Antithyroid Drugs Inhibit Thyroid Hormone Receptor-Mediated Transcription

0021-972X/07/$15.00/0 Printed in U.S.A. The Journal of Clinical Endocrinology & Metabolism 92(3):1066 –1072 Copyright © 2007 by The Endocrine Society doi: 10.1210/jc.2006-1621 Antithyroid Drugs Inhibit Thyroid Hormone ReceptorMediated Transcription Kenji Moriyama, Tetsuya Tagami, Takeshi Usui, Mitsuhide Naruse, Takuo Nambu, Yuji Hataya, Naotetsu Kanamoto, Yu-shu Li, Akihiro Yasoda, Hiroshi Arai, and Kazuwa Nakao Department of Medicine and Clinical Science (K.M., T.N., Y.H., N.K., Y.L., A.Y., H.A., K.N.), Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan; and Division of Endocrinology and Metabolism (K.M., T.T., T.U., M.N.), Clinical Research Institute, Kyoto Medical Center, National Hospital Organization, Kyoto 612-8555, Japan Context: Methimazole (MMI) and propylthiouracil (PTU) are widely used as antithyroid drugs (ATDs) for the treatment of Graves’ disease. Both MMI and PTU reduce thyroid hormone levels by several mechanisms, including inhibition of thyroid hormone synthesis and secretion. In addition, PTU decreases 5⬘-deiodination of T4 in peripheral tissues. ATDs may also interfere with T3 binding to nuclear thyroid hormone receptors (TRs). However, the effect of ATDs on the transcriptional activities of T3 mediated by TRs has not been studied. Objective: The present study was undertaken to determine whether ATDs have an effect on the gene transcription regulated by T3 and TRs in vitro. Methods: Transient gene expression experiments and GH secretion assays were performed. To elucidate possible mechanisms of the antagonistic action of ATDs, the interaction between TR and nuclear cofactors was examined. T HYROID HORMONES REGULATE growth, development, and critical metabolic functions. They exert these effects through complex biological pathways, which offer a wealth of opportunity to intervene pharmacologically in thyroid hormone signaling at numerous steps. These include biosynthesis, cell-specific uptake, or export of thyroid hormone as well as nuclear targeting and actions, which are exerted through thyroid hormone receptor (TR) binding and histone acetylation. Such processes represent potentially important pharmacological targets for the drug therapies of thyroid hormone abnormalities, especially hyperthyroidism. Some compounds having thionamide structure, such as thiourea and thiouracil, inhibit thyroid function. Clinically used antithyroid drugs (ATDs) include methimazole (1methyl-2-mercaptoimidazole; MMI), and propylthiouracil (6-propyl-2-thiouracil; PTU) to treat Graves’ hyperthyroidism (Fig. 1). ATDs have intrathyroidal and extrathyroidal First Published Online December 27, 2006 Abbreviations: AF, Activation function; ATD, antithyroid drug; CoA, coactivator protein; CoR, corepressor protein; DBD, DNA binding domain; FBS, fetal bovine serum; GRIP, glucocorticoid receptor interacting protein; LBD, ligand-binding domain; Luc, luciferase; MMI, methimazole; NCoR, nuclear receptor corepressor; PTU, propylthiouracil; SMRT, silencing mediator of retinoid and thyroid receptor; SRC, steroid receptor coactivator; TK, thymidine kinase; TR, thyroid hormone receptor; TRE, thyroid hormone response element. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community. Results: In the transient gene expression experiments, both MMI and PTU significantly suppressed transcriptional activities mediated by the TR and T3 in a dose-dependent manner. In mammalian twohybrid assays, both drugs recruited one of the nuclear corepressors, nuclear receptor corepressor, to the TR in the absence of T3. In addition, PTU dissociated nuclear coactivators, such as steroid receptor coactivator-1 and glucocorticoid receptor interacting protein-1, from the TR in the presence of T3. Finally, MMI decreased the GH release that was stimulated by T3. Conclusions: ATDs inhibit T3 action by recruitment of transcriptional corepressors and/or dissociation of coactivators. This is the first report to show that ATDs can modulate T3 action at the transcriptional level. (J Clin Endocrinol Metab 92: 1066 –1072, 2007) actions. The chief intrathyroidal actions include inhibition of iodine oxidation and organization and iodotyrosine coupling, among others. The main extrathyroidal action is inhibition of conversion of T4 to T3 by PTU, but not MMI (1, 2). Thus, the reduction in thyroid hormone production induced by the drugs is central to these actions. Furthermore, ATDs are known to influence oxygen consumption, or peripheral metabolic suppression, although the mechanisms are not fully understood (3, 4). To date, a number of studies was performed to elucidate how ATDs suppress peripheral metabolism. ATDs can affect gene expression and modulate functions of some cell types (5). Although ATDs were not effective in the binding affinity of T4 to serum thyroxine binding globulin, they inhibited T3 binding to the hepatic nuclear extracts (6). However, the effect of ATDs on the transcriptional activities of T3 mediated by TRs has not been studied in detail. The present study was undertaken to determine whether ATDs have an effect on the gene transcription regulated by T3 and TRs in vitro. Materials and Methods Reagents The chemical structures of PTU, MMI, and T3 are shown in Fig. 1. ATDs were purchased from Sigma-Aldrich, Corp. (St. Louis, MO). T3 was purchased from Nakalai Tesque Inc. (Kyoto, Japan). Plasmid constructions Expression vectors containing wild-type human TR␤1 (pCMXhTR␤1) and human TR␣1 (pCMX-hTR␣1) were provided by K. Ume- 1066 Moriyama et al. • ATDs and TR-Mediated Transcription J Clin Endocrinol Metab, March 2007, 92(3):1066 –1072 1067 tivity obtained by the T3-specific promoter was divided by the Renilla activity obtained by the nonspecific promoter in each well. Results are expressed as the mean ⫾ sd from at least three transfections, each performed in triplicate. Data were analyzed by ANOVA with post hoc Dunnett’s tests to compare with the control. GH secretion and assay For GH assays, GH3 cells, derived from the rat pituitary tumor cell line, were seeded into 6-well plates at 1.5 ⫻ 104 cells/well. T3 (1 nm) as a physiological concentration and/or 10 ␮m MMI was added on the day after the medium was replaced. In the case of 10 ␮m PTU, cell survival was inhibited and the assay was abandoned. Culture media were collected after 2 d of incubation and GH was measured by ELISA (rat GH enzyme-immunoassay system; GE Healthcare UK Ltd., Buckinghamshire, UK) according to the manufacturer’s instructions. After harvesting the supernatant, cell numbers were counted to evaluate cell proliferation. Results are expressed as the mean ⫾ sd from at least five experiments, each performed in quadricate. Data were analyzed by ANOVA with post hoc Dunnett’s tests to compare with the control. Results ATDs suppressed transcriptional activities mediated by TR␣1 and TR␤1 FIG. 1. The structure of T3 and two a (...truncated)