FoxO1 signaling in pituitary gonadotropes

描述（由申请人提供）：这份为期两年的NRSA个人培训奖学金申请概述了Danalea Skarra博士的培训和研究计划。斯卡拉博士设计了生殖神经内分泌学和生育代谢调节领域的综合强化培训计划。特别是，Skarra博士提出研究FOXO1对促黄体生成素b亚基（Lhb）启动子的抑制与垂体促性腺激素中FOXO1的胰岛素信号调节之间的整合。加州大学圣地亚哥分校拟议的工作和独特的培训环境将使斯卡拉博士实现她的长期目标，即成为生殖内分泌学领域的独立学术研究员，研究代谢紊乱如何影响生育能力。培训和发展将由生殖内分泌学专家Varykina Thackray博士（赞助商）和世界知名胰岛素和葡萄糖代谢研究员Jerrold Olefsky博士（共同赞助商）指导。此外，她还成立了一个由博士组成的指导委员会。Thackray和Olefsky，以及生殖神经内分泌专家Alexander Kauffman博士，GnRH信号专家Mark Lawson博士和FOXO1专家Karen Arden博士。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。本提案的研究部分验证了胰岛素抑制fox01增加Lhb转录和LH产生的假设。Thackray博士的实验室报道FOXO1抑制基础和gnrh诱导的Lhb合成，FOXO1的抑制定位于Lhb启动子的-150/-1。其他研究人员发现，该区域可能通过EGR1介导胰岛素诱导Lhb合成。此外，我们在垂体促性腺激素中检测到胰岛素诱导的FOXO1磷酸化和亚细胞定位变化，这与胰岛素在其他组织中对FOXO1的抑制作用一致。在Aim 1中，我们将使用荧光素酶测定、定量RT-PCR和western blot来确定FOXO1是否抑制EGR1活性，从而抑制Lhb转录。在Aim 2中，将使用垂体原代细胞，通过定量RT-PCR和免疫放射测定LH蛋白，研究FOXO1活性增加或FOXO1消融对Lhb mRNA的影响。目的3研究胰岛素对瘦小鼠和饮食诱导肥胖小鼠垂体FOXO1磷酸化和亚细胞定位的体内调节。本研究结果有可能在垂体促性腺激素水平上确定与多囊卵巢综合征和肥胖相关的不孕机制，并可能为使用潜在的fox01调节药物治疗糖尿病、良性肿瘤和癌症如何影响Lhb的产生提供见解。

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.