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Cell type-dependent modulation of the dominant negative action of human mutant thyroid hormone beta 1 receptors
1Molecular and Cellular Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Background:
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.
Materials And Methods:
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.
Results:
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.
Conclusions:
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.
Insights
Cell type influences the dominant negative effects of thyroid hormone receptor beta (TR beta) mutants. This finding helps explain the varied resistance to thyroid hormone (RTH) symptoms observed in patients.
Area of Science:
- Endocrinology
- Molecular Biology
- Genetics
Background:
- Resistance to thyroid hormone (RTH) is caused by mutations in the thyroid hormone receptor beta (TR beta) gene.
- Mutant TR beta 1 proteins exert a dominant negative effect on normal TR alpha and TR beta alleles, leading to RTH.
- Significant variability in organ resistance exists among RTH patients and families.
Purpose of the Study:
- To investigate the role of cell type in modulating the dominant negative potency of human TR beta 1 (h-TR beta 1) mutants.
- To understand how different cellular environments affect the function of mutant TR beta 1.
Main Methods:
- Transient transfections of HeLa and NIH3T3 cells with wild-type and mutant h-TR beta 1 constructs.
- Immunocytochemistry to assess TR beta 1 expression levels.
- Gel-shift analyses to examine TR beta 1 interactions with cellular partners and DNA.
Main Results:
- Thyroid hormone (T3)-induced transactivation varied between cell types for wild-type h-TR beta 1.
- Mutant TR beta 1 receptors (ED and OK) showed T3-induced dose responsiveness, while mutant PV did not.
- The dominant negative effect magnitude differed between cell types, despite identical receptor expression levels.
- Gel-shift analyses revealed cell type- and TRE motif-dependent differences in hetero- and homodimer formation.
Conclusions:
- Cell type influences the formation of TR beta 1 hetero- and homodimers.
- The interplay between mutation type, TRE motif, and cell type-specific dimerization modulates the dominant negative action of mutant TR beta 1.
- These factors contribute to the heterogeneous clinical characteristics observed in RTH.