Roles and interdependence of calcineurin/NFAT and ERK pathways in pulsatile GnRH effects on gonadotrophin expression

促性腺激素释放激素（GnRH）从大脑以脉冲形式分泌，作用于垂体，控制LH和FSH的合成和分泌。这些激素反过来控制性腺中的性类固醇和生殖细胞的产生，因此GnRH对人类生殖至关重要。GnRH脉冲频率在生理上变化，例如，通过月经周期增加以驱动排卵。脉冲式GnRH给药模拟生理情况，在治疗某些形式的不孕症和IVF中增加LH/FSH分泌。相反，持续刺激导致脱敏，从而使LH/FSH分泌减少。这减少了性类固醇，用于治疗乳腺癌，卵巢癌，前列腺癌等。鉴于其生理和治疗的重要性，人们对了解GnRH的作用有着极大的兴趣。在这里，前提是了解GnRH如何激活其靶细胞（受体和所涉及的生化反应）将提供额外的治疗靶点。然而，这项工作的一个局限性是，大多数实验都使用了恒定的刺激，所以我们仍然对细胞如何对脉动GnRH做出反应知之甚少。这在很大程度上是因为用于监测细胞内反应的技术是非常劳动密集型的，因此为了解决这一研究瓶颈，我们开发了更有效的系统，用于监测基于成像荧光蛋白融合到信号蛋白的GnRH信号传导。GnRH使这些荧光报告分子从细胞质移动到细胞核。这可以通过自动显微镜进行定量，提供激活的活细胞读数。具体地，我们使用细胞外信号调节激酶（ERK）-GFP和活化T细胞的核因子（NFAT）-EFP。这些蛋白质被认为是GnRH对LH和FSH表达影响的介质，并且在其他系统中都解码刺激脉冲频率。我们假设激活这些蛋白质的生化途径介导GnRH对LH和FSH表达的影响，并且它们是相互依赖的（即它们在细胞质中相互影响，并在细胞核中会聚在LH和FSH基因启动子区）。我们已经计划了一系列实验来测试这一点，使用细胞成像来测试细胞质信号的相互依赖性和互补的分子方法来定义LH和FSH基因中的ERK和NFAT靶点以及它们与GnRH作用的相关性。我们还计划扩展我们的成像策略，以监测正常垂体细胞中的GnRH信号传导和转录，这是从未实现过的。

Gonadotrophin-releasing hormone (GnRH) is secreted in pulses from the brain and acts at the pituitary to control synthesis and secretion of LH and FSH. These hormones in turn control sex steroid and germ cell production in the gonads, so GnRH is absolutely essential for human reproduction. GnRH pulse frequency varies physiologically, increasing for example, through the menstrual cycle to drive ovulation. Pulsatile GnRH administration mimics the physiological situation, increasing LH/FSH secretion in treatment of some forms of infertility and in IVF. In contrast, sustained stimulation causes desensitisation so that LH/FSH secretion reduces. This reduces sex steroids and is used to treat hormone-dependent cancers (breast, ovary, prostate etc.) Given its physiological and therapeutic importance there is immense interest in understanding GnRH action. Here, the premise is that knowing how GnRH activates its target cells (the receptors and the biochemical responses involved) will provide additional therapeutic targets. A limitation of this work, however, is that most experiments have used constant stimulation so we still know remarkably little about how cells respond to pulsatile GnRH. This is largely because the techniques used to monitor responses within the cell are very labour intensive, so to resolve this research bottle-neck we have developed much more efficient systems for monitoring GnRH signalling based on imaging fluorescent proteins fused to signalling proteins. GnRH causes these fluorescent reporters to move from the cytoplasm to the nucleus. This can be quantified by automated microscopy, providing live-cell readouts for activation. Specifically, we are using extracellular signal regulated kinase (ERK)-GFP and nuclear factor for activated T-cells (NFAT)-EFP. These proteins are implicated as mediators of GnRH effects on LH and FSH expression and both decode stimulus pulse frequency in other systems. We hypothesise that the biochemical pathways activating these proteins mediate GnRH effects on LH and FSH expression with pulsatile stimulation, and that they are interdependent (i.e. that they influence one-another in the cytoplasm and converge on LH and FSH gene promoter regions in the nucleus). We have planned a series of experiments to test this, using cell imaging to test for interdependence of the cytoplasmic signals and complimentary molecular approaches to defining ERK and NFAT targets in LH and FSH genes as well as their relevance for GnRH action. We also plan to extend our imaging strategies to monitor GnRH signalling and transcription in normal pituitary cells, something that has never yet been achieved.