Department of Pharmacology and the Barshop Institute for Longevity and Aging Studies, University of Texas Health Sciences Center at San Antonio, San Antonio, Texas, USA.
J Neurochem. 2013 Jul;126(1):19-28. doi: 10.1111/jnc.12294. Epub 2013 May 28.
The human tyrosine hydroxylase (hTH) gene has a 42 bp evolutionarily conserved region designated (CR) II at -7.24 kb, which bears 93% homology to the region we earlier identified as containing the glucocorticoid response element, a 7 bp activator protein-1 (AP-1)-like motif in the rat TH gene. We cloned this hTH-CRII region upstream of minimal basal hTH promoter in luciferase (Luc) reporter vector, and tested glucocorticoid responsiveness in human cell lines. Dexamethasone (Dex) stimulated Luc activity of hTH-CRII in HeLa cells, while mifepristone, a glucocorticoid receptor (GR) antagonist, prevented Dex stimulation. Deletion of the 7 bp 5'-TGACTAA at -7243 bp completely abolished the Dex-stimulated Luc activity of hTH-CRII construct. The AP-1 agonist, tetradeconoyl-12,13-phorbol acetate (TPA), also stimulated hTH promoter activity, and Dex and TPA together further accentuated this response. Chromatin immunoprecipitation assays revealed the presence of both GR and AP-1 proteins, especially Jun family members, at this hTH promoter site. Dex did not stimulate hTH promoter activity in a catecholaminergic cell line, which had low endogenous GR levels, but did activate the response when GR was expressed exogenously. Thus, our studies have clearly identified a glucocorticoid-responsive element in a 7 bp AP-1-like motif in the promoter region at -7.24 kb of the human TH gene.
人类酪氨酸羟化酶（hTH）基因在-7.24kb 处有一个 42bp 的进化保守区（CR）II，与我们之前鉴定的含有糖皮质激素反应元件的区域具有 93%的同源性，该区域是大鼠 TH 基因中包含的 7bp 激活蛋白-1（AP-1）样基序。我们将这个 hTH-CRII 区域克隆到最小基础 hTH 启动子上游的荧光素酶（Luc）报告载体中，并在人细胞系中测试了糖皮质激素反应性。地塞米松（Dex）刺激 HeLa 细胞中 hTH-CRII 的 Luc 活性，而米非司酮，一种糖皮质激素受体（GR）拮抗剂，可阻止 Dex 刺激。-7243bp 处的 7bp5'-TGACTAA 缺失完全消除了 hTH-CRII 构建体的 Dex 刺激的 Luc 活性。AP-1 激动剂十四烷酰佛波醇-12,13-乙酸酯（TPA）也刺激 hTH 启动子活性，Dex 和 TPA 共同进一步增强了这种反应。染色质免疫沉淀试验显示，在这个 hTH 启动子位点存在 GR 和 AP-1 蛋白，特别是 Jun 家族成员。在儿茶酚胺能细胞系中，Dex 不会刺激 hTH 启动子活性，因为该细胞系内源性 GR 水平较低，但当 GR 外源性表达时，Dex 确实会激活该反应。因此，我们的研究清楚地鉴定了人类 TH 基因启动子区域-7.24kb 处的 7bpAP-1 样基序中的糖皮质激素反应元件。
