Transcriptional regulation of apoE DNA on dietary hyperlipidemia

Research Project

Project/Area Number	03671128
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	内分泌・代謝学
Research Institution	The University of Tsukuba
Principal Investigator	MATSUSHIMA Teruhiko Univ.of Tsukuba Inst.Clin.Med Assist.Prof., 臨床医学系, 講師 (60199792)
Project Period (FY)	1991 – 1992
Project Status	Completed (Fiscal Year 1992)
Budget Amount *help	¥2,100,000 (Direct Cost: ¥2,100,000) Fiscal Year 1992: ¥700,000 (Direct Cost: ¥700,000) Fiscal Year 1991: ¥1,400,000 (Direct Cost: ¥1,400,000)
Keywords	Apolipoprotein E / DNA / Nuclear protein / Cholesterol / Hyperlipidemia / Guinea pig / Liver / Transcription factor / 高コレステロール血症 / 遺伝子転写 / 調節因子 / CATアッセイ / サウスウェスタンブロット / コレステロ-ル
Research Abstract	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.