Asai Masato, Iwasaki Yasumasa, Yoshida Masanori, Mutsuga-Nakayama Noriko, Arima Hiroshi, Ito Masafumi, Takano Koji, Oiso Yutaka
Department of Endocrinology, Metabolism, and Nephrology, Kochi University, Kochi Medical School, Kohasu, Oko-cho, Nankoku 783-8505, Japan.
Mol Endocrinol. 2004 Dec;18(12):3011-9. doi: 10.1210/me.2003-0471. Epub 2004 Aug 19.
GHRH plays a pivotal role in the regulation of both synthesis and secretion of GH in the anterior pituitary. In this study, we examined the molecular mechanism of depolarization-induced GHRH gene transcription using the hypothalamus cell line, Gsh+/+, revealing the involvement of the transcription factor called nuclear factor of activated T cells (NFAT). GHRH, NFAT1, NFAT4, and related genes were endogenously expressed in Gsh+/+ cells and the rat arcuate nucleus, where NFAT1 and GHRH were colocalized. Cellular excitation with high potassium potently stimulated endogenous GHRH gene 5'-promoter activity as well as the NFAT-mediated gene transcription, the former being further enhanced by coexpression of NFAT. On the other hand, cyclosporin A (a calcineurin-NFAT inhibitor) or EGTA (a calcium chelator) significantly blocked the depolarization-induced GHRH gene transcription. EMSA and site-directed mutagenesis experiments showed the direct binding of NFAT at five sites of the GHRH promoter, among which the relative importance of three distal sites (-417/-403, -402/-387, -317/-301) was suggested. Finally, elimination of all five sites completely abolished the NFAT-induced GHRH gene up-regulation. Altogether, our results suggest that the transcription factor NFAT is involved in the depolarization-induced transcriptional activation of GHRH gene in the neuronal cells.
生长激素释放激素（GHRH）在前脑垂体生长激素（GH）的合成和分泌调节中起关键作用。在本研究中，我们使用下丘脑细胞系Gsh+/+研究了去极化诱导的GHRH基因转录的分子机制，揭示了活化T细胞核因子（NFAT）这种转录因子的参与。GHRH、NFAT1、NFAT4及相关基因在Gsh+/+细胞和大鼠弓状核中内源性表达，其中NFAT1和GHRH共定位。高钾引起的细胞兴奋强烈刺激内源性GHRH基因5'-启动子活性以及NFAT介导的基因转录，前者通过NFAT的共表达进一步增强。另一方面，环孢素A（一种钙调神经磷酸酶-NFAT抑制剂）或乙二醇双四乙酸（EGTA，一种钙螯合剂）显著阻断去极化诱导的GHRH基因转录。电泳迁移率变动分析（EMSA）和定点诱变实验表明NFAT直接结合在GHRH启动子的五个位点，其中三个远端位点（-417/-403、-402/-387、-317/-301）的相对重要性得到提示。最后，消除所有五个位点完全消除了NFAT诱导的GHRH基因上调。总之，我们的结果表明转录因子NFAT参与神经元细胞中去极化诱导的GHRH基因转录激活。
