Role of a pituitary-specific transcription factor (pit-1/GHF-1) or a closely related protein in cAMP regulation of human thyrotropin-beta subunit gene expression.
Role of a pituitary-specific transcription factor (pit-1/GHF-1) or a closely related protein in cAMP regulation of human thyrotropin-beta subunit gene expression.
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垂体特异性转录因子 (pit-1/GHF-1) 或密切相关的蛋白质在 cAMP 调节人促甲状腺素-β 亚基基因表达中的作用。
DOI：
10.1172/jci115600
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Wondisford,FE
中科院分区：
文献类型：
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作者：
Steinfelder,HJ;Radovick,S;Mroczynski,MA;Hauser,P;McClaskey,JH;Weintraub,BD;Wondisford,FE
在人胚胎肾细胞系(293)和大鼠垂体细胞系(GH3)中，研究了人促甲状腺激素-β(TSH-β)基因cAMP对cAMP的调节作用。在293细胞中，腺苷环化酶激活剂Forsklin或cAMP类似物8-溴-cAMP(8-溴-cAMP)不刺激人TSH-β基因的表达。另一方面，这些药物在GH3细胞中诱导人TSHβ基因表达4-12倍。缺失分析表明，在+3~+8bp和-128~-61bp之间都是cAMP刺激所必需的区域。后一个区域包含三个与垂体特异性转录因子Pit-1/GHF-1同源的DNA序列，即DNA结合位点。凝胶迁移率分析表明，放射性标记的人促甲状腺激素β探针(-128~-61bp)与小鼠甲状腺肿瘤核提取液形成了5个特异的DNA-蛋白质复合体，与体外翻译的Pit-1/GHF-1形成了2个特异的复合体。5个四甲基偶氮唑盐复合体中的4个和体外的Pit-1/GHF-1复合体都被大鼠生长激素基因中一个未标记的Pit-1/GHF-1 DNA结合位点还原或消除，但不是同一DNA片段的突变版本，这表明Pit-1/GHF-1或密切相关的促甲状腺激素蛋白与这些DNA序列结合。在293细胞中，共转染含Pit-1/GHF-1基因的表达载体，恢复了对人TSHβ启动子的cAMP反应(8-溴-cAMP和Forsklin的最大刺激分别是5.2和6.6倍)，但不能恢复疱疹病毒胸苷激酶启动子的cAMP反应(这两种药物的最大刺激都是1.2倍)。因此，我们得出结论，人TSHβ基因在GH3细胞中受cAMP正向调节，而在293细胞中不受cAMP正向调节。由于人TSHβ基因在cAMP刺激所必需的区域中至少包含一个Pit-1/GHF-1的高亲和力结合位点，并且cAMP刺激可以通过添加Pit-1/GHF-1恢复到先前无反应的细胞系中的人TSHβ启动子，这表明Pit-1/GHF-1，或甲状腺激素中密切相关的蛋白质，可能是cAMP刺激TSHβ基因的反式作用因子。
cAMP regulation of the human thyrotropin-beta (TSH beta) gene cAMP was studied in two heterologous cell lines, a human embryonal kidney cell line (293) and a rat pituitary cell line (GH3). In 293 cells, human TSH beta gene expression was not stimulated by the adenylate cyclase activator forskolin or the cAMP analogue 8-bromo-cAMP (8-Br-cAMP). On the other hand, these agents induced human TSH beta gene expression 4-12-fold in GH3 cells. Deletion analysis demonstrated that the regions from +3 to +8 bp and from -128 to -61 bp were both necessary for cAMP stimulation. The latter region contains three DNA sequences homologous to a pituitary-specific transcription factor, Pit-1/GHF-1, DNA-binding site. Gel-mobility assays demonstrated that a radiolabeled human TSH beta probe (-128 to -61 bp) formed five specific DNA-protein complexes with mouse thyrotropic tumor (MTT) nuclear extract and two specific complexes with in vitro translated Pit-1/GHF-1. Four of the five MTT complexes and both in vitro Pit-1/GHF-1 complexes were reduced or eliminated by excess of an unlabeled Pit-1/GHF-1 DNA-binding site from the rat growth hormone gene, but not a mutated version of the same DNA fragment, suggesting that Pit-1/GHF-1 or a closely related thyrotroph protein binds to these DNA sequences. In 293 cells, co-transfection of an expression vector containing the Pit-1/GHF-1 cDNA restored cAMP-responsiveness to the human TSH beta promoter (5.2- and 6.6-fold maximal stimulation by 8-Br-cAMP and forskolin, respectively) but not the herpes virus thymidine kinase promoter (1.2-fold maximal stimulation by either agent). Thus we conclude that the human TSH beta gene is positively regulated by cAMP in GH3 but not 293 cells. Since the human TSH beta gene contains at least one high-affinity binding site for Pit-1/GHF-1 in a region necessary for cAMP stimulation and cAMP stimulation could be restored to the human TSH beta promoter in a previously nonresponsive cell line by the addition of Pit-1/GHF-1, this suggests that Pit-1/GHF-1, or a closely related protein in the thyrotroph, may be a trans-acting factor for cAMP stimulation of the TSH beta gene.Images