FoxO1 signaling in pituitary gonadotropes

描述（由申请人提供）：这个为期两年的个人NRSA培训奖学金申请概述了培训和研究计划博士。Skarra博士在生殖神经内分泌学和生育代谢调节领域设计了一个综合和强化的培训计划。特别是，Skarra博士建议研究FOXO 1抑制促黄体激素B亚基（Lh B）启动子和FOXO 1在垂体促性腺激素中的胰岛素信号调节之间的整合。在加州大学圣地亚哥分校的拟议工作和独特的培训环境将使Skarra博士实现她的长期目标，成为生殖内分泌学领域的独立学术研究人员，研究代谢紊乱如何影响生育能力。培训和发展将需要生殖内分泌学领域专家Varykina Thackray博士（申办者）和世界知名的胰岛素和葡萄糖代谢研究人员Jerrold Olefsky博士（共同申办者）的指导。此外，她还成立了一个指导委员会，由Thackray博士和Olefsky博士以及生殖神经内分泌专家亚历山大考夫曼博士、GnRH信号专家马克劳森博士和FOXO 1专家卡伦雅顿博士组成。Skarra博士的综合培训计划基于密集的研究经验，教学指导，负责任的研究行为培训以及关键职业技能的获得。该提案的研究部分测试了胰岛素抑制FOXO 1增加Lhb转录和LH产生的假设。Thackray博士的实验室报告说，FOXO 1抑制基础和GnRH诱导的Lhb合成，FOXO 1的抑制定位于Lhb启动子的-150/-1。其他研究人员已经发现该区域可能通过EGR 1负责胰岛素诱导Lhb合成。此外，我们可以检测到胰岛素诱导的垂体促性腺激素细胞中FOXO 1的磷酸化和亚细胞定位的变化，这与胰岛素对其他组织中FOXO 1的抑制作用一致。在目标1中，我们将使用荧光素酶测定、定量RT-PCR和蛋白质印迹来确定FOXO 1是否抑制EGFR 1活性，从而抑制Lhb转录。在目的2中，原代垂体细胞将被用于研究增加FOXO 1活性或消除FOXO 1对Lhb mRNA的影响，通过定量RT-PCR和LH蛋白定量的免疫放射分析。目的3研究胰岛素对瘦小鼠和饮食诱导肥胖小鼠垂体中FOXO 1磷酸化和亚细胞定位的体内调节。该提案的结果有可能在垂体促性腺激素水平上确定与PCOS和肥胖相关的不孕症机制，并且它可以深入了解使用潜在的FOXO 1调节药物治疗糖尿病，良性肿瘤和癌症如何影响Lhb的产生。

DESCRIPTION (provided by applicant): This two-year individual NRSA training fellowship application outlines the training and research plans for Dr. Danalea Skarra. Dr. Skarra has designed an integrated and intensive training program in the area of reproductive neuroendocrinology and metabolic regulation of fertility. In particular, Dr. Skarra proposes to study the integration between FOXO1 inhibition of the luteinizing hormone b subunit (Lhb) promoter and insulin signaling regulation of FOXO1 in pituitary gonadotropes. The proposed work and unique training environment at the University of California, San Diego will allow Dr. Skarra to achieve her long-term goal of becoming an independent academic researcher in the field of reproductive endocrinology, studying how metabolic disorders impact fertility. Training and development will entail mentoring by Dr. Varykina Thackray (Sponsor), an expert in the field of reproductive endocrinology, and Dr. Jerrold Olefsky (Co-Sponsor), a world-renowned researcher of insulin and glucose metabolism. Furthermore, she has established a Mentoring Committee comprised of Drs. Thackray and Olefsky, as well as Dr. Alexander Kauffman, a reproductive neuroendocrine expert, Dr. Mark Lawson, an expert in GnRH signaling, and FOXO1 expert Dr. Karen Arden. Dr. Skarra's comprehensive training plan is based on an intensive research experience, didactic instruction, training in responsible conduct of research, and acquisition of critical career skills. The research component of this proposal tests the hypothesis that insulin inhibition of FOXO1 increases Lhb transcription and LH production. Dr. Thackray's laboratory has reported that FOXO1 suppresses basal and GnRH-induced Lhb synthesis, and FOXO1's suppression localized to -150/-1 of the Lhb promoter. This region has been found by other researchers to be responsible for insulin induction of Lhb synthesis, potentially through EGR1. Furthermore, we can detect insulin-induced phosphorylation and subcellular localization changes of FOXO1 in pituitary gonadotropes, which is consistent with insulin's inhibitory effect on FOXO1 in other tissues. In Aim 1, we will determine if FOXO1 suppresses EGR1 activity, thereby suppressing Lhb transcription, using luciferase assays, quantitative RT-PCR and western blot. In Aim 2, primary pituitary cells will be used to investigate the effects of increased FOXO1 activity or ablation of FOXO1 on Lhb mRNA by quantitative RT-PCR and LH protein quantitation by immunoradiometric assay. Aim 3 investigates the in vivo regulation of insulin on FOXO1 phosphorylation and subcellular localization in pituitaries from lean and diet-induced obese mice. Results from this proposal have the potential to identify mechanisms of infertility related to PCOS and obesity at the level of the pituitary gonadotrope, and it may provide insight into how the use of potential FOXO1-modulating drug treatments for diabetes, benign tumors, and cancer could impact Lhb production.