Control of nuclear thyroid hormone signaling by cell surface proteins

描述(申请人提供)：甲状腺疾病影响超过2500万美国人，是第二常见的代谢性疾病。甲状腺激素(TH)对生长、发育和新陈代谢至关重要，它通过结合细胞核中的TH受体和改变基因表达来发挥作用。重要的是，TH必须穿过血液的质膜，才能与细胞核中的TH受体结合。这种易位过程中的一种细胞表面蛋白，L型氨基酸转运体LAT1，在TH依赖的发育过程中强烈上调。有趣的是，LAT1的表达也与快速分裂的细胞有关，如激活的淋巴细胞和癌症。尽管已经对LAT1的功能进行了很多体外研究，但LAT1在体内的发育作用还不是很清楚。本项目的目标是研究LAT1在发育过程中如何调节组织对TH的反应。这个项目补充了研究人员的长期目标，即阐明细胞质蛋白控制TH信号的潜在分子机制。为了实现这一目标，计划在具体目标1中开发和表征一种改进的四环素诱导转基因系统。他将设计、生产和鉴定转基因诱导物和报告系，当它们杂交时，将能够产生高水平的转基因诱导。这个改进的系统将使我们能够最大限度地减少泄漏的转基因表达，减少由插入位置引起的转基因表达水平的变异性，并通过不同剂量的诱导化学物质多西环素(Dox)来“拨入”转基因表达水平。此外，该系统还可以扩展以实现组织特异性转基因诱导。初步数据显示，在没有Dox的情况下，最低限度的泄漏表达，而在Dox存在的情况下，转基因诱导很高。然后，《特定目标2》将研究LAT1在TH依赖发育中的体内作用。研究人员将使用目标1的转基因技术来验证LAT1在体内调节TH信号的假设。他将在转基因蝌蚪中比较TH诱导的形态和基因调控变化的速度和幅度，并用Dox和不用Dox来诱导LAT1的过度表达。该项目有望揭示LAT1如何在体内调节TH信号的基本见解。特别是，TH转运体LAT1的过表达预计将增加TH对核TH受体的利用率，这是通过增加TH依赖的基因表达和形态变化来衡量的。这个项目的一个重要的积极影响是，调查者将扩展一个成熟的系统的用途，研究TH依赖的发育，青蛙的变态，并为青蛙群落提供新的转基因资源。这些资源将对各种实验方法有用。此外，当考虑将LAT1作为治疗甲状腺疾病和癌症的药物开发的靶点时，了解LAT1如何调节体内组织对TH的反应是重要的。 项目简介：研究人员正在研究细胞表面蛋白LAT1在发育过程中如何影响甲状腺激素信号。重要的是，LAT1已被确定为治疗甲状腺疾病以及相关疾病和并发症的药物靶点，如肥胖症、心血管疾病和癌症。因此，当考虑使用针对LAT1的药物来评估潜在的益处和副作用时，了解LAT1的发育作用是重要的。

DESCRIPTION (provided by applicant): Thyroid diseases affect over 25 million Americans and constitute the second most common group of metabolic disorders. Thyroid hormone (TH) is critical for growth, development, and metabolism, and works by binding TH receptors in the cell nucleus and changing gene expression. Importantly, TH must cross the plasma membrane from the blood to bind TH receptors in the nucleus. One cell surface protein in this translocation process, the L-type amino acid transporter LAT1, is strongly up-regulated during TH-dependent development. Interestingly, LAT1 expression is also associated with rapidly dividing cells, such as activated lymphocytes and cancer. Even though many in vitro studies on LAT1 function have been carried out, the in-vivo developmental role of LAT1 is not well understood. The objective of the current project is to study how LAT1 regulates tissue responses to TH during development. This project complements the investigator's long-term goal of elucidating molecular mechanisms underlying control of TH signaling by cytoplasmic proteins. To achieve this objective, the plan is to, in Specific Aim 1, develop and characterize an improved tetracycline-inducible transgenic system. He will design, produce, and characterize transgenic inducer and reporter lines that, when crossed, will be capable of high levels of transgene induction. This improved system will enable us to minimize leaky transgene expression, reduce variability in transgene expression levels caused by insertion sites, and "dial in" transgene expression levels with the inducing chemical doxycycline (Dox) administered at different doses. In addition, this system can expand to enable tissue-specific transgene induction. Preliminary data showed minimal leaky expression in the absence of Dox and high transgene induction in the presence of Dox. Specific Aim 2 will then examine the in-vivo role of LAT1 in TH-dependent development. The investigator will use the transgenic technology from Aim 1 to test the hypothesis that LAT1 modulates TH signaling in vivo. He will compare rate and magnitude of TH-induced morphological and gene regulation changes in transgenic tadpoles treated with and without Dox to induce LAT1 overexpression. This project is expected to reveal fundamental insights about how LAT1 regulates TH signaling in vivo. In particular, it is expected that overexpression of the TH transporter LAT1 will increase availability of TH to nuclear TH receptors as measured by increased TH-dependent gene expression and morphological change. A significant positive impact of this project is that the investigator will extend the usefulness of a well-established system for studying TH-dependent development, frog metamorphosis, and provide new transgenic resources to the frog community. These resources will be useful for a wide variety of experimental approaches. In addition, knowledge of how LAT1 regulates tissue responses to TH in-vivo is significant when considering LAT1 as a target for drug development in the treatment of thyroid disorders and cancer. PROJECT NARRATIVE: The investigator is studying how the cell surface protein LAT1 affects thyroid hormone signaling during development. Importantly, LAT1 has been identified as a drug target in the treatment of thyroid diseases and related disorders and complications, such as obesity, cardiovascular disease, and cancer. Thus, knowledge of developmental roles of LAT1 is important when considering use of drugs targeting LAT1 to evaluate potential benefits and side-effects.