MECHANISMS OF REGULATION OF GNRH GENE EXPRESSION

该计划的总体目标是确定和表征 调节GnRH基因表达的细胞和分子机制。 将在永生化小鼠GnRH神经元中进行初步研究。 GT 1 -7细胞模型。 我们将重点关注PKC和Ca++信号 转导系统，因为它们已经被证明可以引起 对GnRH转录、mRNA周转和分泌有显著影响。 此外，Ca++是细胞内激活的主要途径， 兴奋性氨基酸（EAA）谷氨酸，我们以前已经表明， NMDA对GnRH基因表达有非常迅速的影响， 细胞质水平 这些调查结果将扩大到 为了验证观察到的机制是主要的 重要的是在体内。有四个具体目标： 目的1：鉴定小鼠GnRH启动子和反式- 负调节的作用因素 通过PKC和钙通道在GT 1 -7细胞中。两个的功能 还将鉴定小鼠特异性启动子元件。 目的2：先前在GT 1 -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。 Ca ~（++）离子载体对GnRH也有类似的作用 mRNA稳定性 我们的假设是，GnRH mRNA的周转起着重要的作用。 在设定GnRH基因表达水平中起重要作用。 我们将 确定激活PKC和Ca++通路的机制， 降低GT 1 -7细胞中GnRH mRNA的稳定性。 目的3：阐明GnRH mRNA快速变化的机制， 水平在下丘脑中被激发。 我们将使用EAA范式 先前显示在体内显著调节GnRH基因表达 并分析多聚腺苷酸和多聚核糖体载量的相对水平的变化， 大鼠下丘脑神经元GnRH mRNA。 以确定后- 存在于GnRH mRNA中的转录调节元件在体内起作用， 将产生表达突变GnRH mRNA构建体的转基因小鼠， 分析EAA处理的效果。 目的4：利用GT 1细胞灌流，报道了不同的灌流模式， EAA的加入引起GnRH释放或Ca++的不同反应 activation.我们还看到分泌的GnRH肽被切割成 GnRH（1-5）随后拮抗NMDA受体，可能是一种 GnRH发挥抑制性超短环反馈的机制 GnRH神经元。 在这项研究中，我们将确定是否有不同的模式， 用EAA处理GT 1 -7细胞对GnRH基因有不同的影响 转录和/或GnRH mRNA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.