INVOLVEMENT OF A CAMP-RESPONSIVE DNA ELEMENT IN MEDIATING TRH RESPONSIVENESS OF THE HUMAN THYROTROPIN ALPHA-SUBUNIT GENE
INVOLVEMENT OF A CAMP-RESPONSIVE DNA ELEMENT IN MEDIATING TRH RESPONSIVENESS OF THE HUMAN THYROTROPIN ALPHA-SUBUNIT GENE
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DOI：
10.1210/me.8.4.528
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发表时间：
1994-04-01
影响因子：
--
通讯作者：
PARK, SD
中科院分区：
文献类型：
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作者：
KIM, DS;AHN, SK;PARK, SD
已知TRH刺激垂体细胞中TSH基因的转录。为了检测人TSH α亚基基因的TRH反应元件，我们使用瞬时转染GH（3）大鼠垂体瘤细胞。使用该系统，TRH处理刺激了含有846个碱基对的报告基因的表达，该报告基因来自与荧光素酶连接的人糖蛋白激素α-亚基基因的5 '侧翼区。对α-亚基序列5 ′-缺失的分析表明，至少有两个上游限制在-223至-190和-151至-135位置之间的DNA区域对TRH的调节是重要的。更近端的区域包括先前定义的cAMP反应元件（CRE），而更上游的区域包含与垂体转录因子Pit-1的结合位点具有序列相似性的元件。通过在胸苷激酶-荧光素酶报告基因的上游插入片段来测试每个区域的TRH反应性。-151至-100区域具有基础增强子活性，并允许对TRH的3.4倍反应。-223至-168区域不允许TRH反应，但具有基础增强子活性。两个区域的组合导致TRH的五倍刺激。为了评估不同信号转导途径的贡献，检查了各种治疗组合。TRH和毛喉素的联合治疗导致了加性活性。用TRH加佛波醇12-肉豆蔻酸酯-13-乙酸酯治疗导致与单独使用任一种药物相同水平的报告基因活性。钙通道阻滞剂维拉帕米和钙螯合剂EGTA完全阻断了TRH对α亚基报告基因的诱导作用。α-亚基基因的-151至-100区域含有两个共有CRE拷贝。删除一个拷贝的克雷斯导致TRH和钙反应性的丧失。总体而言，这些结果表明，α-亚基基因的TRH反应元件可能涉及先前表征的CRE。TRH对α-亚基基因的作用机制的至少一部分可能涉及细胞内钙水平的变化，导致钙应答蛋白激酶的活化和CRE结合蛋白的磷酸化。
TRH is known to stimulate the transcription of the TSH gene in pituitary cells. To examine TRH-responsive elements of the human TSH alpha-subunit gene, we have used transient transfection of GH(3) rat pituitary tumor cells. Using this system, TRH treatment stimulated expression of a reporter gene containing 846 base pairs from the 5'-flanking region of the human glycoprotein hormone alpha-subunit gene linked to luciferase. Analysis of 5'-deletions of the alpha-subunit sequence revealed that at least two DNA regions with upstream limits between positions -223 to -190 and positions -151 to -135 are important for regulation by TRH. The more proximal region includes a previously defined cAMP-response element (CRE) while the more upstream region contains an element with sequence similarity to the binding site for the pituitary transcription factor, Pit-1. The TRH responsiveness of each individual region was tested by inserting fragments upstream of a thymidine kinase-luciferase reporter gene. The -151 to -100 region had basal enhancer activity and permitted a 3.4-fold response to TRH. The -223 to -168 region did not permit a TRH response, but possessed basal enhancer activity. The combination of both regions resulted in a fi-fold stimulation by TRH. To assess the contributions of different signal transduction pathways, various combinations of treatments were examined. Combined treatment with TRH and forskolin led to an additive activity. Treatment with TRH plus phorbol 12-myristate-13-acetate resulted in the same level of reporter gene activity as with either agent alone. The calcium channel blocker, verapamil, and the calcium chelator, EGTA, completely blocked induction by TRH of the alpha-subunit reporter gene. The -151 to -100 region of the alpha-subunit gene contains two copies of a consensus CRE. Deletion of one copy of the CREs resulted in a loss of TRH and calcium responsiveness. Overall, these results indicate that the TRH responsive element of the alpha-subunit gene likely involves a previously characterized CRE. At least a portion of the mechanism of TRH effects on the alpha-subunit gene likely involves changes in intracellular calcium levels leading to activation of calcium-responsive protein kinases and phosphorylation of CRE-binding proteins.