MECHANISMS OF REGULATION OF GNRH GENE EXPRESSION

该计划的总体目标是确定和描述 调节GnRH基因表达的细胞和分子机制。 初步研究将在永生化的小鼠促性腺激素释放激素神经元中进行 细胞模型，GT1-7细胞。我们将关注PKC和Ca++信号 转导系统，因为它们已经被证明可以诱导 对GnRH转录、信使核糖核酸转换和分泌有显著影响。 此外，钙离子是细胞内激活的主要途径。 兴奋性氨基酸(EAA)和谷氨酸，我们之前已经证明 NMDA对GnRH基因的表达有极快的影响 细胞质水平。这些调查结果将扩展到#年的调查。 动物，以验证观察到的机制是主要的 在活体中的重要性。具体目标有四个： 目的1：鉴定小鼠促性腺激素释放激素启动子中的元件及其反式激动子。 负调控的中介作用因素 通过GT1-7细胞中的PKC和钙通路。两个人的作用 还将确定小鼠特有的启动子元件。 目的2：先前在GT1-7细胞培养中的研究表明佛波醇 酯类导致GnRH mRNA稳定性降低 Poly(A)尾长减少和核糖体数量减少 与GnRH mRNA相关。钙离子载体对促性腺激素释放激素也有类似的作用 信使核糖核酸稳定性。我们的假设是GnRH mRNA的周转对 在决定GnRH基因表达水平方面具有重要作用。我们会 确定PKC和Ca~(++)途径激活的机制 降低GT1-7细胞中GnRH mRNA的稳定性。 目的3：阐明促性腺激素释放激素(GnRH)mRNA快速变化的机制(S 水平在下丘脑中被激发。我们将使用EAA模式 以前被证明在体内显著调节GnRH基因的表达 并分析多聚体和多聚体相对负荷水平的变化。 大鼠下丘脑神经元GnRH基因的表达。以确定POST是否- GnRH基因转录调控元件在体内发挥作用 将创建表达突变型GnRH mRNA结构的转基因小鼠，并 分析了EAA处理的效果。 目的：利用GT1细胞灌流，报道了不同的灌流模式 EaAs对GnRH释放和Ca++释放的不同反应 激活。我们还看到分泌的促性腺激素释放激素多肽被切割成 GnRH(1-5)，随后拮抗NMDA受体，可能是 促性腺激素释放激素抑制超短环反馈的作用机制 促性腺激素释放激素神经元。在这项研究中，我们将确定不同的模式是否 EaaS对GT1-7细胞GnRH基因的影响 转录和/或GnRH mRNA在灌流系统中的稳定性。

The overall goal of this program is to identify and characterize the cellular and molecular mechanisms which regulate GnRH gene expression. Initial studies will be conducted in an immortalized mouse GnRH neuronal cell model, the GT1-7 cell. We will focus on the PKC and Ca++ signal transduction systems since they have already been shown to elicit significant effects on GnRH transcription, mRNA turnover and secretion. Moreover, Ca++ is a major intracellular path way activated by the excitatory amino acid (EAA) glutamate and we have previously shown that NMDA, has extremely rapid effects on GnRH gene expression at the cytoplasmic level. These findings will be extended to investigations in animals in order to verify that the observed mechanisms are of primary importance in vivo. There are four specific aims: Aim 1:Characterize the elements in the mouse GnRH promoter and the trans- acting factors which are responsible for mediating the negative regulation by the PKC and calcium pathways in the GT1-7 cells. The function of two mouse specific promoter elements will also be identified. Aim 2:Previous studies in cultures of GT1-7 cells have shown that phorbol esters cause a decrease in the stability of GnRH mRNA concomitant with a decrease in poly (A) tail length and a decrease in the number of ribosomes associated with GnRH mRNA. Ca++ ionophores have similar effects on GnRH mRNA stability. Our hypothesis is that GnRH mRNA turnover plays an important role in setting the level of GnRH gene expression. We will determine the mechanism by which activation of the PKC and Ca++ pathways decreases the stability of the GnRH mRNA in GT1-7 cells. Aim 3: Elucidate the mechanism(s) by which rapid changes in GnRH mRNA levels are elicited in the hypothalamus. We will use an EAA paradigm previously shown to significantly modulate GnRH gene expression in vivo and analyze changes in the relative level of polyA and polysome loading of GnRH mRNA in rat hypothalamic neurons. To determine if the post- transcriptional regulatory elements present in GnRH mRNA function in vivo, transgenic mice expressing mutant GnRH mRNA constructs will be created and the effects of EAA treatment analyzed. Aim 4: Using perifusion of GT1 cells, it was reported that different modes of addition of EAAs elicit different responses in GnRH release or in Ca++ activation. We have also seen that secreted GnRH peptide is cleaved to GnRH(1-5) which subsequently antagonizes the NMDA receptor, possibly a mechanism by which GnRH exerts inhibitory ultra-short loop feedback on GnRH neurons. In this study, we will determine if different modes of treatment of GT1-7 cells with EAAs will differentially affect GnRH gene transcription and/or GnRH mRNA stability in a perifusion system.