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

钙调磷酸酶/NFAT 和 ERK 通路在促性腺激素表达的脉冲性 GnRH 效应中的作用和相互依赖性

• 批准号: G0901763/1
• 负责人: Craig McArdle
• 金额: $ 50.95万
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2010
• 资助国家: 英国
• 起止时间: 2010 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=G0901763%2F1
• 关键词: Roles; interdependence; calcineurin; NFAT; ERK

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.

促性腺营养蛋白释放激素（GNRH）分泌在大脑的脉冲中，并作用于垂体以控制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对于人类繁殖绝对是必不可少的。 GNRH脉冲频率在生理上有所不同，例如通过月经周期增加排卵。脉冲GNRH给药模仿生理状况，增加了LH/FSH分泌在某些形式的不育和IVF治疗中。相反，持续的刺激会导致脱敏，因此LH/FSH分泌减少。这可以减少性类固醇，并用于治疗激素依赖性癌症（乳房，卵巢，前列腺等），鉴于其生理和治疗意义，人们对理解GNRH作用具有极大的兴趣。在这里，前提是知道GNRH如何激活其靶细胞（受体和涉及的生化反应）将提供其他治疗靶标。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，因此我们对细胞对脉冲GNRH的反应方式知之甚少。这主要是因为用于监测细胞内反应的技术非常密集，因此，为了解决此研究瓶颈，我们开发了更有效的系统来基于成像荧光蛋白与信号蛋白融合的荧光蛋白监测GNRH信号传导。 GNRH导致这些荧光记者从细胞质转移到细胞核。这可以通过自动显微镜来量化，从而提供活细胞读数以进行激活。具体而言，我们正在使用激活的T细胞（NFAT）-EFP使用细胞外信号调节激酶（ERK）-GFP和核因子。这些蛋白被认为是GNRH对LH和FSH表达的效应的介体，并且在其他系统中都解码了刺激脉冲频率。我们假设激活这些蛋白质的生化途径通过脉动刺激介导GnRH对LH和FSH表达的影响，并且它们是相互依存的（即它们在细胞质中影响一个人，并在核中LH和FSH基因启动子上收敛于LH和FSH基因启动子） 。我们已经计划了一系列实验来对此进行测试，并使用细胞成像测试细胞质信号的相互依赖性和免费分子方法来定义LH和FSH基因中的ERK和NFAT靶标，以及它们与GNRH作用的相关性。我们还计划扩展成像策略，以监视正常垂体细胞中的GNRH信号传导和转录，这是从未实现的。