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Corticosteroids, the primary circulating vertebrate stress hormones, are known to potentiate the actions of thyroid hormone in amphibian metamorphosis. Environmental modulation of the production of stress hormones may be one way that tadpoles respond to variation in their larval habitat, and thus control the timing of metamorphosis. Thyroid hormone and corticosteroids act through structurally similar nuclear receptors, and interactions at the transcriptional level could lead to regulation of common pathways controlling metamorphosis. To better understand the roles of corticosteroids in amphibian metamorphosis we analyzed the developmental and hormone-dependent expression of glucocorticoid receptor (GR) mRNA in the brain (diencephalon), intestine and tail of Xenopus laevis tadpoles. We compared the expression patterns of GR with expression of thyroid hormone receptor beta (TRbeta). In an effort to determine the relationship between nuclear hormone receptor expression and levels of ligand, we also analyzed changes in whole-body content of 3,5,3'-triiodothyronine (T(3)), thyroxine, and corticosterone (CORT). GR transcripts of 8, 4 and 2 kb were detected in all tadpole tissues, but only the 4 and 2 kb transcripts could be detected in embryos. The level of GR mRNA was low during premetamorphosis in the brain but increased significantly during prometamorphosis, remained at a constant level throughout metamorphosis, and increased to its highest level in the juvenile frog. GR mRNA level in the intestine remained relatively constant, but increased in the tail throughout metamorphosis, reaching a maximum at metamorphic climax. The level of GR mRNA was increased by treatment with CORT in the intestine but not in the brain or tail. TRbeta mRNA level increased in the brain, intestine and tail during metamorphosis and was induced by treatment with T(3). Analysis of possible crossregulatory relationships between GRs and TRs showed that GR mRNA was upregulated by exogenous T(3) (50 nM) in the tail but downregulated in the brain of premetamorphic tadpoles. Exogenous CORT (100 nM) upregulated TRbeta mRNA in the intestine. Our findings provide evidence for tissue-specific positive, negative and crossregulation of nuclear hormone receptors during metamorphosis of X. laevis. The synergy of CORT with T(3) on tadpole tail resorption may depend on the accelerated accumulation of GR transcripts in this tissue during metamorphosis, which may be driven by rising plasma thyroid hormone titers.
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The syndrome of resistance to thyroid hormone (RTH) is an inherited disorder involving a mutation of the thyroid hormone receptor (TR) gene. Mutant (m) TR inhibits wild-type (wt) TR functions in a dominant negative manner, and this dominant negative effect (DNE) is a crucial factor in RTH pathogenesis. The molecular mechanism of the DNE is still unclear, although several possibilities (including competition between wt- and mTRs at the T(3) response element (TRE), sequestration of TR-associated protein(s) and titration out of functional TR) have been considered. Here we report that the DNE of mTRs is strongly correlated with their binding avidity for the retinoid X receptor (RXR), and especially for corepressor SMRT (silencing mediator for retinoid and thyroid hormone receptor), but not for the nuclear receptor corepressor, NCoR. The DNE of six natural TRs and four artificially constructed mTRs was assayed using a TR reporter gene containing TRE-DR4 (DR=direct repeat), TRE-pal (pal=palindrome) or TRE-lap (lap=inverted palindrome) in CV1 cells treated with 10 nM T(3). Of the mTRs examined, F451X (with a carboxy-terminal 11-amino-acid truncation) identified in a patient with RTH exhibited the strongest DNE on all TREs. The binding affinities between mTRs and corepressors SMRT or NCoR were quantified using a two-hybrid interference assay system consisting of VP16-TR(LBD) (LBD=ligand binding domain) and Gal4(DBD)-SMRT (DBD=DNA binding domain), or Gal4(DBD)-NCoR respectively, together with the Gal4 reporter gene. In this assay, VP16-TR(LBD) and Gal4(DBD)-SMRT (or Gal4 (DBD)-NCoR) interact with each other and trans-activate the Gal4 reporter gene. When an equal amount of mTR is coexpressed, it reduces the transcriptional activity of the reporter gene, depending on its binding avidity for a corepressor. A very strong correlation was observed between the SMRT-binding activity and the potency of the DNE among six natural mTRs and also among all mTRs, including four artificially constructed ones. The relationship between NCoR and DNE, however, was not significant. When we assayed the binding avidity of mTRs for RXR by using a two-hybrid assay system consisting of Gal4(DBD)-RXR(LBD) and VP16-TR(LBD), a significant correlation between DNE and binding avidity for the RXR was also observed. These results suggest that a corepressor plays an important role in DNE pathogenesis.
Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, PO Box 22660, 1100DD, Amsterdam, The Netherlands
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Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, PO Box 22660, 1100DD, Amsterdam, The Netherlands
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Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, PO Box 22660, 1100DD, Amsterdam, The Netherlands
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Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, PO Box 22660, 1100DD, Amsterdam, The Netherlands
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Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, PO Box 22660, 1100DD, Amsterdam, The Netherlands
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Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, PO Box 22660, 1100DD, Amsterdam, The Netherlands
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Introduction Tri-iodothyronine (T 3 ) affects cardiac function mainly by exerting a direct effect on cardiac cells through binding to thyroid hormone receptors (TR), thus regulating several functionally important proteins responsible
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Thyroid hormone receptor alpha 1, beta 1 and beta 2-deficient mice (TR alpha 1-/-beta-/- mice) demonstrate growth retardation and defective ossification in the epiphyses associated with an inhibition of the GH/IGF-I axis. There are differences between TR alpha 1-/-beta-/- mice (receptor deficient) and the hypothyroid animal model (ligand deficient). Such differences include possible repressive actions exerted by unliganded receptors in the ligand-deficient (hypothyroid) model but not in the receptor-deficient model. In the present study we have investigated whether or not GH substitution rescues the skeletal phenotype of TR alpha 1-/-beta-/- mice. TR alpha 1-/-beta-/- and wild-type (WT) mice were treated with GH from day 18 until 10 weeks of age. GH substitution of mutant mice resulted in a significant and sustained stimulatory effect on the body weight that was not seen in WT mice. GH-treated mutant mice but not GH-treated WT mice demonstrated increased length and periosteal circumference of the femur. However, GH substitution did not reverse the defective ossification seen in TR alpha 1-/-beta-/- mice. TR alpha 1-/-beta-/- mice displayed increased width of the proximal tibial growth plate, which was caused by increased width of the proliferative but not the hypertrophic layer. GH substitution did not restore the disturbed morphology of the growth plate in TR alpha 1-/-beta-/- mice. In summary, GH substitution reverses the growth phenotype but not the defective ossification in TR alpha 1-/-beta-/- mice. Our data suggest that TRs are of importance both for the regulation of the GH/IGF-I axis and for direct effects on cartilage.
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Houston Methodist Research Institute, College of Arts and Sciences, Departments of Paediatrics, Children's Health Research Institute, Department of Molecular Physiology and Biophysics, The Third Affiliated Hospital of Guangzhou Medical University, Genomic Medicine Program, 6670 Bertner Ave, Houston, Texas 77030, USA
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Houston Methodist Research Institute, College of Arts and Sciences, Departments of Paediatrics, Children's Health Research Institute, Department of Molecular Physiology and Biophysics, The Third Affiliated Hospital of Guangzhou Medical University, Genomic Medicine Program, 6670 Bertner Ave, Houston, Texas 77030, USA
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Arumanayagam AS Webb P 2014 Genome-wide binding patterns of thyroid hormone receptor β . PLoS ONE 9 e81186 . ( doi:10.1371/journal.pone.0081186 ) Badman MK Pissios P Kennedy AR Koukos G Flier JS Maratos-Flier E 2007 Hepatic
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Human thyroid hormone receptor (TR) is encoded by two distinct genes, TR alpha and TR beta. TR heterodimerizes with retinoid X receptor (RXR) and binds efficiently to the thyroid hormone (T(3)) response element (TRE) of target genes. In the absence of T(3), unliganded TR suppresses the basal promoter activity of positively regulated genes (silencing). Silencing mediator for retinoid and thyroid hormone receptors (SMRT) and nuclear receptor co-repressor (N-CoR) interact with unliganded TR and function as corepressor proteins. Previously, we found beta F451X with carboxyl (C)-terminal 11-amino acid deletion had stronger silencing potency than wild-type TR beta 1 and beta E449X with C-terminal 13-amino acid deletion on a subset of TREs. In the present study, to assess the isoform-specific effects of the C-terminal truncations on TR silencing, we constructed two mutant TR alpha 1s (alpha F397X and alpha E395X) with the same respective C-terminal truncations as beta F451X and beta E449X and analysed their silencing activities. Unlike beta F451X and beta E449X, alpha F397X and alpha E395X showed similarly stronger silencing potency than wild-type TR alpha 1. We further studied the abilities of wild-type and the mutant TR beta 1s and alpha 1s on RXR and co-repressor binding by a two-hybrid interference assay. beta F451X had significantly stronger abilities to bind to RXR and SMRT than did wild-type TR beta 1 and beta E449X. In contrast, wild-type TR alpha 1, alpha F397X and alpha E395X showed similar abilities to bind to RXR and SMRT. beta E449X and alpha E395X, which have identical C-terminal truncation, showed less ability to bind to N-CoR than did wild-type TR beta 1 and beta F451X and wild-type TR alpha 1 and alpha F397X respectively. These results indicate that an identical C-terminal truncation gives rise to different effects on TR beta 1 and alpha1 with respect to silencing potency, RXR binding and SMRT binding. The difference in the silencing potency among wild-type TR beta 1, beta F451X and beta E449X correlated well with the difference in the ability to bind co-repressor SMRT.
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To correlate the differentiation phenotype of two human thyroid cancer cell lines with their expression of various molecular markers, we analyzed the mRNA levels of four thyroid-specific genes, including thyrotropin receptor (TSHR), thyroglobulin (Tg), thyroid transcription factor-1 (TTF-1), and paired-box containing transcription factor-8 (PAX-8) genes. The results showed a differentiation-status-related pattern in which a well-differentiated cell line (WRO) expressed all the four genes, in contrast to an anaplastic cell line (ARO) that expressed TTF-1 and reduced levels of TSHR, but no Tg or PAX-8 genes. Furthermore, to verify the finding of concomitant loss of beta subtype thyroid hormone receptor (TRbeta) and TSHR gene expression in neoplastic thyroid tumors (Bronnegard et al. 1994), we examined the expression levels of TRbeta1 gene in these cell lines. Whereas the WRO cells produced an abundant amount of TRbeta1 protein detectable by immunoprecipitation, the ARO cells produced none. This new observation prompted us to investigate whether overexpression of TRbeta1 protein in ARO cells might produce changes in the differentiation phenotypes. We found that the level of expression of the TSHR gene and the proliferative index of ARO cells were significantly upregulated in the cells stably transfected with wild-type TRbeta1. These findings suggest that TRbeta1 protein overexpression can affect the differentiation phenotypes and induce more efficient cell proliferation of the anaplastic ARO cells.
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Thyroid hormone receptors (TR) are members of the nuclear receptor superfamily. There are at least two TR isoforms, TRalpha and TRbeta, which act as mediators of thyroid hormone in tissues. However, the relative expression of each TR isoform in target tissues is still elusive. Herein, we have developed an RT-PCR and restriction enzyme digestion method to determine the expression of TRalpha1 and TRbeta1. We analyzed the expression of TR isoforms in 3T3-L1 preadipocytes induced to differentiate by an adipogenic cocktail in the presence or absence of 100 nM triiodothyronine (T(3)). The TRalpha1 isoform was predominantly expressed in 3T3-L1 adipocytes, and its expression was increased at the stage of development concomitant with the emergence of lipid droplets. Little, if any, TRbeta1 mRNA was detected in adipocytes. Administration of T(3) to the differentiating 3T3-L1 cells enhanced the accumulation of triglyceride. The expression profile of TRalpha1 in T(3)-treated adipocytes was similar to that in non-treated cells. The transcripts of adipogenic factors, CCAAT/enhancer binding protein beta (C/EBPbeta) and peroxisome proliferator activated receptor gamma (PPARgamma), were not altered by T(3). Lipid binding protein, aP2, that is downstream of these transcription factors was also unaffected by T(3). In contrast, the lipogenic enzyme, glyceraldehyde-3-phosphate dehydrogenase mRNA was significantly increased in the presence of T(3). Therefore, T(3) appears to be a hormone capable of modulating the expression of lipogenic enzyme and augments the accumulation of lipid droplets. We conclude that the TRalpha isoform might play an important role in the generation and maintenance of the mature adipocyte phenotype, regulating the expression of lipogenic enzymes.
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Introduction Thyroid hormone receptors (TRs) belong to the steroid family of nuclear receptor and are expressed as two gene products TRα and TRβ ( Yen 2001 ). TRs typically form heterodimers with another member of the nuclear receptor superfamily
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Tri-iodothyronine (T3) is known to be involved in the regulation of the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis. In previous studies we demonstrated that IGF-I and GH reduced the metabolic response to T3 measured as the activity of two T3-dependent enzymes, mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) and cytosolic malic enzyme (ME) in cultured rat liver cells. In this study we analysed in vivo the effect of IGF-I administered to rats on the activity of alpha-GPD and ME. IGF-I (240 micrograms/100 g body weight (BW) every 12 h for 48 h) significantly diminished alpha-GPD (P < 0.01) and ME (P < 0.05) activities. Serum basal glucose concentration was not significantly modified 12 h after the administration of recombinant human IGF-I (240 and 480 micrograms/100 g BW every 12 h for 48 h). Under similar conditions, no significant change in serum total thyroxine (TT4) concentration was observed, although free thyroxine (FT4) was diminished (P < 0.02) and total T3 (TT3) was increased (P < 0.03). To explore the participation of the nuclear thyroid hormone receptor (THR) in the mechanism of IGF-I action we measured the maximal binding capacity and the affinity constant (Ka) of THR by Scatchard analysis, and concentrations of messenger RNAs (mRNAs) that code for the isoforms of THR present in the liver (beta 1, alpha 1 and alpha 2) by Northern blot. IGF-I (240 micrograms/100 g BW every 12 h for 48 h) significantly reduced maximal binding capacity to 37% of the control value (P < 0.01) without changes in the Ka. beta 1, alpha 1 and alpha 2 THR mRNAs were significantly reduced (P < 0.01) by 120-480 micrograms/100 g BW IGF-I administration every 12 h for 48 h. Time-course studies indicated that this effect was obtained 12 h after the administration of 240 micrograms/100 g BW IGF-I (P < 0.05). These results indicate that IGF-I administration to rats diminishes the metabolic thyroid hormone action in the liver by a mechanism that involves, at least in part, a reduction in the number of THRs and in their level of expression.
