Thyroid hormone conversion in vitro and ex vivo studies

我们实验室目前正在体外和离体研究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。标准生物化学方法用于酶活性测定；蛋白/蛋白和核酸/蛋白相互作用通过免疫沉淀和迁移转移试验检测。DIO2基因5UTR区域的常见多态性（258 A/G DIO2）与循环T3/T4比率的变化有关，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 DIO2变体通过取代假定的抑制因子诱导基因转录增加。目前的努力旨在表征与多态性相互作用的推定抑制因子。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。

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 FY 10 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 allows 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. 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. 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.