A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand.

The thyroid hormone 3,3,5-triiodo-L-thyronine (T3) is essential for growth, differentiation, and development. Its biological activities are mediated by T3 nuclear receptors (TRs). At present, how T3 regulates TR proteins and the resulting functional consequences are still unknown. Immunofluorescence analyses of endogenous TR in the growth hormone-producing GC cells showed that the T3-induced rapid degradation of TR was specifically blocked by lactacystin, a selective inhibitor of the ubiquitin-proteasome degradation pathway. Immunoblots demonstrated that the transfected TR␤1 was ubiquitinated and that the ubiquitination was T3 independent. Studies with a series of truncated TR␤1 showed that the hormone-binding domain was sufficient for the T3-induced rapid degradation of TR␤1 by the proteasome degradation pathway. T3 also induced rapid degradation of TR␤2 and TR␣1. In contrast, the stability of the non-T3-binding TR␣2 and naturally occurring TR␤1 mutants that do not bind T3 was not affected by T3 treatment, indicating that hormone binding to receptor was essential for the degradation of the wild-type receptors. In the presence of proteasome protease inhibitors, the levels of both total and ubiquitinated TR␤1 protein increased, yet T3-dependent transcriptional activation and the expression of the growth hormone gene were diminished, suggesting that proteasome-mediated degradation played a novel role in modulating transcriptional activation by TR. The present study reveals a role of T3 in modulating the functions of TR by regulating its receptor level via the ubiquitin-proteasome degradation pathway. T he thyroid hormone 3,3Ј,5-triiodo-L-thyronine (T3) is essential in metabolic-energetic homeostasis, development, and differentiation. Its actions are mediated by thyroid hormone nuclear receptors (TRs), which regulate the expression of T3-targeted genes. TRs belong to a superfamily of hormone nuclear receptors functioning as ligand-activated transcription factors, which include receptors for steroid hormones, vitamin D3, and the retinoids (1). TR consists of domains including the Nterminal A͞B domain, the DNA-binding domain C, and the hormone-binding domain (domains D and E). Recent studies indicate that the transcriptional activity of TR depends not only on the type of the thyroid hormone response elements located on the promoter regions of T3 target genes but also on a host of corepressors and coactivators (2). In the absence of T3, TR binds to corepressors, such as N-CoR. Binding of T3 leads to the release of N-CoR from TR and recruitment of coactivators leading to gene activation (2). In this model, how TR proteins are regulated and the role of T3 in this process are unknown.Using biochemical methods, Samuels and Casanova have previously reported that T3 down-regulates its endogenous TR in growth hormone (GH)-producing GC cells (3). However, the underlying molecular mechanisms by which T3 downregulates the TR have not been elucidated. In this report, we demonstrated that the T3-induced degradation of TR was via ...

Ponseti clubfoot treatment has become more popular during the last decade because of its high initial correction rate. But the most common problem affecting the long-term successful outcome is relapse of the deformity. Non-compliance with Ponseti brace protocol is a major problem associated with relapse. Although more comfortable braces have been reported to improve the compliance, they all have the same design and no significant changes have been made to the protocols. After refinement in the Ponseti method and emphasizing the importance of brace to parents, the relapse rate has been markedly decreased. Nevertheless, there are patients who do not have any recurrence although they are not completely compliant with the brace treatment, whereas other patients have a recurrence even though they are strictly compliant with the brace treatment. The aim of this article is to review the relapse of clubfoot and the function of the brace and to develop an individualized brace protocol for each patient by analyzing the mechanism of the brace and the biomechanical properties of muscles, tendons, and ligaments.

Thyroid-stimulating hormone (TSH)-secreting tumors (TSH-omas) are pituitary tumors that constitutively secrete TSH. The molecular genetics underlying this abnormality are not known. We discovered that a knockin mouse harboring a mutated thyroid hormone receptor (TR) ␤ (PV; TR␤ PV/PV mouse) spontaneously developed TSH-omas. TR␤ PV/PV mice lost the negative feedback regulation with highly elevated TSH levels associated with increased thyroid hormone levels (3,3,5-triiodo-L-thyronine [T3]). Remarkably, we found that mice deficient in all TRs (TR␣1 ؊/؊ TR␤ ؊/؊ ) had similarly increased T3 and TSH levels, but no discernible TSH-omas, indicating that the dysregulation of the pituitary-thyroid axis alone is not sufficient to induce TSH-omas. The thyroid hormone T3 (3,3Ј,5-triiodo-L-thyronine) is critical for growth, differentiation, and development and for maintenance of metabolic homeostasis. Thyroid hormone receptors (TRs) act as ligand-activated transcription factors and occupy a central position in mediating the functions of T3. TRs are encoded by two genes, TR␣ and TR␤, located on human chromosomes 17 and 3, respectively (4, 8). Alternative splicing of the primary transcripts gives rise to four major T3-binding TR isoforms: TR␣1, ␤1, ␤2, and ␤3. These isoforms differ in their length and amino acid sequence at the amino terminal A/B domain but bind T3 with high affinity to mediate gene regulatory activity. Like other nuclear receptors, these isoforms have an amino terminal A/B domain, a central DNA-binding domain, and a carboxyl-terminal ligand-binding domain. The carboxyl-terminal region also contains multiple contact surfaces that are important for dimerization with its partner, the retinoid X receptor, and for interactions with corepressors and coactivators (4,8,20). The expression of TR isoforms is tissue dependent and developmentally regulated (4,8).Thyroid-stimulating hormone (TSH)-secreting pituitary tumors (TSH-omas) represent about 2% of all pituitary adenomas in humans. TSH-omas are usually large at diagnosis and are associated with headaches and visual field disturbances. Because diagnosis occurs late in the natural course, the rate of curative surgical resection of TSH-omas remains under 50% (5, 6).The molecular genetics underlying this abnormality are not well understood. Somatic mutations of the TR␤ gene have been found in several patients with TSH-omas. Safer et al. reported R438H mutation in the TR␤ gene in a patient with pituitary adenoma (26). This mutation has also been reported in other patients with resistance to thyroid hormone (10). The R438H mutant has impairment in T3 binding and exhibits dominant negative activity (10). Recently, Ando et al. identified mutated TR␤ in the TSH-omas of two patients (2, 3). One patient had a somatic mutation in the ligand-binding domain of TR␤2 (His450Tyr) (2), and the other patient had a 165-bp deletion within the sixth exon of the ligand-binding domain of TR␤2 (3). Both TR␤ mutants had impaired T3 binding and abnormal regulation of TSH␤ and ␣-glycoprotein co...

The molecular genetic events underlying thyroid carcinogenesis are poorly understood. Mice harboring a knock-in dominantly negative mutant thyroid hormone receptor b (TRb PV/PV mouse) spontaneously develop follicular thyroid carcinoma similar to human thyroid cancer. Using this mutant mouse, we tested the hypothesis that the peroxisome proliferator-activated receptor c (PPARc) could function as a tumor suppressor in thyroid cancer in vivo. Using the offspring from the cross of TRb PV/ þ and PPARc þ /À mice, we found that thyroid carcinogenesis progressed significantly faster in TRb PV/PV mice with PPARc insufficiency from increased cell proliferation and reduced apoptosis. Reduced PPARc protein abundance led to the activation of the nuclear factor-jB signaling pathway, resulting in the activation of cyclin D1 and repression of critical genes involved in apoptosis. Treatment of TRb PV/PV mice with a PPARc agonist, rosiglitazone, delayed the progression of thyroid carcinogenesis by decreasing cell proliferation and activation of apoptosis. These results suggest that PPARc is a critical modifier in thyroid carcinogenesis and could be tested as a therapeutic target in thyroid follicular carcinoma.

Inactivation and silencing of PTEN have been observed in multiple cancers, including follicular thyroid carcinoma. PTEN (phosphatase and tensin homologue deleted from chromosome 10) functions as a tumour suppressor by opposing the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signalling pathway. Despite correlative data, how deregulated PTEN signalling leads to thyroid carcinogenesis is not known. Mice harbouring a dominant-negative mutant thyroid hormone receptor β (TRβPV/PV mice) spontaneously develop follicular thyroid carcinoma and distant metastases similar to human cancer. To elucidate the role of PTEN in thyroid carcinogenesis, we generated TRβPV/PV mice haploinsufficient for Pten (TRβPV/PVPten+/− mouse). PTEN deficiency accelerated the progression of thyroid tumour and increased the occurrence of metastasis spread to the lung in TRβPV/PVPten+/− mice, thereby significantly reducing their survival as compared with TRβPV/PVPten+/+ mice. AKT activation was further increased by two-fold in TRβPV/PVPten+/− mice thyroids, leading to increased activity of the downstream mammalian target of rapamycin (mTOR)–p70S6K signalling and decreased activity of the forkhead family member FOXO3a. Consistently, cyclin D1 expression was increased. Apoptosis was decreased as indicated by increased expression of nuclear factor-κB (NF-κB) and decreased caspase-3 activity in the thyroids of TRβPV/PVPten+/− mice. Our results indicate that PTEN deficiency resulted in increased cell proliferation and survival in the thyroids of TRβPV/PVPten+/− mice. Altogether, our study provides direct evidence to indicate that in vivo, PTEN is a critical regulator in the follicular thyroid cancer progression and invasiveness.

We previously created a knock-in mutant mouse harboring a dominantly negative mutant thyroid hormone receptor ␤ (TR␤ PV/PV mouse) that spontaneously develops a follicular thyroid carcinoma similar to human thyroid cancer. We found that ␤-catenin, which plays a critical role in oncogenesis, was highly elevated in thyroid tumors of TR␤ PV/PV mice. We sought to understand the molecular basis underlying aberrant accumulation of ␤-catenin by mutations of TR␤ in vivo. Cell-based studies showed that thyroid hormone (T3) induced the degradation of ␤-catenin in cells expressing TR␤ via proteasomal pathways. In contrast, no T3-induced degradation occurred in cells expressing the mutant receptor (TR␤PV). In vitro binding studies and cell-based analyses revealed that ␤-catenin physically associated with unliganded TR␤ or TR␤PV. However, in the presence of T3, ␤-catenin was dissociated from TR␤-␤-catenin complexes but not from TR␤PV-␤-catenin complexes. ␤-Catenin signaling was repressed by T3 in TR␤-expressing cells through decreasing ␤-catenin-mediated transcription activity and target gene expression, whereas sustained ␤-catenin signaling was observed in TR␤PV-expressing cells. The stabilization of ␤-catenin, via association with a mutated TR␤, represents a novel activating mechanism of the oncogenic protein ␤-catenin that could contribute to thyroid carcinogenesis in TR␤ PV/PV mice.

Overexpression of pituitary tumor-transforming 1 (PTTG1) is associated with thyroid cancer. We found elevated PTTG1 levels in the thyroid tumors of a mouse model of follicular thyroid carcinoma (TRβ PV/PV mice). Here we examined the molecular mechanisms underlying elevated PTTG1 levels and the contribution of increased PTTG1 to thyroid carcinogenesis. We showed that PTTG1 was physically associated with thyroid hormone β receptor (TRβ) as well as its mutant, designated PV. Concomitant with thyroid hormone-induced (T3-induced) degradation of TRβ, PTTG1 proteins were degraded by the proteasomal machinery, but no such degradation occurred when PTTG1 was associated with PV. The degradation of PTTG1/TRβ was activated by the direct interaction of the liganded TRβ with steroid receptor coactivator 3 (SRC-3), which recruits proteasome activator PA28γ. PV, which does not bind T3, could not interact directly with SRC-3/PA28γ to activate proteasome degradation, resulting in elevated PTTG1 levels. The accumulated PTTG1 impeded mitotic progression in cells expressing PV. Our results unveil what we believe to be a novel mechanism by which PTTG1, an oncogene, is regulated by the liganded TRβ. The loss of this regulatory function in PV led to an aberrant accumulation of PTTG1 disrupting mitotic progression that could contribute to thyroid carcinogenesis. IntroductionFollicular and papillary thyroid carcinomas are the most common thyroid malignancies. Although both are well-differentiated cancers, each has distinguishable morphological and pathological characteristics (1). Follicular thyroid carcinoma has a greater tendency than does papillary cancer to metastasize to distant sites. Such distant metastasis predicts a poor response to treatment and subsequent progression and mortality from thyroid cancer. Despite recent progress in the identification of key genetic alterations and aberrant molecular pathways, the precise mechanisms underlying the initiation and progression of follicular thyroid cancer are not fully understood.The development of a mouse model of follicular thyroid cancer (TRβ PV/PV mice) has provided a valuable tool to elucidate the molecular basis underlying thyroid carcinogenesis (2). The TRβ PV/PV mouse was created by a targeted mutation of thyroid hormone β receptor (TRβ) via homologous recombination and the Cre-LoxP system (3). The TRβ mutant (referred to here as PV) was identified in a patient with resistance to thyroid hormone (RTH) (4). RTH is caused by mutations of the TRβ gene and manifests symptoms as a result of decreased sensitivity to