Thyroid hormone conversion in vitro and ex vivo studies

基于实验室的研究的总体努力是提供从我们小组开发和实施的临床方案中获得的结果的机械解释。具体而言，基本的研究目标是表征甲状腺激素作用的作用及其在时间和组织特异性信号传递中的局部组织特异性调制。 我们实验室目前正在体外和离体表征2型脱碘酶基因常见多态性的功能活性。为了实现这一目标，我们建立了一个基于细胞培养的2型脱碘酶表达系统。采用标准生物化学方法进行酶活性测定;通过免疫沉淀和迁移率变动测定检测蛋白质/蛋白质和核酸/蛋白质相互作用。DIO 2基因的5 UTR区域中的常见多态性（258 A/G DIO 2）与循环T3/T4比率的变化相关，表明体内酶活性的增加导致反应平衡的变化。我们的体外和离体数据，与其他基因型/表型关联研究一致，表明258 A/G DIO 2变体通过置换推定的阻遏物诱导基因转录增加。目前的努力旨在表征与多态性相互作用的假定阻遏因子。对数据的初步分析表明有多个候选转录因子。我们目前的努力旨在表征阻遏物。 前脂肪细胞分化和培养。在过去的两个财政年度中，我们集中精力开发一种从脂肪组织活检中获得的基质细胞再分化人类脂肪细胞的系统。该系统将成为以受控方式测试特定基因型对脂肪组织功能的影响的平台，而与采样时受试者的代谢状态无关。我们目前正在使用这个实验模型来表征甲状腺激素在脂肪细胞分化和调节其转录体中的作用。进一步的努力是针对表征该系统的转录模式在整个分化过程中，特别关注棕色脂肪特异性基因的表达。我们现在正在表征从不同解剖位置收获的前脂肪细胞的差异分子特征，以及它们在棕色脂肪细胞中分化的潜力。 原代培养甲状腺滤泡细胞2型脱碘酶测定。与Gershengorns博士团队（NIDDK-CEB）开展的合作工作开发了一种可靠的测定方法，用于测量甲状腺细胞原代培养物中2型脱碘酶活性，作为TSH-cAMP途径的读数。该系统已成功地用于测试TSH受体激动剂的活性。 开发基于细胞的系统来表征调节2型脱碘酶活性的物质的作用。我们目前正在开发稳定表达2型脱碘酶的细胞系和甲状腺激素反应元件（TRE）驱动的报告构建体，以评估调节2型脱碘酶活性的小物质的活性。 常见多态性对甲状腺激素抵抗综合征（RTH）临床表现的影响在与Forreset博士的合作中，我们描述了β-2甲状腺激素受体基因内含子控制区中常见SNP的作用。我们已经证明，一个共同的SNP导致垂体，组织特异性，增加基因的转录。在垂体选择性RTH的指示病例中，该SNP与β受体的致病性R338 W突变顺式。因此，这种组合导致突变基因的组织选择性过度表达，最终产生垂体选择性显性负性状态，重现了索引病例的特殊表型。这项研究的结果最近发表了，我们正在与道格拉斯·福雷斯特博士合作开发一种翻译方案，旨在描述β-2甲状腺激素受体基因调节区多态性的作用。RTH综合征患者的感觉系统。

The overall effort of the laboratory-based research is to provide mechanistic interpretation of the findings obtained from the clinical protocols developed and implements by our group. Specifically, the underlying research goal is the characterization of the role of thyroid hormone action and its local, tissue-specific modulation in the delivery of time- and tissue specificity of the signal. Our laboratory is currently characterizing in vitro and ex vivo the functional activity of common polymorphisms of type-2 deiodinase gene. In order to achieve this goal, we have established a cell culture-based type-2 deiodinase expression system. Standard biochemistry methods are utilized for the enzymatic activity assay; protein/protein and nucleic acids/protein interactions are tested by immunoprecipitation and mobility shift assay. A common polymorphism in the 5UTR region of the DIO2 gene (258 A/G DIO2) has been associated with a shift in the ratio of circulating T3/T4, suggesting an increased in the activity of the enzyme in vivo leading to a shift of the reaction equilibrium. Our in vitro and ex vivo data, consistent with others genotype/phenotype association studies, indicate that the 258 A/G DIO2 variant induces an increase in the transcription of the gene by displacing a putative repressor. Current efforts are aimed to characterize the putative repressor factor interacting with the polymorphism. Preliminary analysis of the data has indicated multiple candidate transcription factors. Our current efforts are aimed to the characterization of the repressor. Pre-adipocyte differentiation and culture. During the past two FYs we have focused our effort in developing a system of re-differentiation of human adipocytes from stromal cells obtained during adipose tissue biopsy. This system will become the platform to test in a controlled fashion the effects of specific genotypes on adipose tissue function independently of the metabolic status of the subject at the time of the sampling. We are currently using this experimental model to characterize the role of thyroid hormones in the differentiation of the adipocytes and in the modulation of their transcriptosome. Further effort is directed toward characterizing the transcriptional pattern of this system throughout the differentiation process with a particular focus on the expression of brown-fat specific genes. We are now characterizing the differential molecular signature of pre-adipocytes harvested from different anatomical locations, as well as their potential for differentiation in brown adypocytes. Type-2 deiodinase assay in primary culture of follicular thyroid cells. Collaborative work carried out with Dr. Gershengorns group (NIDDK-CEB) has led to the development of a reliable assay for the measurement of type-2 deiodinase activity in primary cultures of thyroid cells as a read-out of TSH-cAMP pathway. This system has been successfully utilized to test the activity of agonists of the TSH receptor. Development of a cell-based system to characterize the effects of substances modulating the type-2 deiodinase activity. We are currently developing cell line stably expressing type-2 deiodinase and a Thyroid Hormone Responsive Element (TRE)-driven reporter construct in order to evaluate the activity of small substances modulating the type-2 deiodinase activity. Effects of common polymorphisms in the modulation of the clinical presentation of the Resistance to Thyroid Hormone syndrome (RTH). In collaboration with Dr. Forreset we have characterized the role of common SNPs in the intron control region of the beta-2 thyroid hormone receptor gene. We have demonstrated that a common SNP causes a pituitary, tissue-specific, increase in transcription of the gene. This SNP is in cis with a pathogenic R338W mutation of the beta-receptor in the index case of pituitary selective RTH. Thus this combination causes a tissue selective over expression of the mutant gene ultimately generating a pituitary-selective dominant-negative state, recapitulating the peculiar phenotype of the index case. The results of this study have been recently published, and we are developing a translational protocol in collaboration with Dr. Douglas Forrest aimed to perform a haracterization of the role of polymorphisms in the regulatory region of the beta-2 thyroid hormone receptor gene in RTH syndrome patients with respect to their sensory system.