Transcriptional regulation of apoE DNA on dietary hyperlipidemia

为探讨载脂蛋白E(ApoE)生物合成的调控机制，将豚鼠apoE基因组DNA的5‘调控区克隆到CAT载体中，导入原代培养的豚鼠肝细胞，用CAT检测系统分析其转录活性。用凝胶位移分析、DNaseI足迹分析和Southwest blotting分析DNA与核蛋白的相互作用。在CAT分析中，apoE基因在胆固醇喂养的豚鼠肝细胞中表现出比正常动物肝细胞更强的转录活性。凝胶位移分析显示，该基因5‘上游区与豚鼠肝细胞核提取液形成DNA-蛋白质复合体。正常肝脏和高胆固醇饲料喂养的脂肪肝的DNA结合模式不同。在后者中，apoE mRNA的含量已被证实增加，这表明DNA-蛋白质相互作用的改变可能导致转录增强。用32P标记的DNA 5‘上游区域对核蛋白进行西南印迹分析。在110千道尔顿和33千道尔顿处探测到了信号。在33kD处，正常肝脏提取液的信号强于脂肪肝提取液。33kD蛋白可能参与了载脂蛋白E基因在胆固醇负荷下的正转录调控。提示胆固醇负荷可在DNA转录水平上调载脂蛋白E的合成。

In order to investigate the control mechanism of apolipoprotein E (apoE) biosynthesis, 5' regulatory region of guinea pig apoE genomic DNA was cloned in CAT vector, transfected into the primary cultured guinea pig hepatocytes and the transcriptional activity was analyzed by CAT assay system. DNA-nuclear protein interaction was analyzed by gel shift analysis, DNaseI footprint analysis and Southwestern blotting. On the CAT assay analysis, the apoE gene showed stronger transcriptional activity in hepatocytes from cholesterol-fed guinea pig than in the cells from normal animals. Gel shift analysis showed DNA-protein complex of the 5' upstream region of the gene with nuclear extract of the guinea pig liver. The binding patterns of the DNA were different between to the normal liver and to the fatty liver from animals fed with high cholesterol diet. In the latter the content of apoE mRNA had been confirmed to be increased, suggesting that the change of DNA-protein interaction may cause enhanced transcription. Southwestern blotting analysis was performed on the nuclear protein with 32P-labeled 5' upstream region of the DNA as a prove. Signals were detected at the 110 and 33 kilodaltons. The signal at 33kD was observed stronger in the extract from normal liver than in that from fatty liver. The 33kD protein may be involved in the positive transcriptional regulation of apoE gene at the cholesterol loading. It was suggested that apoE synthesis is up-regulated at the level of DNA transcription by cholesterol loading.