Wong R, Zhu X G, Pineda M A, Cheng S Y, Weintraub B D
Molecular and Cellular Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Mol Med. 1995 Mar;1(3):306-19.
Mutations in the ligand-binding domain of the thyroid hormone receptor beta (TR beta) gene cause the syndrome of resistance to thyroid hormone (RTH). The clinical phenotype results from the antagonism of the normal TR alpha and the non-mutated TR beta alleles by the TR beta 1 mutants, via a dominant negative effect. There is, however, marked heterogeneity of organ resistance within and among kindreds with RTH. This study examines the potential role of cell type in modulating the dominant negative potency of human TR beta 1 (h-TR beta 1) mutants.
Transient transfections were performed in HeLa and NIH3T3 cells, using a wild type (WT) and three naturally occurring mutant h-TR beta 1 constructs, and three natural thyroid hormone response elements (TREs). Immunocytochemistry was performed to detect levels of TR beta 1 expression in these two cell types. In order to determine how TR beta 1 interacts with other cellular partners, gel-shift analyses using HeLa and NIH3T3 nuclear extracts were performed.
Transfection studies using WT h-TR beta 1 in HeLa and NIH3T3 cells, showed that the 3,3',5-triiodothyronine (T3)-induced transactivation of the different TREs varied between cell types. Unlike the non-T3-binding h-TR beta 1 mutant, PV, mutants ED and OK displayed the expected T3-induced dose responsiveness in these two cell types. For each TRE examined, the magnitude of the dominant negative effect varied between the cell types. The levels of receptor expression in HeLa and NIH3T3 cells were identical, as determined by immunocytochemistry. Gel-shift analyses showed differences in the formation of hetero- and homodimers depending on both the cell type and TRE motif.
The cell type in which a mutant receptor operates affects the relative amounts of hetero- and homodimers. Together with the nature of the mutation and the TRE-motif, this could modulate the dominant negative action of mutant receptors in different tissues, which, in turn, could contribute to the variable phenotypic characteristics of RTH.
甲状腺激素受体β（TRβ）基因配体结合域的突变会导致甲状腺激素抵抗综合征（RTH）。临床表型是由TRβ1突变体通过显性负效应拮抗正常的TRα和未突变的TRβ等位基因所致。然而，RTH家系内部和家系之间存在明显的器官抵抗异质性。本研究探讨细胞类型在调节人TRβ1（h-TRβ1）突变体显性负效应中的潜在作用。
使用野生型（WT）和三种天然存在的突变型h-TRβ1构建体以及三种天然甲状腺激素反应元件（TRE），在HeLa和NIH3T3细胞中进行瞬时转染。进行免疫细胞化学检测这两种细胞类型中TRβ1的表达水平。为了确定TRβ1如何与其他细胞伴侣相互作用，使用HeLa和NIH3T3核提取物进行凝胶迁移分析。
在HeLa和NIH3T3细胞中使用WT h-TRβ1进行转染研究表明，3,3',5-三碘甲状腺原氨酸（T3）诱导的不同TRE的反式激活在细胞类型之间存在差异。与非T3结合的h-TRβ1突变体PV不同，突变体ED和OK在这两种细胞类型中表现出预期的T3诱导的剂量反应性。对于所检测的每种TRE，显性负效应的大小在细胞类型之间有所不同。通过免疫细胞化学确定，HeLa和NIH3T3细胞中的受体表达水平相同。凝胶迁移分析表明异源二聚体和同源二聚体的形成取决于细胞类型和TRE基序。
突变受体发挥作用的细胞类型会影响异源二聚体和同源二聚体 的相对数量。与突变的性质和TRE基序一起，这可能会调节突变受体在不同组织中的显性负作用，进而可能导致RTH可变的表型特征。
