help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-1621
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
92/3/1066    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Moriyama, K.
Right arrow Articles by Nakao, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moriyama, K.
Right arrow Articles by Nakao, K.
Related Collections
Right arrow Thyroid
The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 3 1066-1072
Copyright © 2007 by The Endocrine Society

Antithyroid Drugs Inhibit Thyroid Hormone Receptor-Mediated 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

Address all correspondence and requests for reprints to: Tetsuya Tagami, M.D., Ph.D., Division of Endocrinology and Metabolism, Clinical Research Institute, Kyoto Medical Center, National Hospital Organization, Kyoto 612-8555, Japan. E-mail: ttagami{at}kyotolan.hosp.go.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.

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 two-hybrid 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID 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 (1-methyl-2-mercaptoimidazole; MMI), and propylthiouracil (6-propyl-2-thiouracil; PTU) to treat Graves’ hyperthyroidism (Fig. 1Go). ATDs have intrathyroidal and extrathyroidal 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.


Figure 1
View larger version (13K):
[in this window]
[in a new window]

  		FIG. 1. The structure of T3 and two antithyroid drugs, propylthiouracil and methimazole.

 
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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

The chemical structures of PTU, MMI, and T3 are shown in Fig. 1Go. 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 TRß1 (pCMX-hTRß1) and human TR{alpha}1 (pCMX-hTR{alpha}1) were provided by K. Umesono (Salk Institute, San Diego, CA) (7). The ligand-binding domain (LBD) of TR{alpha}1 or TRß1 was fused to the DNA binding domain (DBD) of Gal4 in-frame in pSG424 (8). The Gal4-NCoR (residues 1552–2453), Gal4-SRC1 (residues 213-1061), and Gal4-GRIP1 (residues 480-1462) constructs contain the indicated TR interaction domains of these proteins (9). The VP16 construct for TRß contains the LBD of the receptor downstream of the VP16 activation domain in-frame in pCMX (9). The reporter plasmids, TRE-TK-Luc (9) and ME-TK-Luc (10), contain two copies of a palindromic thyroid hormone response elements (TRE) and malic enzyme, respectively, upstream of the thymidine kinase (TK) promoter in the pA3-luciferase vector (Luc). TSH{alpha}-Luc contains 846 bp of the 5'-flanking sequence and 44 bp of exon I from the human glycoprotein hormone {alpha}-subunit gene in pA3-Luc (9). The Gal4 reporter plasmid, UAS-E1BTATA-Luc, contains five copies of the Gal4 recognition sequence (UAS) upstream of E1BTATA in pA3-Luc (11). The pRL-TK vector (Promega Corp., Madison, WI) comprised of the tk promoter and Renilla luciferase cDNA was used as an internal control.

Cell culture

TSA201, a clone of human embryonic kidney 293 cells (12), human hepatoblastoma HepG2 cells, and rat pituitary GH3 cells were maintained in DMEM, containing penicillin (100 U/ml) and streptomycin (100 µg/ml) with 10% fetal bovine serum (FBS; Invitrogen, Corp., Carlsbad, CA). For hormone and drug treatment, the medium was changed to phenol red-free DMEM (Nikken Biomedical Laboratory, Kyoto, Japan) containing 10% charcoal-stripped FBS (13).

Transient expression assays

TSA201 and HepG2 cells were grown in phenol red-free DMEM (Nikken Biomedical Laboratory) with 10% charcoal-stripped FBS and were transfected using the calcium precipitation method (14). After exposure to the calcium phosphate-DNA precipitate for 8 h, phenol red-free DMEM with charcoal-stripped FBS was added, in the absence or presence of compounds and/or T3. Cells were harvested for measurements of luciferase activity, according to the manufacturer’s instructions (dual-luciferase reporter assay system; Promega). The transfection efficiencies were corrected with the internal control. Both firefly and Renilla luciferase activities were measured to monitor the transfection efficiency and cytotoxicity of the added materials. The firefly activity 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 x 104 cells/well. T3 (1 nM) as a physiological concentration and/or 10 µM MMI was added on the day after the medium was replaced. In the case of 10 µM 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ATDs suppressed transcriptional activities mediated by TR{alpha}1 and TRß1

The chemical structure of T3, PTU, and MMI are shown in Fig. 1Go. ATDs are known as thionamides, which contain a sulfhydryl group and a thiourea moiety within a heterocyclic structure. Takagi et al. (6) reported that ATDs inhibit specific binding of T3 to its receptor, perhaps due to an interaction with cysteine residues of the receptor.

We examined whether ATDs antagonize T3-induced TR activation. Transient expression experiments were performed using TSA201 cells, which are a derivative of human embryonic kidney 293 cells. The LBD of TR{alpha}1 or TRß was fused to the DNA binding domain of the yeast transcription factor, Gal4, and was cotransfected with a Gal4 reporter gene, UAS-E1BTATA-Luc. We determined the effects of ATDs on various physiological concentrations of T3. In the presence of 10 µM PTU, increasing amounts of T3 were added to the medium, and transcriptional activity was measured. PTU had no significant effects on TR-mediated transcriptional activity in the absence of T3 (Fig. 2Go) and in the absence of Gal4-TR (see Fig. 6AGo). However, PTU suppressed the activity mediated by Gal4-TR{alpha}1 up to 36% and Gal4-TRß up to 39% of the respective control levels in the presence of T3. The maximum suppression by PTU was obtained at the concentration of 1 nM of T3. Similar results were obtained using MMI (data not shown).


Figure 2
View larger version (22K):
[in this window]
[in a new window]

  		FIG. 2. ATDs suppress TR-mediated transcription in the presence of a physiological range of T3. Gal4-TR{alpha}1 (A) or Gal4-TRß1 (B; 50 ng) was cotransfected into TSA-201 cells with 100 ng of the reporter gene, UAS-E1BTATA-Luc, in the absence or presence of 10 µM PTU. RLU, Relative light units. The firefly activity obtained by UAS-E1BTATA-Luc was normalized to the Renilla activity obtained by PRL-TK-Luc.

 

Figure 6
View larger version (37K):
[in this window]
[in a new window]

  		FIG. 6. The effects of ATDs on the interaction between TR and cofactors. The format of the mammalian two-hybrid experiment is shown between panels A and D. Gal4 fusion plasmids of indicated cofactors (50 ng) were cotransfected into TSA-201 cells with 100 ng of VP16-TRß together with 100 ng of the reporter gene, UAS-E1BTATA-Luc, in the absence or presence of 1 nM T3. Increasing amounts (1 nM, 100 nM, and 10 µM) of PTU or MMI were added. A, Gal4-DBD. B and C, Gal4-NCoR and Gal4-SMRT. D and E, Gal4-SRC1 and Gal4-GRIP1. RLU, Relative light units. The firefly activity obtained by UAS-E1BTATA-Luc was normalized to the Renilla activity obtained by PRL-TK-Luc. GTF, General transcription factor.

 
The inhibitory effects of ATDs were also examined in the context of native receptors. A T3-responsive reporter gene, TRE-TK-Luc, was cotransfected with full-length TRs (Fig. 3AGo). ATDs did not affect the activity of TRE-TK-Luc alone without TR. In the presence of 1 nM T3, dose-dependent inhibition of transcription mediated by TR{alpha}1 and TRß1 was observed with both ATDs. PTU suppressed the TR{alpha}1-mediated activity by 45% and TRß1-mediated activity by 39% and MMI suppressed TR{alpha}1-mediated activity by 45% and TRß1-mediated activity by 53% of the respective T3 effect.


Figure 3
View larger version (25K):
[in this window]
[in a new window]

  		FIG. 3. The inhibitory effects of ATDs on the gene transcription mediated by native TR. A, The CMX, CMX-TR{alpha}1, or CMX-TRß1 (50 ng) was cotransfected into TSA-201 cells with 100 ng of the reporter gene, TRE-TK-Luc, in the absence or presence of 1 nM T3 and increasing amount of ATDs. B, The CMX or CMX-TRß1 (50 ng) was cotransfected into TSA-201 cells with 100 ng of the reporter gene, TSH{alpha}-Luc, in the absence or the presence of 1 nM T3 and increasing amount of PTU (1 nM, 100 nM, and 10 µM). RLU, Relative light units. The firefly activity obtained by TRE-TK-Luc or TSH{alpha}-Luc was normalized to the Renilla activity obtained by PRL-TK-Luc. *, P < 0.05.

 
In a reciprocal manner, another group of negatively regulated genes was stimulated by TRs in the absence of T3 and was repressed in response to T3 (14). The effects of ATDs on the TSH{alpha} promoter were examined as a model of a negatively regulated gene. As shown in Fig. 3BGo, although PTU did not affect the activity of TSH{alpha}-Luc alone without TR, PTU increased the transcriptional activity, which was suppressed by 1 nM T3 up to the control level. Similar results were obtained using MMI (data not shown).

The inhibitory effects of PTU were also examined at high concentrations of T3. In the presence of 100 nM of T3, which is almost 20 times greater than concentrations found in blood samples of patients with severe hyperthyroidism, the effects of PTU were eliminated (Fig. 4Go). Similar results were obtained using MMI (data not shown).


Figure 4
View larger version (22K):
[in this window]
[in a new window]

  		FIG. 4. High amount of T3 blocks the inhibitory effects of ATDs on the gene transcription mediated by native TR. The CMX or CMX-TRß1 (50 ng) was cotransfected into TSA-201 cells with 100 ng of the reporter gene, TRE-TK-Luc, in the absence or the presence of 1 or 100 nM T3 and an increasing amount of PTU (1 nM, 100 nM, and 10 µM). RLU, Relative light units. The firefly activity obtained by TRE-TK-Luc was normalized to the Renilla activity obtained by PRL-TK-Luc. *, P < 0.05.

 
ATDs suppressed transcriptional activities mediated by endogenous TRs

We next studied the effects of ATDs using a cell line that contains physiological amounts of endogenous TRs. The reporter gene regulated by the ME-TRE, ME-TK-Luc, was transfected into human hepatoblastoma, HepG2. No significant effects were observed by 48 h incubation with 10 µM PTU or MMI on the ME-TK-Luc activity in the absence of T3. One nanomole T3 stimulated the activity by 1.8-fold, compared with that without T3, and ATDs inhibited its increase by 70% (Fig. 5Go).


Figure 5
View larger version (18K):
[in this window]
[in a new window]

  		FIG. 5. ATDs suppress transcriptional activities mediated by endogenous TRs. The ME-TK-Luc (100 ng) was transfected into HepG2 cells and incubated with or without 1 nM T3 and/or 10 µM PTU or MMI for 48 h. RLU, Relative light units. The firefly activity obtained by ME-TK-Luc was normalized to the Renilla activity obtained by PRL-TK-Luc. *, P < 0.05.

 
ATDs recruit corepressor and dissociate coactivator

Transcriptional repression of the positively regulated genes by unliganded TR is mediated by interacting with corepressor proteins (CoRs) such as nuclear receptor corepressor (NCoR) (15) and silencing mediator of retinoid and thyroid receptors (SMRT) (16). CoRs might also be involved in the basal activation of negatively regulated genes (14). In the other hand, in the presence of T3, liganded TR activates transcription of the positively regulated genes and inhibits that of the negatively regulated genes by interacting with coactivator proteins (CoAs) (17) such as steroid receptor coactivator (SRC)-1 (18) and glucocorticoid-interacting protein (GRIP)-1 (19). Using a mammalian two-hybrid assay, the effect of ATDs on the TR-CoR or TR-CoA interaction was examined. The TR interaction domain of CoRs or CoAs was fused to the Gal4-DBD. The LBD of TRß was fused to the transcriptional activation domain of VP16 to allow detection of the interaction between the Gal4-CoR/CoA and VP16-TR. As shown in Fig. 6AGo, both PTU and MMI had no significant effects on the activity mediated by Gal4-DBD and VP16-TR. PTU enhanced TR-NCoR interaction but not TR-SMRT interaction in a dose-dependent manner (Fig. 6BGo). In contrast, PTU inhibited TR-SRC1 interaction and, to a lesser degree, TR-GRIP1 interaction in a dose-dependent manner (Fig. 6DGo). Similarly, MMI enhanced TR-NCoR interaction but not TR-SMRT interaction and inhibited TR-GRIP1 interaction but not TR-SRC1 interaction (Fig. 6Go, C and E).

ATDs inhibited endogenous hormone secretion induced by T3

T3 stimulates endogenous GH gene transcription mediated by endogenous TRs in rat pituitary tumor cells (20, 21, 22). The production of GH was measured by ELISA in the culture media of GH3 cells. Forty-eight-hour incubation with 1 nM T3 stimulated the GH production by 2.7-fold (Fig. 7Go). Addition of 10 µM MMI significantly decreased the GH release to 82% of that with 1 nM T3 alone. In the case of 10 µM PTU, cell death was induced (data not shown).


Figure 7
View larger version (17K):
[in this window]
[in a new window]

  		FIG. 7. The inhibitory effects of ATDs on the GH secretion induced by T3. GH3 cells were incubated with or without 1 nM T3 and/or 10 µM MMI for 48 h. Culture media were collected and GH was measured by ELISA. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The ATDs have been a mainstay of treatment of patients with Graves’ hyperthyroidism (23). The ATDs are heterocyclic compounds known as thionamides that contain a thioureylene group (Fig. 1Go). Two kinds of drugs of this type are available at present: one is MMI and carbimazole (1-methyl-2-thio-3-carbethxy-imidazole), which is rapidly metabolized to MMI, and the other is PTU. These drugs cause goiter in animals, which is due to the stimulation of the thyroid by the pituitary, consequent to pharmacological inhibition of thyroid hormone production (24, 25). Thus, derivatives of thiourea and thiouracil have been used as antithyroid drugs for more than 60 yr.

The main effect of ATDs is to inhibit the synthesis of T4 and T3 as an intrathyroidal action, including inhibition of iodine oxidation and organization, inhibition of iodotyrosine coupling, possible alteration of thyroglobulin structure, and inhibition of thyroglobulin biosynthesis. As an extrathyroidal action, PTU but not MMI inhibits conversion of T4 to T3. During the last 3 decades, reports have been accumulating on extrathyroidal actions of the ATDs, especially of the thionamide group, causing several undesirable side effects. Bandyopadhyay et al. (5) reviewed extrathyroidal actions of antithyroid thionamides in animals and humans. Thionamides inhibit lactoperoxidase, which contributes to the antibacterial activities of a number of mammalian exocrine gland secretions that protect a variety of mucosal surfaces. These drugs stimulate both gastric acid and pepsinogen secretions, thereby augmenting the severity of gastric ulcers and preventing wound healing. Severe abnormalities may develop in blood cells and the immune system after thionamide therapy. They may cause agranulocytosis, aplastic anemia, and purpura along with immune suppression. Olfactory and auditory systems are also affected by these drugs. Thionamide affects the sense of smell and taste and may also cause loss of hearing. Thionamide also affects gene expression and modulate the functions of some cell types.

After administration of ATDs to patients with Graves’ disease, it usually takes 2 or 3 d to start decreasing serum level of T4 and T3. However, O2 consumption or other peripheral metabolic indexes indicated that ATDs exerted an immediate effect in vivo (4, 5). Besides a specific effect of PTU as a 5'-deiodinase inhibitor effectively preventing T3 generation, those actions were reported to persist in the presence of both of ATDs. Because thyroid hormones directly activate the expression of the human and mouse uncoupling protein-3 genes through a thyroid response element in the proximal promoter region, peripheral metabolism was controlled most partly by thyroid hormone level (26). An immediate effect of both of ATDs might be involved in the inhibition of transcriptional activities by thyroid hormone.

Induction of cell proliferation by mitogen or growth factor stimulation leads to the specific and sequential expression of a large number of genes. To date several reports showed that ATDs change mRNA expression level of certain genes. MMI and PTU increase thyroglobulin gene expression and increase thyroid-specific mRNA concentration in human thyroid FRTL-5 cells (27, 28, 29). The stimulatory effects of MMI and PTU can be suppressed by iodide and do not occur when protein synthesis is inhibited by cycloheximide. MMI and PTU increased thyroid peroxidase mRNA in cultured porcine thyroid follicles (30). MMI can suppress the interferon-{gamma}-induced increase in HLA-DR{alpha} gene expression (31). Fas ligand expression is induced by MMI in follicular cells of thyroid glands obtained from Graves’ patients and cultured thyrocytes, resulting in Fas ligand-dependent apoptosis of thyrocytes (32). In the thyroid, the TR expression has been reported (33, 34, 35).

ATDs are mainly concentrated in the thyroid gland but more than 70% of ATDs are unevenly distributed in the whole body (36). Therefore, the transcriptional inhibition by ATDs in the periphery may be dependent on the concentration of ATDs in each tissue. The peak serum concentrations of MMI are in the range of 300 ng/ml (2.6 mM) after a 15-mg oral dose (37), and those of PTU are about 3 mg/ml (18 mM) after a 150-mg oral dose (38). As we have shown here, inhibition of T3 action was observed for GH expression (20, 21, 22) in rat pituitary GH3 cells and for malic enzyme expression (10) in human hepatic HepG2 cells. However, the inhibitory effects on cell proliferation of these cells were also seen under the therapeutic concentrations of ATDs.

Takagi et al. (6) demonstrated that thionamides inhibit specific T3 binding to the hepatic nuclear receptor. MMI and PTU at a pharmacological dose (2 mM) reduced specific T3 binding to chromatographed nuclear receptors by 84 and 85%, respectively. Scatchard analyses indicated that neither MMI nor PTU significantly altered the affinity constant, whereas they both decreased maximal binding capacity. We obtained similar results using rat liver nuclear extract (data not shown) (13). Thereafter, there is no study of the actions of ATDs on the transcriptional regulation mediated by T3 and TR.

In this study we demonstrated that ATDs could impair thyroid hormone action by suppressing its transcriptional activity. Gene suppression is attributed partly to the recruitment of NCoR and dissociation of GRIP1 or SRC1. In the case of positively regulated promoter, TRE-TK, the effect of T3 was supposed to be inhibited by recruiting NCoR and dissociating GRIP1 or SRC1 by ATDs. In contrast, in the negatively regulated promoter, TSH{alpha}, the opposite effect was observed because corepressors may be involved in basal stimulation in the absence of T3 (14) and coactivators may be involved in T3-dependent inhibition in the presence of T3 (17). A number of nuclear cofactors have been cloned, but most of their specific functions are unclear (39). These cofactors were initially studied in the context of the TR and other nuclear hormone receptors. However, every cofactor has multiple interaction domains comprised of subtly distinct LXXLL motif and its combination and seems to interact with multiple receptors in a different way. Each specific ligand also contributes conformational change of the receptor and modulates the cofactor binding to the receptor as an agonist or an antagonist. Indeed, PTU and MMI enhanced recruitment of NCoR but not SMRT to the TR and both drugs dissociated GRIP1, but dissociation of SRC1 occurred solely by PTU. NCoR preferentially bind TR homodimer over TR-RXR heterodimer (40) and TR prefers to recruit NCoR, and retinoid acid receptor prefers to recruit SMRT (41). These preferences are likely due to sequence differences in interacting domains of corepressors (42).

Conformational change of TR induced by ATDs may be subtly different from that induced by T3, and it may enhance the interaction with specific domain of NCoR. The functional specificity is also reported among SRC family members. For example, progesterone receptor interacts preferentially with SRC1, which recruits cAMP response element-binding protein (CREB) binding protein (CBP) and enhances acetylation of histone H4, whereas glucocorticoid receptor interacts preferentially with SRC2 (GRIP1), which recruits p300/CBP-associated factor and results in histone H3 acetylation (43). PTU and MMI may have some different effect on the interaction between TR and SRC family members via their subtly distinct LXXLL motifs. Every receptor also has more than one activation domain [activation function (AF)-1 and AF-2]. Coactivators may preferentially use specific activation domains, depending on the receptor or activation function (AF-1 vs. AF-2) that is mediating the response to hormone (44). AF2 is conserved among nuclear hormone receptor superfamily as ligand-inducible transcription factors (45). We also tested the effects of ATDs on transcription mediated by other nuclear hormone receptors such as estrogen receptor-{alpha} and -ß. ATDs had no effects on estrogen action (data not shown).

In summary, our findings demonstrate that ATDs, which are the most prevalent drugs to treat Graves’ diseases, suppress transcriptional activity by modulating the cofactor recruitment to the TR. Although the clinical and therapeutic significance of our findings remains to be established, our data provide a mechanistic basis for one of the extrathyroidal actions of ATDs. It might be helpful in designing new therapeutic compounds with modifications of existing ATDs to enhance transcriptional inhibition against T3 action.


   Acknowledgments
 
The authors thank Ms. K. Matsuda, Ms. M. Kouchi, and Ms. H. Hiratani for their excellent secretarial assistance and Dr. S. Sasaki for technical assistance. We are also grateful to Dr. J. Larry Jameson for comments and suggestions on the manuscript.


   Footnotes
 
This work was supported in part by the grants from The Smoking Research Foundation (to K.M.) and The Japanese Ministry of Education and Science (11671103 and 17590973; to T.T.).

Disclosure Statement: The authors have nothing to disclose.

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.

Received July 27, 2006.

Accepted December 14, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cooper DS 1984 Antithyroid drugs. N Engl J Med 311:1353–1362[Abstract]
  2. Cooper DS 2005 Antithyroid drugs. N Engl J Med 352:905–917[Free Full Text]
  3. Meredish JH, Rogers H, Johnston FR 1961 Immediate influence of propylthiouracil on oxygen consumption in the dog. Surg Forum 12:5–7[Medline]
  4. Bray GA, Hildreth S 1967 Effect of propylthiouracil and methimazole on the oxygen consumption of hypothyroid rats receiving thyroxine or triiodothyronine. Endocrinology 81:1018–1020[Medline]
  5. Bandyopadhyay U, Biswas K, Banerjee RK 2002 Extrathyroidal actions of antithyroid thionamides. Toxicol Lett 128:117–127[CrossRef][Medline]
  6. Takagi S, Hummel BC, Walfish PG 1990 Thionamides and arsenite inhibit specific T3 binding to the hepatic nuclear receptor. Biochem Cell Biol 68:616–621[Medline]
  7. Umesono K, Murakami KK, Thompson CC, Evans RM 1990 Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D receptors. Cell 65:1255–1266
  8. Sadowski I, Ma J, Tiezenberg S, Ptashne M 1988 Gal4-VP16 is an unusually potent transcriptional activator. Nature 335:563–564[CrossRef][Medline]
  9. Tagami T, Gu W, Peairs PT, West BL, Jameson JL 1998 A novel natural mutation in the thyroid hormone receptor defines a dual functional domain that exchanges nuclear receptor corepressors and coactivators. Mol Endocrinol 12:1888–1902[Abstract/Free Full Text]
  10. Desvergne B, Petty KJ, Nikodem VM 1991 Functional characterization and receptor binding studies of the malic enzyme thyroid hormone response element. J Biol Chem 266:1008–1013[Abstract/Free Full Text]
  11. Tagami T, Lutz WH, Kumar R, Jameson JL 1998 The interaction of the vitamin D receptor with nuclear receptor corepressors and coactivators. Biochem Biophys Res Commun 253:353–363
  12. Margolskee RF, McHendry-Rinde B, Horn R 1993 Panning transfected cells for electrophysiological studies. Biotechniques 15:906–911[Medline]
  13. Moriyama K, Tagami T, Akamizu T, Usui T, Saijo M, Kanamoto N, Hataya Y, Simatsu A, Kuzuya H, Nakao K 2002 Thyroid hormone action is disrupted by Bisphenol A as an antagonist. J Clin Endocrinol Metab 87:5185–5190[Abstract/Free Full Text]
  14. Tagami T, Madison LD, Nagaya T, Jameson JL 1997 Nuclear receptor corepressors activate rather than suppress basal transcription of genes that are negatively regulated by thyroid hormone. Mol Cell Biol 17:2642–2648[Abstract]
  15. Horlein AJ, Naar AM, Heinzel T, Torchia J, Gloss B, Kurokawa R, Ryan A, Kamei Y, Soderstrom M, Glass CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor. Nature 377:397–404[CrossRef][Medline]
  16. Chen JD, Evans RM 1995 A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature 377:454–457[CrossRef][Medline]
  17. Tagami T, Park Y, Jameson JL 1999 Mechanisms that mediate negative regulation of the thyroid stimulating hormone gene by the thyroid hormone receptor. J Biol Chem 274:22345–22353[Abstract/Free Full Text]
  18. Onate SA, Tsai SY, Tsai MJ, O’Malley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract/Free Full Text]
  19. Hong H, Kohli K, Garabedian MJ, Stallcup MR 1997 GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors. Mol Cell Biol 17:2735–2744[Abstract]
  20. Glass CK, Franco R, Weinberger C, Albert VR, Evans RM, Rosenfeld MG 1987 A c-erb-A binding site in rat growth hormone gene mediates trans-activation by thyroid hormone. Nature 329:738–741[CrossRef][Medline]
  21. Koenig RJ, Brent GA, Warne RL, Larsen PR, Moore DD 1987 Thyroid hormone receptor binds to a site in the rat growth hormone promoter required for induction by thyroid hormone. Proc Natl Acad Sci USA 84:5670–5674[Abstract/Free Full Text]
  22. Koenig RJ, Warne RL, Brent GA, Harney JW, Larsen PR, Moore DD 1988 Isolation of a cDNA clone encoding a biologically active thyroid hormone receptor. Proc Natl Acad Sci USA 85:5031–5035[Abstract/Free Full Text]
  23. Astwood EB 1943 Treatment of hyperthyroidism with thiourea and thiouracil. JAMA 122:78
  24. Mackinzie JB, Mackinzie CG, McCollum EV 1941 The effect of sulfanilylguanidine on the thyroid of the rat. Science 94:518
  25. Mackinzie CG, Mackinzie JB 1943 The effect of sulfonamides and thiouracil on the thyroid gland and basal metabolism. Endocrinology 32:185
  26. Solanes G, Pedraza N, Calvo V, Vidal-Puig A, Lowell BB, Villarroya F 2005 Thyroid hormones directly activate the expression of the human and mouse uncoupling protein-3 genes through a thyroid response element in the proximal promoter region. Biochem J 386:505–513[CrossRef][Medline]
  27. Leer LM, Cammenga M, van der Vorm ER, de Vijlder JJ 1991 Methimazole increases thyroid-specific mRNA concentration in human thyroid cells and FRTL-5 cells. Mol Cell Endocrinol 78:221–228[CrossRef][Medline]
  28. Leer LM, Cammenga M, de Vijlder JJ 1991 Methimazole and propylthiouracil increase thyroglobulin gene expression in FRTL-5 cells. Mol Cell Endocrinol 82:R25–R30
  29. Isozaki O, Tsushima T, Emoto N, Saji M, Tsuchiya Y, Demura H, Sato Y, Shizume K, Kimura S, Kohn LD 1991 Methimazole regulation of thyroglobulin biosynthesis and gene transcription in rat FRTL-5 thyroid cells. Endocrinology 128:3113–3121[Abstract]
  30. Sugawara M, Sugawara Y, Wen K 1999 Methimazole and propylthiouracil increase cellular thyroid peroxidase activity and thyroid peroxidase mRNA in cultured porcine thyroid follicles. Thyroid 9:513–518[Medline]
  31. Montani V, Shong M, Taniguchi SI, Suzuki K, Giuliani C, Napolitano G, Saito J, Saji M, Fiorentino B, Reimold AM, Singer DS, Kohn LD 1998 Regulation of major histocompatibility class II gene expression in FRTL-5 thyrocyte: opposite effects of interferon and methimazole. Endocrinology 139:290–302[Abstract/Free Full Text]
  32. Mitsiades N, Poulaki V, Tseleni-Balafouta S, Chrousos GP, Koutras DA 2000 Fas ligand expression in thyroid follicular cells from patients with thionamide-treated Graves’ disease. Thyroid 10:527–532[Medline]
  33. Machia E, Nakai A, Janiga A, Sakurai A, Fisfaren ME, Gardner P, Soltani K, DeGroot LJ 1990 Characterization of site-specific polyclonal antibodies to c-erb A peptides recognizing human thyroid hormone receptors {alpha}1, {alpha}2, ß and native 3,5,3'-triiodothyronine receptor, and study of tissue distribution of the antigen. Endocrinology 126:3232–3239[Abstract]
  34. Tagami T, Nakamura H, Sasaki S, Mori T, Yoshioka H, Yoshida H, Imura H 1990 Immunohistochemical localization of nuclear 3,5,3'-triiodothyronine receptor proteins in rat tissues studied with antiserum against c-erb A/T3 receptor. Endocrinology 127:1727–1734[Abstract]
  35. Bronnegard M, Torring O, Boos J, Sylven C, Marcus C, Wallin G 1994 Expression of thyrotropin receptor and thyroid hormone receptor messenger ribonucleic acid in normal, hyperplastic, and neoplastic human thyroid tissue. J Clin Endocrinol Metab 79:384–389[Abstract]
  36. Lazarus JH, Marchant B, Alexander WD, Clark DH 1975 35-S-antithyroid drug concentration and organic binding of iodine in the human thyroid. Clin Endocrinol (Oxf) 4:609–615[Medline]
  37. Okamura Y, Shigemasa C, Tatsuhara T 1986 Pharmacokinetics of methimazole in normal subjects and hyperthyroid patients. Endocrinology 33:605–615
  38. Cooper DS, Saxe VC, Meskell M, Maloof F, Ridgway EC 1982 Acute effects of propylthiouracil (PTU) on thyroidal iodine organization and peripheral iodothyronine deiodination: correlation with serum PTU levels measured by radioimmunoassay. J Clin Endocrinol Metab 54:101–107[Medline]
  39. McKenna NJ, Lanz RB, O’Malley BW 1999 Nuclear receptor coregulators: cellular and molecular biology. Endocr Rev 20:321–344[Abstract/Free Full Text]
  40. Cohen RN, Wondisford FE, Hollenberg AN 1998 Two separate NCoR (nuclear receptor corepressor) interaction domains mediate corepressor action on thyroid hormone response elements. Mol Endocrinol 12:1567–1581[Abstract/Free Full Text]
  41. Cohen RN, Putney A, Wondisford FE, Hollenberg AN 2000 The nuclear corepressors recognize distinct nuclear receptor complexes. Mol Endocrinol 14:900–914[Abstract/Free Full Text]
  42. Cohen RN, Brzostek S, Kim B, Chorev M, Wondisford FE, Hollenberg AN 2001 The specificity of interactions between nuclear hormone receptors and corepressors is mediated by distinct amino acid sequences within the interacting domains. Mol Endocrinol 15:1049–1061[Abstract/Free Full Text]
  43. Li X, Wong J, Tsai SY, Tsai MJ, O’Malley BM 2003 Progesterone and glucocorticoid receptors recruit distinct coactivator complexes and promote distinct patterns of local chromatin modification. Mol Cell Biol 23:3763–3773[Abstract/Free Full Text]
  44. Onate SA, Boonyaratanakornkit V, Spencer TE, Tsai SY, Tsai MJ, Edwards DP, O’Malley BW 1998 The steroid receptor coactivator-1 contains multiple receptor interacting and activation domains that cooperatively enhance the activation function 1 (AF1) and AF2 domains of steroid receptors. J Biol Chem 273:12101–12108[Abstract/Free Full Text]
  45. Mangelsdorf DJ, Evans RM 1995 The RXR heterodimers and orphan receptors. Cell 83:841–850[CrossRef][Medline]



This article has been cited by other articles:


Home page
 IOVSHome page
A. H. Conrad, J. M. Strafuss, M. D. Wittman, S. Conway, and G. W. Conrad
Thyroxine Increases the Rate but Does Not Alter the Pattern of Innervation during Embryonic Chick Corneal Development
Invest. Ophthalmol. Vis. Sci., January 1, 2008; 49(1): 139 - 153.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
92/3/1066    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Moriyama, K.
Right arrow Articles by Nakao, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moriyama, K.
Right arrow Articles by Nakao, K.
Related Collections
Right arrow Thyroid

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals