The Hypothalamic-Pituitary-Thyroid axis, including the roles of thyrotropin releasing hormone (TRH), thyroid stimulating hormone (TSH), thyroxine (T 4 ) and triiodothyronine (T 3 ). Other forms of thyroid hormones are not included (e.g. T 2 and rT 3 ). Minus indicates a negative feedback loop. Reproduced with modifications from (Boas et al., 2006).

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... thyroid hormones (TH), triiodothyronine (T 3 ) and thyroxine (T 4 ) (Figure 1), are tyrosine-based hormones produced by the thyroid gland, a butterfly-shaped gland which in humans is located in front of the larynx just below the Adams apple. Low circulating TH levels are detected by the hypothalamus, that responds by releasing thyrotropin-releasing hormone (TRH). The released TRH, stimulates the pituitary gland to produce thyrotropin (also known as thyroid-stimulating hormone, TSH). In turn, TSH stimulates the thyroid to produce TH until levels in the blood return to normal. Circulating TH exerts a negative feedback control over the hypothalamus and pituitary, eventually controlling the release of TRH from the hypothalamus and TSH from the pituitary gland. This hormone system that involves the hypothalamus, pituitary, and thyroid gland, is known as the hypothalamus- pituitary-thyroid (HPT) axis (Figure 2) and regulates TH synthesis by a negative feedback regulatory ...

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... represent mean ± SD of triplicate experiments. Figure 2B), reaching a maximum induction of approximately ...

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... the 1280 compounds from the LOPAC library, 6 (0.5%) were identified as potential TR agonists (Figure 2), with the positive hit cut off being compounds that gave a ...

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... of the TRE based luciferase reporter gene assay with natural and synthetic thyroid hormones. All experimental conditions for culturing, transfecting, shifting to serum free media and measuring luciferase activity with the stable GH3 cells were thoroughly optimized before the final method for transfection and screening compounds was decided upon. For instance, transfection efficiency is optimal when transfecting GH3 cells with 2 $l/well of Lipofectamine 2000 and 800 ng of total DNA (Figure 2). When testing TR-mediated luciferase induction by compounds it is particularly important to use serum free PCM medium instead of culture medium supplemented with stripped serum to avoid background induction by low levels of thyroid hormones that might still be in the serum (Figure 3). For quantification of cell proliferation the resazurine method was preferred as it proved to be more sensitive than total protein ( Figure 4A). Also, 24 h of exposure to increasing concentrations of T 3 barely induced cell proliferation as opposed to 96 h exposure in the T-screen (Figure ...

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... TR" dominant GH3.TRE-Luc cell line was stably transfected with the same TR- (Figure 2A and 3A), with the GH3.TRE-Luc cells slightly more sensitive than the TR!.HeLa-Luc cells. In agreement with other studies, while the binding affinity of TR! or TR" is virtually identical, TR!-mediated responses induced by T 3 are consistently more sensitive (at the EC 50 level) than TR"-mediated responses in our transient transfection assays. Thus, the higher sensitivity of the TR" dominant GH3.TRE-Luc line is most likely due to differences in cellular context, such as coregulator expression, deiodinase activity or TH transport. were consistent with previous reports ...

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... most active agonists with EC 50 values lower than 10 "M were the retinoids (13- cis-retinoic acid and 13-cis-retinal) and the positive allosteric modulators of the GABA receptors GCP-7930 and GCP-13501 (Figure 2). The 13-cis-retinoids are known ligands of the TR heterodimer partner retinoid X receptor RXR ( Li et al., 2002). RXR has been described both as a permissive heterodimer partner, i.e. activity at a thyroid receptor response element can be driven by RXR ligands, and as a non-permissive ( Li et al., 2002). It may be that the status of permissive versus non-permissive is defined by the that they also lack a functional RXR and this would corroborate the conclusion that the agonist activity detected in the GH3.TRE-luc cells may reflect RXR rather than direct TR agonist activity. This would also explain the retinoids antagonistic like behavior in the GH3.TRE-Luc assay without any structural resemblance to Miller, S.C., Huang, R., Sakamuru, S., Shukla, S.J., Attene-Ramos, M.S., Shinn, P., ...

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... of T3, GC-1 and CO23 on GH3 cell proliferation. As shown in Figure 1, T 3 was the most potent in inducing proliferation, with an EC 50 of 0.27 nM. Proliferation concentration-response curves for the relatively TR" selective ligand GC-1 and the relatively TR! selective ligand CO23, resulted in EC 50 's of 1.5 nM and 18 nM for GC-1 and CO23, indicating they were approximately 5-fold and 67 times less potent than the T 3 EC 50 , respectively. Effects of T3, GC-1 and CO23 on a TRE-regulated reporter gene and endogenous growth hormone gene expression. Next, we used T 3 , GC-1 and CO23 in gene expression studies to provide a direct comparison to the T-screen results shown in Figure 1, and to uncover any potential isotype selectivity in target gene regulation in GH3 cells. The response curves of the luciferase reporter in GH3.TRE-Luc cells were remarkably similar to the T-screen based proliferation results. GH expression in the parental GH3 cells in response to T 3 , GC-1 and CO23 (Figure 2) is somewhat more sensitive than the T-screen and reporter gene assays, but overall, the same pattern of relative potencies exists (T 3 >GC-1>>CO23). Thus, the proliferation and transcriptional responses to GC-1 (as measured by reporter gene activity and GH expression) are much closer to the natural ligand T 3 than they are for CO23. The EC 50 values for proliferation, reporter gene, and GH gene expression assays in response to T 3 , GC-1 and CO23 are summarized in Table 1. The effect of putative environmental TR antagonists on reporter gene and GH gene expression in GH3 cells. In order to assess the reliability of the reporter gene assay to detect compounds that inhibit TR activity, we chose three environmentally To determine a full set of genes differentially regulated by T 3 , we expanded our analysis of the array to focus on genes that were significantly expressed and changed at least 2-fold up or down compared to the vehicle treated control cells. We identified 207 genes two-fold up-regulated and 312 genes two-fold down-regulated by T 3 , including several previously known TH response genes from other cell types and tissues including Dio1. Genes induced over four-fold are shown in Table ...