Lin X, Conn P M
Oregon Regional Primate Research Center, Beaverton 97006, USA.
Endocrinology. 1998 Sep;139(9):3896-902. doi: 10.1210/endo.139.9.6214.
GnRH appears to regulate messenger RNA levels and synthesis of its own receptor (GnRHR). In this study, we examined the regulation of GnRHR gene transcription by GnRH and cAMP in the GGH3 cell line (GH3 cells stably expressing GnRHR). Transient transfection of GGH3 cells with luciferase reporter gene vector (GnRHR-pXP2) containing a 1226-bp promoter fragment (-1164 to +62, relative to the major transcription start site) of mouse GnRHR gene resulted in an increase in reporter gene (GnRHR-Luc) activity (11- to 22-fold) compared with the promoterless vector. GnRH or a GnRH agonist (Buserelin) significantly stimulated the GnRHR-Luc activity in a dose-dependent manner. Time-course studies using 10(-7) M Buserelin revealed that GnRHR-Luc activity increased progressively from 1.5-6 h, with a peak at 6 h. The increase in GnRHR-Luc activity was lower at 12 and 24 h. Both cholera toxin and dBcAMP significantly stimulated GnRHR-Luc activity. Pretreatment with dBcAMP also enhanced the extent of stimulation of GnRHR-Luc activity in response to Buserelin. Pertussis toxin did not induce basal or Buserelin-stimulated GnRHR-Luc activity. Treatment of GGH3 cells with 10(-9) or 10(-7) M Buserelin for 6 h was sufficient to stimulate a significant increase in cAMP release. An adenylate cyclase inhibitor SQ 22536 did not affect the basal GnRHR-Luc activity but significantly reduced Buserelin-activated GnRHR-Luc activity. These results suggest that GnRH and cAMP activate transcriptional activity of the GnRHR gene and that GnRH activates GnRHR transcriptional activity, in part, through the cAMP pathway. Progressive 5'-deletion analysis revealed that basal and Buserelin- or dBcAMP-stimulated GnRHR-Luc activity were consistently retained after 5'-deletion at position -456, -381, or -331 relative to the major transcription start site but were significantly decreased after subsequent truncation of the promoter from -331 to -255 relative to the major transcription start site. However, the -255 construct still retained responsiveness to Buserelin and dBcAMP, and the relative activity remained similar under both stimulation conditions. These results suggest that elements located between -331 and -255 necessary for transcriptional activity of the GnRHR gene in GGH3 cells, and that the response elements on the mouse GnRHR gene for both GnRH and cAMP reside at two different sites: between -331 and -255 and between -255 and +62.
促性腺激素释放激素（GnRH）似乎可调节其自身受体（GnRHR）的信使核糖核酸水平及合成。在本研究中，我们检测了GnRH和环磷酸腺苷（cAMP）对GGH3细胞系（稳定表达GnRHR的GH3细胞）中GnRHR基因转录的调控作用。用含有小鼠GnRHR基因1226碱基对启动子片段（相对于主要转录起始位点为-1164至+62）的荧光素酶报告基因载体（GnRHR-pXP2）对GGH3细胞进行瞬时转染，与无启动子载体相比，报告基因（GnRHR-Luc）活性增加了（11至22倍）。GnRH或GnRH激动剂（布舍瑞林）以剂量依赖方式显著刺激GnRHR-Luc活性。使用10⁻⁷M布舍瑞林进行的时间进程研究显示，GnRHR-Luc活性在1.5至6小时逐渐增加，6小时达到峰值。在12小时和24小时时，GnRHR-Luc活性增加幅度较小。霍乱毒素和二丁酰环磷腺苷（dBcAMP）均显著刺激GnRHR-Luc活性。用dBcAMP预处理也增强了对布舍瑞林刺激的GnRHR-Luc活性的刺激程度。百日咳毒素未诱导基础或布舍瑞林刺激的GnRHR-Luc活性。用10⁻⁹或10⁻⁷M布舍瑞林处理GGH3细胞6小时足以刺激cAMP释放显著增加。腺苷酸环化酶抑制剂SQ 22536不影响基础GnRHR-Luc活性，但显著降低布舍瑞林激活的GnRHR-Luc活性。这些结果表明，GnRH和cAMP激活GnRHR基因的转录活性，且GnRH部分通过cAMP途径激活GnRHR转录活性。逐步5'端缺失分析显示，相对于主要转录起始位点在-456、-381或-331位置进行5'端缺失后，基础及布舍瑞林或dBcAMP刺激的GnRHR-Luc活性始终得以保留，但在随后将启动子从相对于主要转录起始位点的-331截短至-255后，活性显著降低。然而，-255构建体仍保留对布舍瑞林和dBcAMP的反应性，且在两种刺激条件下相对活性保持相似。这些结果表明，位于-331和-255之间的元件对于GGH3细胞中GnRHR基因的转录活性是必需的，且小鼠GnRHR基因上对GnRH和cAMP的反应元件位于两个不同位点：-331和-255之间以及-255和+62之间。
