Glucocorticoid responsiveness of the rat phenylethanolamine N-methyltransferase gene
Glucocorticoid responsiveness of the rat phenylethanolamine N-methyltransferase gene
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DOI：
10.1124/mol.61.6.1385
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发表时间：
2002-06-01
影响因子：
3.6
通讯作者：
Wong, DL
中科院分区：
文献类型：
--
作者：
Tai, TC;Claycomb, R;Wong, DL
在大鼠苯乙醇胺N-甲基转移酶（PNMT; EC 2.1.1.28）基因启动子中的-759和-773 bp处的两个新鉴定的重叠（1 bp）糖皮质激素应答元件（GRE）主要负责其糖皮质激素敏感性，而不是最初鉴定的-533-bp GRE。在用野生型PNMT启动子-荧光素酶报告基因构建体转染并用地塞米松处理的RS 1细胞中观察到PNMT启动子活性的剂量依赖性增加（在0.1 μ M时最大活化）。II型糖皮质激素受体拮抗剂RU 38486（10 μ M）完全抑制地塞米松（1 μ M）对PNMT启动子的激活，这与经典的糖皮质激素受体介导皮质激素刺激的转录活性一致。凝胶迁移率变动竞争测定的相对IC 50值表明，-759-bp GRE对糖皮质激素受体的亲和力比-773-bp GRE高2倍。单独或串联的-533-、-759-和-773-bp GRE的定点突变表明，-759- bp GRE在功能上也更重要，但最大糖皮质激素反应需要-759-和-773-bp GRE。此外，-533- bp GRE，而不是增加启动子的糖皮质激素敏感性，可能限制通过-759-和-773-bp GRE介导的皮质类固醇反应性。最后，糖皮质激素受体结合到-759-和-773-bp的GRES与Egr-1和/或AP-2相互作用，以刺激用地塞米松处理的RS 1细胞中的PNMT启动子活性。相反，糖皮质激素受体结合的-533- bp的GRE似乎只参与协同激活的PNMT启动子通过与激活蛋白2的相互作用。
Two newly identified, overlapping (1 bp) glucocorticoid response elements (GREs) at -759 and -773 bp in the promoter of the rat phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) gene are primarily responsible for its glucocorticoid sensitivity, rather than the originally identified -533-bp GRE. A dose-dependent increase in PNMT promoter activity was observed in RS1 cells transfected with a wild-type PNMT promoter-luciferase reporter gene construct and treated with dexamethasone (maximum activation at 0.1 muM). The type II glucocorticoid receptor antagonist RU38486 (10 muM) fully inhibited dexamethasone (1 muM) activation of the PNMT promoter, consistent with classical glucocorticoid receptors mediating corticosteroid-stimulated transcriptional activity. Relative IC50 values from gel mobility shift competition assays showed that the -759-bp GRE has a 2-fold greater affinity for the glucocorticoid receptor than the -773-bp GRE. Site-directed mutation of the -533-, -759-, and -773-bp GREs alone or in tandem demonstrated that the -759- bp GRE was also functionally more important, but both the -759- and -773-bp GREs are required for maximum glucocorticoid responses. Moreover, the -533- bp GRE, rather than increasing glucocorticoid sensitivity of the promoter, may limit corticosteroid responsiveness mediated via the -759- and -773-bp GREs. Finally, the glucocorticoid receptor bound to the -759- and -773-bp GREs interacts cooperatively with Egr-1 and/or AP-2 to stimulate PNMT promoter activity in RS1 cells treated with dexamethasone. In contrast, glucocorticoid receptors bound to the -533- bp GRE only seem to participate in synergistic activation of the PNMT promoter through interaction with activator protein 2.