Tamura K, Tanimoto K, Ishii M, Murakami K, Fukamizu A
Institute of Applied Biochemistry, University of Tsukuba, Ibaraki, Japan.
J Biol Chem. 1993 Jul 15;268(20):15024-32.
Angiotensinogen is abundantly expressed in adipose tissue as well as in liver where it is mainly produced. To address the mechanism of this adipogenic expression, promoter regions of the mouse angiotensinogen gene are fused to the chloramphenicol acetyltransferase reporter gene and stably transfected into 3T3-L1 preadipocytes. Promoter activity correlates well with an increase of mRNA levels during adipogenic differentiation, thereby demonstrating that the induction is primarily due to transcriptional activation. Deletion analysis indicates that the proximal promoter region from -96 to +22 is able to mediate the chloramphenicol acetyltransferase induction and identifies two transcriptionally active regions: AGE1 (position -399 to -139) and AGE2 (position -96 to -52). Heterologous promoter assay reveals that AGE1 behaves with a constitutive enhancer-like property and that AGE2 functions as a differentiation-inducible activator. Gel shift experiments show that AGE2 specifically binds a novel factor (AGF2), which is induced upon differentiation. Furthermore, a constitutive factor (AGF3) binds to the core promoter region including the exon 1 (from -6 to +22, AGE3). Mutations within either AGE2 or AGE3 that disrupt nuclear factors binding in vitro dramatically reduced the chloramphenicol acetyltransferase activation in the native promoter context. These results suggest that both AGE2 and AGE3 are necessary for mediating efficient activation of the mouse angiotensinogen promoter during adipogenic differentiation.
血管紧张素原在脂肪组织以及主要产生它的肝脏中大量表达。为了探究这种脂肪生成表达的机制，将小鼠血管紧张素原基因的启动子区域与氯霉素乙酰转移酶报告基因融合，并稳定转染到3T3-L1前脂肪细胞中。启动子活性与脂肪生成分化过程中mRNA水平的增加密切相关，从而表明这种诱导主要是由于转录激活。缺失分析表明，从-96到+22的近端启动子区域能够介导氯霉素乙酰转移酶的诱导，并确定了两个转录活性区域：AGE1（位置-399至-139）和AGE2（位置-96至-52）。异源启动子分析表明，AGE1表现出组成型增强子样特性，而AGE2作为分化诱导激活剂发挥作用。凝胶迁移实验表明，AGE2特异性结合一种新型因子（AGF2），该因子在分化时被诱导。此外，一种组成型因子（AGF3）与包括外显子1（从-6到+22，AGE3）的核心启动子区域结合。在AGE2或AGE3内破坏体外核因子结合的突变在天然启动子环境中显著降低了氯霉素乙酰转移酶的激活。这些结果表明，AGE2和AGE3对于在脂肪生成分化过程中介导小鼠血管紧张素原启动子的有效激活都是必需的。
