Shupnik M A, Weck J, Hinkle P M
Department of Internal Medicine, University of Virginia, Charlottesville 29203, USA.
Mol Endocrinol. 1996 Jan;10(1):90-9. doi: 10.1210/mend.10.1.8838148.
TRH stimulates rat (r) TSH beta gene promoter activity at two distinct response elements, which also respond to protein kinase C-signaling pathways. The dependence of TRH-stimulated transcription of the TSH beta gene on a rise in intracellular calcium [Ca2+]i, and on the necessity for Ca2+ influx through L-type voltage-gated calcium channels was investigated in two transfected cell lines and in normal thyrotropes. The transcription rate of the homologous gene in normal thyrotropes was measured by nuclear run-off assays. Bay K8644, an L channel agonist, stimulated TSH beta gene transcription 6-fold, and TRH stimulation of TSH beta gene transcription was partially blocked by nimodipine, an L channel antagonist, while phorbol 12-myristate-13-acetate (PMA)-stimulated transcription was not. Bay K8644 plus TRH had a greater effect than either treatment alone. Constructs of the 5'-flanking region of the TSH beta gene fused to the luciferase reporter (TSH beta LUC) were then transfected into excitable GH3 pituitary cells. TSH beta LUC was stimulated 2- to 5-fold by 1 nM TRH or 100 nM Bay K8644, and the TRH effect was nearly abolished by nimodipine or chelation of external Ca2+. Constructs containing isolated TRH-responsive elements fused to a heterologous promoter responded similarly. The protein kinase C activator, PMA (100 nM) also stimulated TSH beta LUC transcription, but its effect was not inhibited by nimodipine. A stable heterologous cell line containing the mouse TRH receptor was constructed by transfection of nonexcitable 293 cells, which lack L channel activity. In the resultant 301 cells, TSH beta LUC activity was increased 2- to 3-fold by TRH or PMA; nimodipine, Bay K8644, and removal of extracellular Ca2+ had no effect. We conclude that TRH stimulation of TSH beta gene transcription requires Ca2+ release from inositol triphosphate-sensitive stores and Ca2+ influx via L-type calcium channels in GH3 cells, but in transfected 293 cells TRH activation of protein kinase C plays a predominant role in activating TSH beta. Both mechanisms appear to be operative in normal thyrotropes.
促甲状腺激素释放激素（TRH）通过两个不同的反应元件刺激大鼠（r）促甲状腺激素β基因启动子活性，这两个元件也对蛋白激酶C信号通路有反应。在两种转染细胞系和正常促甲状腺细胞中，研究了TRH刺激促甲状腺激素β基因转录对细胞内钙[Ca2+]i升高的依赖性，以及通过L型电压门控钙通道的Ca2+内流的必要性。通过核转录分析测量正常促甲状腺细胞中同源基因的转录速率。L通道激动剂Bay K8644刺激促甲状腺激素β基因转录6倍，L通道拮抗剂尼莫地平部分阻断TRH对促甲状腺激素β基因转录的刺激，而佛波醇12-肉豆蔻酸酯-13-乙酸酯（PMA）刺激的转录则不受影响。Bay K8644加TRH的效果比单独任何一种处理都要好。然后将与荧光素酶报告基因融合的促甲状腺激素β基因5'侧翼区构建体（促甲状腺激素β-LUC）转染到可兴奋的GH3垂体细胞中。1 nM TRH或100 nM Bay K8644可使促甲状腺激素β-LUC刺激2至5倍，尼莫地平或螯合细胞外Ca2+几乎消除了TRH的作用。含有与异源启动子融合的分离的TRH反应元件的构建体有类似反应。蛋白激酶C激活剂PMA（100 nM）也刺激促甲状腺激素β-LUC转录，但其作用不受尼莫地平抑制。通过转染缺乏L通道活性的非可兴奋293细胞构建了含有小鼠TRH受体的稳定异源细胞系。在所得的301细胞中，TRH或PMA可使促甲状腺激素β-LUC活性增加2至3倍；尼莫地平、Bay K8644和去除细胞外Ca2+均无作用。我们得出结论，TRH刺激促甲状腺激素β基因转录在GH3细胞中需要从肌醇三磷酸敏感储存库释放Ca2+并通过L型钙通道内流Ca2+，但在转染的293细胞中，TRH激活蛋白激酶C在激活促甲状腺激素β中起主要作用。这两种机制在正常促甲状腺细胞中似乎都起作用。
