Jung N, Sun W, Lee H, Cho S, Shim C, Kim K
Department of Molecular Biology and Research Center for Cell Differentiation, College of Natural Sciences, Seoul National University, Seoul 151-742, South Korea.
Brain Res Mol Brain Res. 1998 Oct 30;61(1-2):162-9. doi: 10.1016/s0169-328x(98)00227-7.
The present study examined the regulatory mechanisms of GnRH gene expression by N-methyl-d-aspartic acid (NMDA) in immortalized hypothalamic GnRH neurons (GT1-1 cells). NMDA (100 microM) stimulated GnRH mRNA levels transiently at 2 h after treatment. Dose-response experiment showed that there was a biphasic action of NMDA on GnRH mRNA levels: GnRH mRNA levels were increased by NMDA at lower concentrations (10 and 100 microM), but not at higher concentrations (1 and 10 mM). NMDA (100 microM)-induced GnRH mRNA levels were efficiently blocked by pre-treatment with NMDA receptor antagonists, MK-801 and AP-5. We next examined the signal transduction pathways involved in NMDA-induced GnRH gene expression based on previous findings that NMDA signal propagates into the cell through Ca2+ and nitric oxide (NO) pathways in many neurons. While ionomycin, a Ca2+ ionopore, application failed to alter GnRH gene expression, treatment of GT1-1 cells with sodium nitroprusside (SNP), an NO donor, increased GnRH gene expression with a similar time course to NMDA treatment. Moreover, application of GT1-1 cells with nitric oxide synthase (NOS) inhibitors (l-NAME, d-NAME, and NA) prior to NMDA treatment, inhibited NMDA-induced GnRH gene expression. These results indicate that the effect of NMDA is mediated by the NO signalling cascade. The mouse GnRH promoter activity was also increased by NMDA at low concentration (100 microM), but not at high concentration (1 microM), confirming the biphasic action of NMDA on GnRH mRNA levels. Since NMDA (100 microM) and SNP (1 microM) markedly induced c-jun expression, but not c-fos expression, we hypothesized that Jun activation is responsible for the transcriptional activation of GnRH gene expression. To examine this, we performed two different experiments. Treatment of NMDA greatly increased the activity of heterologous promoter of Fos/Jun responsive sequence (-187/-69) from the mouse GnRH promoter fused to hsv-tk minimal promoter. Moreover, overexpression of c-jun induced GnRH promoter activity, while c-fos overexpression decreased GnRH promoter activity. Taken together, this study indicates that NMDA regulates GnRH gene expression in GT1-1 cells through the NO-Jun signal transduction pathway.
本研究检测了N-甲基-D-天冬氨酸（NMDA）对永生化下丘脑促性腺激素释放激素（GnRH）神经元（GT1-1细胞）中GnRH基因表达的调控机制。NMDA（100微摩尔）在处理后2小时短暂刺激GnRH mRNA水平。剂量反应实验表明，NMDA对GnRH mRNA水平有双相作用：较低浓度（10和100微摩尔）的NMDA可增加GnRH mRNA水平，但较高浓度（1和10毫摩尔）则无此作用。用NMDA受体拮抗剂MK-801和AP-5预处理可有效阻断NMDA（100微摩尔）诱导的GnRH mRNA水平。基于之前的研究结果，即NMDA信号在许多神经元中通过Ca2+和一氧化氮（NO）途径传入细胞，我们接下来检测了参与NMDA诱导的GnRH基因表达的信号转导途径。虽然应用离子霉素（一种Ca2+离子载体）未能改变GnRH基因表达，但用NO供体硝普钠（SNP）处理GT1-1细胞可增加GnRH基因表达，其时间进程与NMDA处理相似。此外，在NMDA处理前用一氧化氮合酶（NOS）抑制剂（L-NAME、D-NAME和NA）处理GT1-1细胞，可抑制NMDA诱导的GnRH基因表达。这些结果表明，NMDA的作用是由NO信号级联介导的。低浓度（100微摩尔）的NMDA可增加小鼠GnRH启动子活性，但高浓度（1微摩尔）则无此作用，这证实了NMDA对GnRH mRNA水平的双相作用。由于NMDA（100微摩尔）和SNP（1微摩尔）显著诱导c-jun表达，但不诱导c-fos表达，我们推测Jun激活负责GnRH基因表达的转录激活。为了验证这一点，我们进行了两个不同的实验。NMDA处理极大地增加了与单纯疱疹病毒胸苷激酶（hsv-tk）最小启动子融合的小鼠GnRH启动子中Fos/Jun反应序列（-187/-69）的异源启动子活性。此外，c-jun的过表达诱导GnRH启动子活性，而c-fos的过表达则降低GnRH启动子活性。综上所述，本研究表明，NMDA通过NO-Jun信号转导途径调节GT1-1细胞中的GnRH基因表达。
