Thyroid hormone conversion in vitro and ex vivo studies

我们实验室目前正在体外鉴定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。最近，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，表明该酶的活性增加。我们的数据与基因型/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。

Our laboratory is currently characterizing in vitro 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. Very recently, 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. Our data, consistent with the 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.. Pathophysiology of thyrotoxicosis in patients affected by McCune-Albright syndrome. The clinical observation that McCune-Albright syndrome (MAS) is associated with hyperthyroidism with a shift in the ratio of circulating T3/T4 has led to the hypothesis that this finding could be at least in part explained by a an intra-thyroidal activation of the type-2 deiodinase gene. This is in keeping with the molecular pathology of MAS, i.e. activating mutations in GNAS1 gene resulting in ligand-independent inappropriately elevated levels of intracellular cAMP. We thus speculated that activation of the cAMP-driven deiodinase type-2 gene could explain at least in part these findings. Our in vitro and ex-vivo data indicate that, consistent with our experimental hypothesis, the type-2 deiodinase is constitutively activated in MAS. Collaborative efforts with Dr. Collins (NIDCR) are aimed to develop an in vitro system to test specific inhibitors of the mutant alleles of GNAS. Pre-adipocyte differentiation and culture. We are currently in the development phase of a system of re-differentiation of human adipocytes from stromal cells obtained during adipose tissue biopsy. This system will allow to test in a controlled fashion the effects of specific genotypes on adipose tissue function relatively independent of the metabolic status of the subject at the time of the sampling.