| Summary: | 博士 === 長庚大學 === 基礎醫學研究所 === 94 === Thyroid hormones (T3) regulate growth, development, differentiation, and metabolic processes by interacting with and activating thyroid hormone receptors or associated pathways. Both thyroid hormone receptor (TR) and testicular orphan receptor 4 (TR4) belong to the nuclear hormone receptor superfamily and are ligand-dependent transcription factors. Because of TR4 and TR sharing similar response element configurations, known as a direct repeat with four nucleotide spacings, this study investigated the possibility of cross-talk between the two ligand-dependent signal transduction pathways. The transcriptional activity of TR mediated via various thyroid hormone response elements (TREs) reporter construct was repressed by TR4 by up to 92%. The glutathione S-transferase (GST) pull-down assay was performed to determine whether the TR4 interacts with TR to account for the TR4 repressed the TR trans-activation. And the results indicate that both the C (DNA binding) domain of TRα1 and the A/B and C domain of TRβ1 interact with the TR4 protein while the TR4 interacts with the TRα1 or TRβ1 proteins through the DNA and ligand binding domains. To further understand the mechanism of TR4 suppresses T3-signalling, electrophoretic mobility shift assay (EMSA) was carried out. TR homodimer or TR/RXR (retinoid X receptor) heterodimer reduced up to 65% by adding increasing amounts of TR4 protein. Therefore, EMSA and GST pull-down assays demonstrated the direct binding of TR proteins to TR4 and revealed that the interaction is important to the TR4-mediated suppression of TR-transactivation. Additionally, furin was elevated approximately 3.4-fold and 2.8-fold in HepG2-TRα1 cells at the protein and mRNA levels, respectively, after 48 hr of 100 nM T3 treatment. Increasing expression of TR4 repressed approximately 20 to 60% of furin promoter activities, which were stimulated roughly 5-fold by 10 nM T3 for 24 hr. Similarly, the up-regulated furin protein and mRNA levels by T3 were gradually suppressed by increasing amounts of TR4. In addition, α-fetoprotein (AFP) is negatively regulated by T3. And the suppression of AFP by T3 was gradually reversed by co-transfection with increasing amounts of TR4 expression plasmid. Taken together, interaction of TR4 with TR proteins may reduce TR binding to its cognate TRE. Subsequently, the interaction between TR4 and TR alters TR target gene stimulation or repression by T3. These findings suggest that TR4 may influence furin and AFP metabolism regulated by T3/TR at the transcriptional level. This study proposes a mechanism for cross-talk and potential antagonism between TRs and TR4.
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