The Role of AP-1 in the Gonadotrope

描述（由申请方提供）：雌性的配子发生由垂体的促卵泡激素（FSH）控制，而FSH又由下丘脑的促性腺激素释放激素（GnRH）调节。GnRH通过诱导立即早期基因c-Fos调节FSH合成，c-Fos在与c-Jun异源二聚化后产生AP-1转录因子，结合FSH 2亚基基因的启动子。本申请的总体目标是确定c-Fos的GnRH诱导机制，并阐明AP-1在体内促性腺细胞中的作用。AP-1是已知参与GnRH诱导FSH 2亚基基因的唯一因子。c-Fos是促性腺激素释放激素诱导最高的基因，是调节最多的AP-1亚型，而c-Jun表达随激素处理变化较小。促性腺激素释放激素在促性腺细胞中诱导c-Fos的分子机制尚不清楚。AP-1在体内生殖中的作用也没有确定。在此，我将利用新的方法来阐明GnRH调节c-Fos表达的机制。在第一个目标中，我将确定c-Fos由GnRH诱导的分子机制。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是具有非常不稳定的信息和不稳定的蛋白质的立即早期基因，并且其细胞浓度在周转水平上受到控制，就像在表达水平上一样。在本提案的第三个目的中，我将使用转基因动物确定AP-1在体内生殖中的功能。首先，我将评估c-Fos缺陷动物的生殖功能和促性腺激素表达，然后作为补充方法，使用垂体促性腺激素中缺乏c-Jun的小鼠。Jun同种型是Fos同种型的强制性异二聚体伴侣，其相互作用产生结合DNA的AP-1转录因子。尤其是c-Jun，对于发育是必不可少的，因为敲除动物在子宫内死亡，并且使用CRE-lox系统，我将分析c-Jun在体内促性腺激素中的功能。从这些研究中获得的结果将为我们理解影响促性腺细胞基因表达的分子机制做出重大贡献，并为哺乳动物生殖系统的生理学和病理生理学提供见解。我建议在细胞培养模型中扩大我们对促性腺激素细胞中GnRH诱导c-Fos基因的分子机制的理解，这将导致更好地理解GnRH对垂体中基因表达的调节，以及利用遗传修饰小鼠体内AP-1的功能。公共卫生相关性：哺乳动物的生育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垂体中促性腺激素细胞群的作用来调节。促性腺激素、促卵泡激素和促黄体激素作用于性腺，刺激配子发生和类固醇生成。该建议将阐明促性腺激素释放激素在促性腺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.