Jenab S, Inturrisi C E
Department of Pharmacology, Cornell University Medical College, New York, N.Y. 10021, USA.
Biochem Biophys Res Commun. 1995 May 16;210(2):589-99. doi: 10.1006/bbrc.1995.1700.
We used gel shift assays to determine the affinities of a 15 base glucocorticoid response element (GRE) derived from the rat proenkephalin (Penk) gene for the glucocorticoid receptor. A DNA binding domain of the glucocorticoid receptor interacted with a rat GRE (RGRE) with an apparent affinity intermediate between that of a consensus positive GRE oligo (GRE+) and a mismatch GRE oligo (GRE-). When inserted in front of a chloramphenicol acyl transferase (CAT) construct that is driven by a Penk promoter containing a cAMP response element, dexamethasone (10 microM) produced a 5-fold increase with GRE+, a small increase with RGRE, and no change with GRE- or the Penk promoter alone. Forskolin (20 microM) stimulated CAT activity 4- to 9-fold with each construct. However, dexamethasone plus forskolin caused a synergistic induction of CAT expression with the GRE+ oligo, no effect with the RGRE and an antagonistic effect with the Penk promoter alone and the GRE- oligo. These results demonstrate that GRE+, and to a lesser degree RGRE, can mimic the response of the endogenous Penk gene to dexamethasone and forskolin. Furthermore, a dexamethasone activated glucocorticoid receptor may inhibit cAMP mediated transcription of the Penk gene when a GRE+ or RGRE is not present.
我们使用凝胶迁移试验来确定源自大鼠前脑啡肽（Penk）基因的15个碱基的糖皮质激素反应元件（GRE）对糖皮质激素受体的亲和力。糖皮质激素受体的一个DNA结合结构域与大鼠GRE（RGRE）相互作用，其表观亲和力介于共有阳性GRE寡核苷酸（GRE+）和错配GRE寡核苷酸（GRE-）之间。当插入由含有cAMP反应元件的Penk启动子驱动的氯霉素乙酰转移酶（CAT）构建体之前时，地塞米松（10 microM）使GRE+导致CAT活性增加5倍，RGRE导致小幅增加，GRE-或单独的Penk启动子则无变化。福斯高林（20 microM）使每个构建体的CAT活性提高4至9倍。然而，地塞米松加福斯高林对GRE+寡核苷酸导致CAT表达的协同诱导，对RGRE无影响，对单独的Penk启动子和GRE-寡核苷酸则有拮抗作用。这些结果表明，GRE+，以及程度较轻的RGRE，可以模拟内源性Penk基因对地塞米松和福斯高林的反应。此外，当不存在GRE+或RGRE时，地塞米松激活的糖皮质激素受体可能抑制Penk基因的cAMP介导的转录。
