胞显示出更高水平的syndecan-4 mRNA。除了核心蛋白的增加外,syndecan-4蛋白 (具有硫酸乙酰肝素链的蛋白聚糖) 的糖基化也平行增加。对于细胞合成的硫酸乙酰肝素蛋白聚糖和分泌到培养基中的硫酸乙酰肝素蛋白聚糖,都观察到这种增加。通过细胞的代谢标记,syndecan-4的免疫沉淀和肝素酶处理观察到硫酸乙酰肝素合成的这种增强。此外,如亲本细胞系所观察到的那样,在G(1)-S相变期间,EJ-ras转染细胞不会显示出硫酸乙酰肝素的合成减少。此外,如亲代内皮细胞所显示的,PMA不会刺激硫酸乙酰肝素的合成。在EJ-ras转染的细胞中观察到硫酸乙酰肝素的显着结构变化,例如O-硫酸化降低。还观察到与硫酸乙酰肝素的生物合成途径有关的某些酶 (葡萄糖醛酸C-5差向异构酶,iduronosyl-2-O-sulfotransferase,glucosaminyl-6-O-sulfotransferase-1和N-脱乙酰基酶/N-sulfotransferase-1) 的mRNA水平降低。结果表明,EJ-ras癌基因的过表达会改变细胞周期,通过信号转导级联,上调syndecan-4的表达,并下调与链修饰有关的硫酸乙酰肝素生物合成的酶,导致内皮细胞中硫酸乙酰肝素syndecan-4蛋白聚糖的结构变化。