Sheue-yann Cheng

Hormone-induced Translocation of Thyroid Hormone Receptors in Living Cells Visualized Using a Receptor Green Fluorescent Protein Chimera

Thyroid hormone nuclear receptors (TRs) are ligand-dependent transcription factors that regulate growth, differentiation, and development. To understand the role of the hormone, 3,3′,5-triiodo-l-thyronine (T3), in the nuclear translocation and targeting of TRs to the regulatory sites in chromatin, we appended green fluorescent protein (GFP) to the human TR subtype β1 (TRβ1). The fusion of GFP to the amino terminus of TRβ1 protein did not alter T3 binding or transcriptional activities of the receptor. The subcellular localization of GFP-TRβ1 in living cells was visualized by laser-scanning confocal microscopy. In the presence of T3, the expressed GFP-TRβ1 was predominately localized in the nucleus, exhibiting a nuclear/cytoplasmic ratio of ∼5.5. No GFP-TRβ1 was detected in the nucleolus. In the absence of T3, more GFP-TRβ1 was present in the cytoplasm, exhibiting a nuclear/cytoplasmic ratio of ∼1.5. In these cells, cytoplasmic GFP-TRβ1 could be induced to enter the nucleus by T3. The T3-induced translocation was blocked when Lys184-Arg185 in domain D of TRβ1 was mutated to Ala184-Ala185. Furthermore, the inability of the mutant TR to translocate to the nucleus correlated with the loss of most of its transcriptional activity. These results suggest that TR functions may, in part, be regulated by T3-induced nuclear entry.

Cyclin D1 Is a Ligand-independent Co-repressor for Thyroid Hormone Receptors

Thyroid hormone receptors (TRs) are critical regulators of growth, differentiation, and homeostasis. TRs function by regulating the expression of thyroid hormone (T3) target genes in both ligand-dependent and -independent pathways. Distinct classes of co-regulatory proteins modulate these two pathways. We show here a novel role of cyclin D1 as a T3-independent co-repressor for TRs. Cyclin D1 interacted with TR in vitro and in cells in a ligand-independent manner. Cyclin D1 acted to repress both the silencing activity of the unliganded TR and the transcriptional activity of the liganded TR. The repression was not due to the inhibition of the binding of TR to the thyroid hormone response element but by serving as a ligand-independent bridging factor to selectively recruit HDAC3 to form ternary complexes. The repression was augmented by increasing expression of HDAC3 but not by HDAC1 and was alleviated by trichostatin A. Thus, cyclin D1 is a novel ligand-independent co-repressor that opens a new paradigm to understand the molecular basis of the silencing action of TR.

Impaired adipogenesis caused by a mutated thyroid hormone α1 receptor

ABSTRACT Thyroid hormone (T3) is critical for growth, differentiation, and maintenance of metabolic homeostasis. Mice with a knock-in mutation in the thyroid hormone receptor α gene (TRα1PV) were created previously to explore the roles of mutated TRα1 in vivo. TRα1PV is a dominant negative mutant with a frameshift mutation in the carboxyl-terminal 14 amino acids that results in the loss of T3 binding and transcription capacity. Homozygous knock-in TRα1PV/PV mice are embryonic lethal, and heterozygous TRα1PV/+ mice display the striking phenotype of dwarfism. These mutant mice provide a valuable tool for identifying the defects that contribute to dwarfism. Here we show that white adipose tissue (WAT) mass was markedly reduced in TRα1PV/+ mice. The expression of peroxisome proliferator-activated receptor γ (PPARγ), the key regulator of adipogenesis, was repressed at both mRNA and protein levels in WAT of TRα1PV/+ mice. Moreover, TRα1PV acted to inhibit the transcription activity of PPARγ by competition with PPARγ for binding to PPARγ response elements and for heterodimerization with the retinoid X receptors. The expression of TRα1PV blocked the T3-dependent adipogenesis of 3T3-L1 cells and repressed the expression of PPARγ. Thus, mutations of TRα1 severely affect adipogenesis via cross talk with PPARγ signaling. The present study suggests that defects in adipogenesis could contribute to the phenotypic manifestation of reduced body weight in TRα1PV/+ mice.

Expression of thyroid hormone receptors is disturbed in human renal clear cell carcinoma

Human renal clear cell carcinoma (RCCC) accounts for up to 2% of human cancers. To find out if thyroid hormone (T3) and its receptors (TRs) play a role in tumorigenesis of RCCC, the expression of TRs was evaluated on mRNA and protein level. It was found that TRalpha (both alpha1 and alpha2) mRNA amount was significantly decreased in tumors while compared with healthy kidney tissue, and this decrease was deepest in G1 (well differentiated) RCCCs. In contrast, TRalpha1 protein was 1.6x overexpressed in tumors. TRbeta1 mRNA amount was overexpressed in 30% and significantly decreased in 70% of examined tumors. On the protein level, TRbeta1 amount was 1.7x lower in tumors than in healthy controls.

Tumor Suppressor p53 Is a Negative Regulator in Thyroid Hormone Receptor Signaling Pathways

Thyroid hormone nuclear receptors (TRs) are ligand-dependent transcription factors which regulate growth, differentiation, and development. The molecular mechanisms by which TRs mediate these diverse effects are unclear. One emerging hypothesis suggests that TRs could mediate these diverse effects via cooperation with different transcription factors/receptors. Indeed, we have recently shown that the human TR subtype β1 (h-TRβ1) interacts with the tumor suppressor p53. p53 is a transcription factor that plays a critical role in cell cycle regulation and tumor development. To assess the physiological relevance of the interaction of h-TRβ1 with p53, the present study addressed the question as to whether the functions of h-TRβ1 could be modulated by p53. We first compared the h-TRβ1-mediated transcriptional activity in two pairs of isogenic cell lines, RKO/RKO E6 and MCF-7/MCF-7 E6. RKO and MCF-7 cells are colon and breast carcinoma cell lines, respectively, that contain p53 but lack TRβ1. The isogenic RKO E6 and MCF-7 E6 cells are stable clones expressing high levels of papillomavirus type 16 E6 protein. In these cells, the level of p53 protein was lower than the parental cells. The impairment of p53 functions in these E6-containing cells led to an activation of TRβ1-mediated transcriptional activity. Furthermore, in a growth hormone-producing cell line in which the expression of the growth hormone gene is positively regulated by TRs, overexpression of the wild-type p53 led to repression in the expression of the growth hormone gene. Thus, TRs could cross-talk with p53 in its signaling pathways to regulate gene regulatory functions. The present findings further strengthen the hypothesis that mediation of the pleiotropic effects of T3 requires the cooperation of TRs with a large network of transcription factors.

Transgenic mice bearing a human mutant thyroid hormone beta 1 receptor manifest thyroid function anomalies, weight reduction, and hyperactivity.

BackgroundResistance to thyroid hormone (RTH) is a syndrome characterized by refractoriness of the pituitary and/or peripheral tissues to the action of thyroid hormone. Mutations in the thyroid hormone receptor β (TRβ) gene result in TRβl mutants that mediate the clinical phenotype by interfering with transcription of thyroid hormone-regulated genes via a dominant negative effect. In this study, we developed transgenic mice harboring PV, a potent dominant negative human mutant TRβl devoid of thyroid hormone binding and transcriptional activation, as an animal model to understand the molecular basis of this human disease.Materials and MethodsStandard molecular biology approaches were used to obtain a cDNA fragment containing mutant PV which was injected into the pronucleus of fertilized egg. Founders were identified by Southern analysis and the expression of PV in tissues was determined by RNA and immunohistochemistry. Thyroid function was determined by radioimmunoassays of the hormones and the behavior of mice was observed using standard methods.ResultsThe expression of mutant PV was directed by the β-actin promoter. Mutant PV mRNA was detected in all tissues of transgenic mice, but the levels varied with tissues and with different lines of founders. Thyroid function tests in transgenic mice with high expression of mutant PV showed a significantly (∼1.5-fold) higher mean serum total of L-thyroxine levels (p < 0.01) than those of nontransgenic mice. Moreover, thyroid-stimulating hormone levels were not significantly different from those of nontransgenic mice. In addition, these mice displayed decreased weights and a behavioral phenotype characterized by hyperactivity.ConclusionsThese mice have phenotypic features consistent with the commonly observed clinical features of RTH and could be used as a model system to better understand the action of mutant TRβl in a physiological context, which could lead to better treatment for this disease.

Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling

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.

Knock-in mouse model for resistance to thyroid hormone (RTH): an RTH mutation in the thyroid hormone receptor beta gene disrupts cochlear morphogenesis.

Thyroid hormone and the beta isoform of its receptor, Trb, are essential for normal development of the mammalian auditory system. We have analyzed auditory system function and structure in a mouse strain with a targeted Thrb mutation, ThrbPV, which leads to the loss of binding of thyroid hormone (T3) to the Trb protein. Heterozygosity for the orthologous human THRBPV mutation and other similar mutations in human THRB cause resistance to thyroid hormone (RTH), which is occasionally associated with mild sensorineural hearing impairment. Auditory brainstem response analysis of heterozygous ThrbPV/+ mice demonstrates that they develop normal hearing. In contrast, ThrbPV/ThrbPV mice have severe hearing impairment that is already present at 3 weeks of age. This hearing loss is associated with disruption of postnatal morphogenesis of the tectorial membrane and organ of Corti. Comparison with the previously described phenotype of a Thrb ?/? knockout strain suggests that ThrbPV disrupts the function of other genes that are critical for development and/or maintenance of these structures.

Tissue-specific Stabilization of the Thyroid Hormone β1 Nuclear Receptor by Phosphorylation

The present study evaluated the expression and regulation of endogenous thyroid hormone receptors (TRs) in cultured cells. In COS-1 cells, the endogenous TR, subtype β1 (TRβ1), but not subtype β2 or α1, was induced to express by okadaic acid (OA) in a concentration-dependent manner. The induced TRβ1 had immunoreactivity and partial V8 proteolytic maps similar to those of the transfected and in vitro translated human TRβ1 (h-TRβ1). The OA-induced expression of endogenous TRβ1 was, however, not observed in a variety of other cultured cell lines tested, indicating that the induction was cell type-dependent. TRβ1 induced by OA was a multisite phosphorylated protein, in which serine and threonine in a ratio of 10:1 were phosphorylated. The induced TRβ1 was functional as it could mediate the thyroid hormone-dependent transcriptional activity via several thyroid hormone response elements. The induction of endogenous TRβ1 expression by OA was not accompanied by an increase in mRNA levels but was the result of an increase in the stability of the TRβ1 protein. This is the first report to indicate that one of the mechanisms by which the TR isoforms are differentially expressed is via the tissue-specific stabilization of the TR isoform proteins. Furthermore, this selective stability of TRβ1 could be conferred by phosphorylation.

Modulation of hormone-dependent transcriptional activity of the glucocorticoid receptor by the tumor suppressor p53

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor which regulates growth, development and metabolic functions. To test the hypothesis that the pleiotropic effect of the GR could be mediated by other transcription factors/oncogenes, the present study assessed its interaction with the tumor suppressor p53. p53 is a transcription factor which is involved in cell cycle regulation and apoptosis. We found that the wild-type p53 physically interacted with the GR and repressed the glucocorticoid-dependent transcriptional activity. In contrast, mutant p53 had no or a lesser effect depending on the type of p53 mutant. These findings raised the possibility that p53 may play an important role in modulating the activities of glucocorticoids in cells.