Liu J L, Papachristou D N, Patel Y C
Fraser Laboratories, Department of Medicine, McGill University, Royal Victoria Hospital, Montreal, Quebec, Canada.
Biochem J. 1994 Aug 1;301 ( Pt 3)(Pt 3):863-9. doi: 10.1042/bj3010863.
The somatostatin (SS) gene is transcriptionally regulated via the cyclic AMP (cAMP) response element (CRE), located in the proximal promoter (-41 to -48 bp). We have previously reported that glucocorticoids induce dose-dependent cell-specific alterations in the steady-state SS mRNA level. Here we have investigated direct transcriptional control of the SS gene by glucocorticoids. We have examined transcriptional interaction between glucocorticoids and the cAMP signalling pathway and mapped the 5' upstream regulatory region of the SS gene involved in glucocorticoid transactivation. Transcriptional regulation was determined by analysis of chloramphenicol acetyltransferase (CAT) activity in PC12 rat pheochromocytoma cells and A126-1B2 (protein kinase A-deficient mutant PC12) cells, by acute transfection of 5' flanking SS DNA (- 750, -250 and -71 bp) ligated to the reporter (CAT) gene. Dexamethasone (DEX) induced a dose-dependent 2.2-fold stimulation of SS gene transcription in PC12 cells, but not in A126-1B2 cells. Other steroid and thyroid hormones tested, and retinoic acid, were ineffective, while cAMP and forskolin stimulated gene transcription 4-5-fold in PC12 cells but not in A126-1B2 cells. DEX exerted an additive effect on cAMP-induced gene transcription. Deletion of the promoter from -750 to -71 bp (but not from -750 to -250 bp) abolished all stimulatory effects of DEX without affecting cAMP responsiveness. Mutation of the CRE abrogated both DEX- and cAMP-dependent gene enhancement. Gel electrophoretic mobility shift assays confirmed that the -250 to -71 bp region of the SS promoter (but not the -71 to +55 bp domain) binds specifically to a glucocorticoid response element-sensitive nuclear protein(s) from PC12 cells, suggesting a putative glucocorticoid receptor interaction with SS promoter DNA. We conclude that glucocorticoids regulate SS gene transcription positively. Glucocorticoid-induced transactivation shows dependence on protein kinase. A activity, and may be mediated via protein-protein interaction between the glucocorticoid receptor and the CRE binding protein. DNA sequences upstream from the CRE between -250 and -71 bp in the SS promoter appear to be the target of glucocorticoid action.
生长抑素（SS）基因通过位于近端启动子（-41至-48 bp）的环磷酸腺苷（cAMP）反应元件（CRE）进行转录调控。我们之前报道过，糖皮质激素可诱导稳态SS mRNA水平出现剂量依赖性的细胞特异性改变。在此，我们研究了糖皮质激素对SS基因的直接转录调控。我们检测了糖皮质激素与cAMP信号通路之间的转录相互作用，并绘制了参与糖皮质激素反式激活的SS基因5'上游调控区域图谱。通过在PC12大鼠嗜铬细胞瘤细胞和A126 - 1B2（蛋白激酶A缺陷型突变PC12）细胞中分析氯霉素乙酰转移酶（CAT）活性来确定转录调控，方法是将5'侧翼SS DNA（-750、-250和-71 bp）与报告基因（CAT）连接后进行急性转染。地塞米松（DEX）在PC12细胞中诱导了剂量依赖性的2.2倍SS基因转录刺激，但在A126 - 1B2细胞中未出现。所测试的其他类固醇和甲状腺激素以及视黄酸均无效，而cAMP和福斯高林在PC12细胞中刺激基因转录4 - 5倍，但在A126 - 1B2细胞中无此作用。DEX对cAMP诱导的基因转录发挥了相加作用。将启动子从-750 bp缺失至-71 bp（但不是从-750 bp缺失至-250 bp）消除了DEX的所有刺激作用，而不影响cAMP反应性。CRE的突变消除了DEX和cAMP依赖性的基因增强作用。凝胶电泳迁移率变动分析证实，SS启动子的-250至-71 bp区域（但不是-71至+55 bp结构域）与来自PC12细胞的糖皮质激素反应元件敏感核蛋白特异性结合，提示糖皮质激素受体与SS启动子DNA之间存在假定的相互作用。我们得出结论，糖皮质激素对SS基因转录起正向调控作用。糖皮质激素诱导的反式激活显示出对蛋白激酶A活性的依赖性，并且可能通过糖皮质激素受体与CRE结合蛋白之间的蛋白质 - 蛋白质相互作用介导。SS启动子中CRE上游-250至-71 bp之间的DNA序列似乎是糖皮质激素作用的靶点。
