The Role of AP-1 in the Gonadotrope

描述(申请人提供)：女性的配子发生由来自脑下的卵泡刺激素(FSH)控制，而卵泡刺激素又由来自下丘脑的促性腺激素释放激素(GnRH)调节。GnRH通过诱导即刻早期基因c-Fos来调节FSH的合成，c-Fos与c-Jun异源二聚后，产生一个AP-1转录因子，与FSH 2亚单位基因的启动子结合。这一应用的总体目标是确定GnRH诱导c-Fos的机制，并阐明AP-1在体内促性腺激素中的作用。AP-1是已知的唯一参与GnRH诱导FSH2亚单位基因的因子。C-Fos是促性腺激素释放激素诱导作用最强的基因，是AP-1亚型中最受调控的基因，而c-jun的表达受激素处理的影响较小。GnRH诱导促性腺激素细胞c-Fos的分子机制尚不清楚。AP-1在体内生殖中的作用还没有确定。在这里，我将利用新的方法来描述GnRH调节c-Fos表达的潜在机制。在第一个目的中，我将确定GnRH诱导c-Fos的分子机制。在第二个目标中，我将确定蛋白质稳定性和降解的调节，因为c-Fos是一个即刻早期基因，具有非常不稳定的信息和不稳定的蛋白质，其细胞浓度受周转水平和表达水平的控制。在这项提议的第三个目的中，我将利用转基因动物来确定AP-1在体内繁殖中的功能。首先，我将评估c-fos缺陷动物的生殖功能和促性腺激素的表达，然后，作为补充方法，使用在脑下垂体促性腺激素中缺乏c-jun的小鼠。Jun异构体是Fos异构体必需的异二聚体伙伴，它们的相互作用产生了一种与DNA结合的AP-1转录因子。尤其是c-jun对发育至关重要，因为基因敲除的动物在子宫中死亡，我将利用Cre-lox系统来分析c-jun在体内促性腺激素中的作用。这些研究的结果将有助于我们理解影响促性腺激素基因表达的分子机制，并为深入了解哺乳动物生殖系统的生理学和病理生理学提供帮助。我建议在细胞培养模型中扩大我们对GnRH诱导促性腺激素c-Fos基因表达的分子机制的理解，这将有助于更好地理解GnRH对垂体基因表达的调控，以及利用转基因小鼠体内AP-1的功能。公共卫生相关性：哺乳动物的生育能力主要由促性腺激素释放激素通过调节促性腺激素基因的表达和分泌对脑下垂体促性腺激素细胞群的影响来调节。促性腺激素，卵泡刺激素和黄体生成素，作用于性腺，刺激配子发生和类固醇生成。这项建议将解决GnRH在促性腺激素细胞中基因调控的分子机制。

DESCRIPTION (provided by applicant): Gametogenesis in females is controlled by follicle-stimulating hormone (FSH) from the pituitary, which is, in turn, regulated by gonadotropin-releasing hormone (GnRH) from the hypothalamus. GnRH regulates FSH synthesis through induction of the immediate early gene, c-Fos, which, upon heterodimerization with c-Jun, creates an AP-1 transcription factor that binds the promoter of the FSH 2-subunit gene. The overall goal of this application is to ascertain the mechanism of GnRH induction of c-Fos and to elucidate the role of AP-1 in the gonadotrope in vivo. AP-1 is the only factor known to be involved in the induction of the FSH 2-subunit gene by GnRH. c-Fos, the most highly GnRH-induced gene in the gonadotrope, is the most regulated AP-1 isoform, while c-Jun expression varies less with hormone treatment. The molecular mechanism of c-Fos induction by GnRH in the gonadotrope cell is not known. Neither has a role for AP-1 in reproduction in vivo been determined. Herein, I will utilize novel approaches to delineate the mechanisms underlying the regulation of c- Fos expression by GnRH. In the first aim, I will determine the molecular mechanism of c-Fos induction by GnRH. In the second aim, I will determine the regulation of protein stability and degradation, since c-Fos is an immediate-early gene with a very unstable message and labile protein, and its cellular concentration is controlled on the level of turnover, as much as on the level of expression. In the third aim of this proposal, I will determine the function of AP-1 in reproduction in vivo with the use of genetically modified animals. First, I will assess reproductive function and gonadotropin expression in c-Fos deficient animals and then, as complementary approach, use mice that lack c-Jun specifically in the pituitary gonadotrope. Jun isoforms are obligatory heterodimeric partners for Fos isoforms, whose interaction creates an AP-1 transcription factor that binds DNA. c-Jun, in particular, is essential for development, since knock-out animals die in utero, and with the use of CRE-lox system, I will analyze the function of c-Jun in gonadotrope in vivo. Results obtained from these studies will make substantial contributions to our understanding of the molecular mechanisms that affect gene expression in the gonadotrope and provide insight into the physiology and pathophysiology of the mammalian reproductive system. I propose to expand our understanding of molecular mechanisms of GnRH induction of c- Fos gene in the gonadotrope in a cell culture model, which will lead to a better understanding of GnRH regulation of gene expression in the pituitary, and the function of AP-1 in vivo, utilizing genetically modified mice. PUBLIC HEALTH RELEVANCE: Fertility in mammals is regulated primarily by gonadotropin-releasing hormone effect on the gonadotrope cell population in the pituitary gland, through modulation of gonadotropin gene expression and secretion. Gonadotropins, follicle-stimulating hormone and luteinizing hormone, work on the gonads to stimulate gametogenesis and steroidogenesis. This proposal will address the molecular mechanism of gene regulation by GnRH in the gonadotrope cell.