Cho S, Cho H, Geum D, Kim K
Department of Molecular Biology, Seoul National University, South Korea.
Brain Res Mol Brain Res. 1998 Feb;54(1):74-84. doi: 10.1016/s0169-328x(97)00325-2.
The present study attempts to examine the possible involvement of retinoic acid (RA) in the regulation of gonadotropin-releasing hormone (GnRH) release and gene expression in the rat hypothalamic fragments and GT1-1 neuronal cells in vitro. During a short-term period (2h), RA (0.01-1 microM) increased GnRH release in a dose-related manner. Time-course experiments showed that RA rapidly increased GnRH release by 30 min in both cells. RA-induced GnRH release was slowly attenuated in the next incubation period in hypothalamic fragments, but rapidly returned to control levels in GT-1 cells. In hypothalamic fragments, GnRH mRNA levels decreased, but in GT1-1 cells, no change in GnRH mRNA levels was observed. We then extended the incubation time to see any changes in GnRH mRNA levels by RA in GT1-1 cells. In a long term (up to 48 h), RA increased GnRH mRNA levels in a dose- and time-related manner. Significant increase in GnRH mRNA levels by RA (at higher than 10 nM) was observed within 12h. Transient transfection experiments with a luciferase reporter vector containing more than 3 kb of the rat GnRH 5'-flanking region (-3002 to +88) revealed that RA also increased the rat GnRH promoter activity in a similar dose-and time-dependent manner, suggesting that increases in GnRH mRNA levels are attributable, at least in part, to the enhanced gene transcription. The promoter analysis with the 5'-deletional constructs demonstrated that cis-elements responsible for the RA action may reside within -1640/-1438 of the rat GnRH promoter, where multiple direct or palindromic arrangements of the AGGTCA-related sequences exist. We also showed that GT1-1 cells as well as the hypothalamic tissues express mRNA for multiple subtypes of retinoid receptors, and that reporter plasmids with three copies of the strong retinoic acid response element (RARE) were activated by 80 folds upon treatment with RA in GT1-1 cells, suggesting that retinoid receptors in GT1-1 cells are functional. Taken together, the present study strongly suggests that RA is an important regulator of the GnRH neurons.
本研究旨在探讨视黄酸（RA）在体外对大鼠下丘脑片段和GT1-1神经元细胞中促性腺激素释放激素（GnRH）释放及基因表达的调控作用。在短期（2小时）内，RA（0.01 - 1微摩尔）以剂量相关的方式增加GnRH的释放。时间进程实验表明，RA在30分钟内迅速增加了两种细胞中GnRH的释放。在下丘脑片段中，RA诱导的GnRH释放在接下来的孵育期缓慢减弱，但在GT-1细胞中迅速恢复到对照水平。在下丘脑片段中，GnRH mRNA水平下降，但在GT1-1细胞中，未观察到GnRH mRNA水平的变化。然后我们延长孵育时间，观察RA对GT1-1细胞中GnRH mRNA水平的影响。在长期（长达48小时）内，RA以剂量和时间相关的方式增加GnRH mRNA水平。在12小时内观察到RA（高于10纳摩尔）使GnRH mRNA水平显著增加。用含有大鼠GnRH 5'-侧翼区域超过3千碱基（-3002至+88）的荧光素酶报告载体进行瞬时转染实验表明，RA也以类似的剂量和时间依赖性方式增加大鼠GnRH启动子活性，这表明GnRH mRNA水平的增加至少部分归因于基因转录增强。用5'-缺失构建体进行的启动子分析表明，负责RA作用的顺式元件可能位于大鼠GnRH启动子的-1640 / -1438区域内。在该区域存在多个与AGGTCA相关序列的直接或回文排列。我们还表明，GT1-1细胞以及下丘脑组织表达多种视黄酸受体亚型的mRNA，并且在GT1-1细胞中，用RA处理后，含有三个拷贝强视黄酸反应元件（RARE）的报告质粒被激活了80倍，这表明GT1-1细胞中的视黄酸受体具有功能。综上所述，本研究强烈表明RA是GnRH神经元的重要调节因子。
