Region-specific effects of hypothyroidism on the relative expression of thyroid hormone receptors in adult rat brain

The aim of this study was to determine whether changes in the circulating thyroid hormone (TH) and brain synaptosomal TH content affected the relative levels of mRNA encoding different thyroid hormone receptor (TR) isoforms in adult rat brain. Northern analysis of polyA⁺RNA from cerebral cortex, hippocampus and cerebellum of control and hypothyroid adult rats was performed in order to determine the relative expression of all TR isoforms. Circulating and synaptosomal TH concentrations were determined by radioimmunoassay. Region-specific quantitative differences in the expression pattern of all TR isoforms in euthyroid animals and hypothyroid animals were recorded. In hypothyroidism, the levels of TRα2 mRNA (non-T₃-binding isoform) were decreased in all brain regions examined. In contrast the relative expression of TRα1 was increased in cerebral cortex and hippocampus, whereas in cerebellum remained unaffected. The TRβ1 relative expression in cerebral cortex and hippocampus of hypothyroid animals was not affected, whereas this TR isoform was not detectable in cerebellum. The TR isoform mRNA levels returned to control values following T₄ intraperitoneal administration to the hypothyroid rats. The obtained results show that in vivo depletion of TH regulates TR gene expression in adult rat brain in a region-specific manner. (Mol Cell Biochem 278: 93–100, 2005)     

Thyroid hormone-dependent gene expression as a biomarker of short-term 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) exposure in European common frog (Rana temporaria) tadpoles

The effects on thyroid hormone-dependent gene biomarker responses of the persistent organochlorine pesticide metabolite 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) were investigated after exposure of 4-week-old European common frog (Rana temporaria) (stage 36) tadpoles to two (0.001 and 0.01 ppm) DDE concentrations. Total body weight, total length, and tail length and width increased after 3-day exposure to DDE. Expression patterns of genes encoding for growth hormone, thyroid-stimulating hormone (TSHβ) and thyroid hormone receptor (TRα and TRβ) isoforms were evaluated in the head, body and tail regions using a validated real-time polymerase chain reaction (PCR) method. The mRNA expression of growth hormone in the body, and TSHβ in the head showed significant DDE concentration-dependent decreases. While DDE caused variable effects on TRα mRNA steady-state, the expression of TRβ was significantly decreased in the tail by DDE in a concentration-specific manner. The effect of DDE exposure on TRβ mRNA expression showed a negative correlation with tail length and width during the exposure period. The unique pattern of a DDE-induced decrease of tail TRβ expression probably reflects the significant role of this thyroid hormone receptor isoform in tail re-absorption and overall metamorphosis in anuran species. Therefore, the present study shows that the evaluation of thyroid hormone-dependent genes may represent quantitative biomarkers of acute exposure to organochlorine pesticides in anuran species during critical developmental periods such as metamorphosis. Given the widespread environmental levels of DDT and its metabolites, these pollutants will remain a subject of concern and their effects on anuran species should be studied in more detail.     

Molecular cloning and mRNA tissue expression of thyroid hormone receptors in yellow catfish Pelteobagrus fulvidraco and Javelin goby Synechogobius hasta

Thyroid hormones (THs) play a pivotal role in many physiological functions in vertebrates, including fish. Their effects are mediated by thyroid hormone receptors (TRs), which are members of the nuclear hormone receptor superfamily. In this study, full-length cDNA sequences of TRs from yellow catfish Pelteobagrus fulvidraco and Javelin goby Synechogobius hasta were cloned and their mRNA tissue expression profiles were determined. In P. fulvidraco, the validated cDNAs encoding for TRα and TRβ were 1789 and 1848 bp in length, encoding peptides of 401 and 378 amino acid residues, respectively. In addition, a TRβ spliced variant (named P. fulvidraco-TRβv), containing a 60-bp insertion, was detected. In S. hasta, cDNAs encoding for TRαA, TRαB and TRβ were 1827, 2295 and 2258 bp in length, encoding peptides of 401, 409 and 393 amino acid residues, respectively. The phylogenetic analysis revealed that TRα and TRβ cDNAs grouped into two separate clusters with other vertebrate counterparts and two TRα sequences grouped separately, suggesting that the two TRαs derived from paralogous genes that might arise during a teleost-specific genome duplication event. All TR mRNAs were detected in various tissues sampled. The mRNA levels of both TRα and TRβ from P. fulvidraco were the highest in brain, followed by liver, and lowest in heart, intestine, muscle, gill and spleen. However, in S. hasta, TRαA, TRαB and TRβ showed the highest mRNA levels in brain and lowest in muscle. Identification and mRNA tissue expression of TR genes from P. fulvidraco and S. hasta provide an initial step towards understanding their biological roles in the two fish species.     

Thyroid hormone receptor subtype specificity for hormone-dependent neurogenesis in Xenopus laevis

Thyroid hormone (T₃) influences cell proliferation, death and differentiation during development of the central nervous system (CNS). Hormone action is mediated by T₃ receptors (TR) of which there are two subtypes, TRα and TRβ. Specific roles for TR subtypes in CNS development are poorly understood. We analyzed involvement of TRα and TRβ in neural cell proliferation during metamorphosis of Xenopus laevis. Cell proliferation in the ventricular/subventricular neurogenic zones of the tadpole brain increased dramatically during metamorphosis. This increase was dependent on T₃ until mid-prometamorphosis, after which cell proliferation decreased and became refractory to T₃. Using double labeling fluorescent histochemistry with confocal microscopy we found TRα expressed throughout the tadpole brain, with strongest expression in proliferating cells. By contrast, TRβ was expressed predominantly outside of neurogenic zones. To corroborate the histochemical results we transfected living tadpole brain with a Xenopus TRβ promoter-EGFP plasmid and found that most EGFP expressing cells were not dividing. Lastly, treatment with the TRα selective agonist CO23 increased brain cell proliferation; whereas, treatment with the TRβ-selective agonists GC1 or GC24 did not. Our findings support the view that T₃ acts to induce cell proliferation in the tadpole brain predominantly, if not exclusively, via TRα.     

Sequential up-regulation of thyroid hormone β receptor,ornithine transcarbamylase,and carbamyl phosphate synthetase mRNAs in the liver of Rana catesbeiana tadpoles during spontaneous and thyroid hormone-induced metamorphosis

During both spontaneous and thyroid hormone (TH)-induced metamorphosis, the Rana catesbeiana tadpole undergoes postembryonic developmental changes in its liver which are necessary for its transition from an ammonotelic larva to a ureotelic adult. Although this transition ultimately results from marked increases in the activities and/or de novo synthesis of the urea cycle enzymes, the precise molecular means by which TH exerts this tissue-specific response are presently unknown. Recent reports, using RNA from whole Xenopus laevis tadpole homogenates and indirect means of measuring TH receptor (TR) mRNAs, suggest a correlation between the up-regulation of TRβ-mRNAs and the general morphological changes occurring during amphibian metamorphosis. To assess whether or not this same relationship exists in a TH-responsive tissue, such as liver, we isolated and characterized a cDNA clone containing the complete nucleotide sequence for a R. catesbeiana urea cycle enzyme, ornithine transcarbamylase (OTC), as well as a genomic clone containing a portion of the hormone-binding domain of a R. catesbeiana TRβ gene. Through use of these homologous sequences and a heterologous cDNA fragment encoding rat carbamyl phosphate synthetase (CPS), we directly determined the relative levels of the TRβ, OTC, and CPS mRNAs in liver from spontaneous and TH-induced tadpoles. Our results establish that TH affects an up-regulation of mRNAs for its own receptor prior to up-regulating CPS and OTC mRNAs. Moreover, results with cultured tadpole liver demonstrate that TH, in the absence of any other hormonal influence, can affect an up-regulation of both the TRβ and OTC mRNAs. © 1992 Wiley-Liss, Inc.     

Short-term effects of thyroid hormones during development: Focus on signal transduction

Extranuclear or nongenomic effects of thyroid hormones are mediated by receptors located at the plasma membrane or inside cells, and are independent of protein synthesis. Recently the αVβ3 integrin was identified as a cell membrane receptor for thyroid hormones, and a wide variety of nongenomic effects have now been shown to be induced through binding of thyroid hormones to this receptor. However, also other thyroid hormone receptors can produce nongenomic effects, including the cytoplasmic TRα and TRβ receptors and probably also a G protein-coupled membrane receptor, and increasing importance is now given to thyroid hormone metabolites like 3,5-diiodothyronine and reverse T₃ that can mimick some nongenomic effects of T₃ and T₄. Signal transduction from the αVβ3 integrin may proceed through at least three independent pathways (protein kinase C, Src or mitogen-activated kinases) but the details are still unknown. Thyroid hormones induce nongenomic effects on at least three important Na⁺-dependent transport systems, the Na⁺/K⁺-ATPase, the Na⁺/H⁺ exchanger, and amino acid transport System A, leading to a mitogenic response in embryo cells; but modulation of the same transport systems may have different roles in other cells and at different developmental stages. It seems that thyroid hormones in many cases can modulate nongenomically the same targets affected by the nuclear receptors through long-term mechanisms. Recent results on nongenomic effects confirm the old theory that the primary role of thyroid hormones is to keep the steady-state level of functioning of the cell, but more and more mechanisms are discovered by which this goal can be achieved.     

Thyroid hormone receptor α and regulation of type 3 deiodinase

Mice deficient in thyroid hormone receptor α (TRα) display hypersensitivity to thyroid hormone (TH), with normal serum TSH but diminished serum T(4). Our aim was to determine whether altered TH metabolism played a role in this hypersensitivity. TRα knockout (KO) mice have lower levels of rT(3), and lower rT(3)/T(4) ratios compared with wild-type (WT) mice. These alterations could be due to increased type 1 deiodinase (D1) or decreased type 3 deiodinase (D3). No differences in D1 mRNA expression and enzymatic activity were found between WT and TRαKO mice. We observed that T(3) treatment increased D3 mRNA in mouse embryonic fibroblasts obtained from WT or TRβKO mice, but not in those from TRαKO mice. T(3) stimulated the promoter activity of 1.5 kb 5'-flanking region of the human (h) DIO3 promoter in GH3 cells after cotransfection with hTRα but not with hTRβ. Moreover, treatment of GH3 cells with T(3) increased D3 mRNA after overexpression of TRα. The region necessary for the T(3)-TRα stimulation of the hD3 promoter (region -1200 to -1369) was identified by transfection studies in Neuro2A cells that stably overexpress either TRα or TRβ. These results indicate that TRα mediates the up-regulation of D3 by TH in vitro. TRαKO mice display impairment in the regulation of D3 by TH in both brain and pituitary and have reduced clearance rate of TH as a consequence of D3 deregulation. We conclude that the absence of TRα results in decreased clearance of TH by D3 and contributes to the TH hypersensitivity.     

Influence of ration level and rearing temperature on hepatic GHR1 and 2, and hepatic and intestinal TRα and TRβ gene expression in late stages of rainbow trout embryos

The study examined whether the early life-history temperature experience of rainbow trout Oncorhynchus mykiss embryos affects subsequent growth and expression of growth-related genes in the growing-up juveniles in response to variations in ration levels. Embryos were reared in a Heath incubator at either 8·5° C (E_8·5) or 6·0° C (E_6·0) until hatching, at which time they were transferred to grow-up tanks supplied with water at 8·5° C. At swim-up, the late stage embryos were subsequently fed a salmonid starter diet at levels of 5, 2 or 0·5% of live body mass per day. The body mass and proximate composition of the juveniles was examined when yolk absorbance was complete (21 days after the fish commenced feeding). Quantitative RT-PCR was used to examine the expression of mRNA encoding for growth hormone receptors 1 and 2 (GHR1 and GHR2) in the liver, and the two isoforms of thyroid hormone receptor (TRα and TRβ) in the liver and intestinal tract. Final body mass and total length, liver and intestinal masses, and total lipid content of the E_8·5 treatment group were directly related to increased ration size. These variables in the E_6·0 treatment group fed the 5% ration were significantly lower than for the comparable E_8·5 treatment group, suggesting an effect of embryo rearing temperature on the subsequent growth of these late stage embryos as they undergo the transition from embryo to early juvenile. Intestinal TRα and TRβ mRNA abundance was directly related to ration size in the E_8·5 treatment group, but not in the E_6·0 treatment group. Conversely, hepatic TRα and TRβ mRNA abundance was significantly affected by ration size only in the E_6·0 group, with TRβ and TRα abundance showing direct and inverse relationships with ration size, respectively. Hepatic GHR1 mRNA abundance was significantly and directly related to ration size in both the E_8·5 and E_6·0 treatment groups, but there were no differences in the abundance of hepatic GHR2 mRNA among any treatments.     

3D-QSAR,molecular docking and molecular dynamics studies of a series of RORγt inhibitors

The discovery of clinically relevant inhibitors of retinoic acid receptor-related orphan receptor-gamma-t (RORγt) for autoimmune diseases therapy has proven to be a challenging task. In the present work, to find out the structural features required for the inhibitory activity, we show for the first time a three-dimensional quantitative structure–activity relationship (3D-QSAR), molecular docking and molecular dynamics (MD) simulations for a series of novel thiazole/thiophene ketone amides with inhibitory activity at the RORγt receptor. The optimum CoMFA and CoMSIA models, derived from ligand-based superimposition I, exhibit leave-one-out cross-validated correlation coefficient (R²_cv) of .859 and .805, respectively. Furthermore, the external predictive abilities of the models were evaluated by a test set, producing the predicted correlation coefficient (R²_pred) of .7317 and .7097, respectively. In addition, molecular docking analysis was applied to explore the binding modes between the inhibitors and the receptor. MD simulation and MM/PBSA method were also employed to study the stability and rationality of the derived conformations, and the binding free energies in detail. The QSAR models and the results of molecular docking, MD simulation, binding free energies corroborate well with each other and further provide insights regarding the development of novel RORγt inhibitors with better activity.     

Distinct thyroid hormone‐dependent expression of trkB and p75NGFR in nonneuronal cells during the critical TH‐dependent period of the cochlea

Analyzing the thyroid hromone (TH)‐dependent period of the inner ear, we observed that the presence of triiodothyronine (T3) between postnatal day 3 (P3) and P12 is sufficient for functional maturation of the auditory system. Within this short time period, an unusual transient TH‐dependent expression of nonneuronal neurotrophin receptors (NT‐R) trkB and p75^NGFR was observed in correlation with neuronal and morphogenetic processes. The availability of thyroid hormone was revealed to be invariably correlated with (a) a transient expression of full‐length trkB in TRα1‐, TRα2‐ and TRβ1‐expressing hair cells concomitant to the segregation of afferent fibers and the synaptogenesis of efferent fibers; and (b) a transient expression of p75^NGFR in TRα1‐ and TRβ1‐expressing great epithelia ridge cells in direct spatiotemporal correlation with the appearance of apoptotic cells and morphogenetic maturation of the organ. For the first time, these data suggest a TH dependency of the expression of neurotrophin receptors in nonneuronal cells. A potential role of these peculiar neurotrophin receptor expression for the conversion of the biological function of TH on innervation patterning and morphogenesis during the critical TH‐dependent period of the inner ear may be considered. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 338–356, 1999     

Molecular modelling studies on d-annulated benzazepinones as VEGF-R2 kinase inhibitors using docking and 3D-QSAR

Chemotypes comprising the d-annulated 1,3-dihydro-2H-1-benzazepin-2-one scaffold derived from the paullone structure were found to be potent vascular endothelial growth factor receptor 2 (VEGF-R2) kinase inhibitors. Three-dimensional quantitative structure-activity relationship (3D-QSAR) and docking studies were performed on a series of d-annulated benzazepinones with VEGF-R2 kinase inhibition activities. The comparative molecular field analysis and comparative molecular similarity indices analysis models using 32 molecules in the training set gave r²_cv values of 0.811 and 0.769, r² values of 0.962 and 0.953, respectively. 3D contour maps generated from the two models revealed that the electron-withdrawing groups at R₁ and the bulky, electron-withdrawing as well as hydrogen bond donor groups at R₂ position are favourable; the bulky, hydrogen bond acceptor substituent at R₃ and the minor groups at R₄ position may benefit the potency. We have designed a series of novel VEGF-R2 inhibitors by utilizing the SAR results revealed in the present study, which were predicted with excellent potencies in the developed models. The results may aid in designing of potential VEGF-R2 inhibitors with better activities.     

Serum copper as a novel biomarker for resistance to thyroid hormone

Thyroid hormone action is mediated by the thyroid hormone receptors TRα1 and TRβ. Defects in TRβ lead to RTH (resistance to thyroid hormone) β, a syndrome characterized by high levels of thyroid hormone and non-suppressed TSH (thyroid-stimulating hormone). However, a correct diagnosis of RTHβ patients is difficult as the clinical picture varies. A biochemical serum marker indicative of defects in TRβ signalling is needed and could simplify the diagnosis of RTHβ, in particular the differentiation to TSH-secreting pituitary adenomas, which present with clinically similar symptoms. In the present paper we show that serum copper levels are regulated by thyroid hormone, which stimulates the synthesis and the export of the hepatic copper-transport protein ceruloplasmin into the serum. This is accompanied by a concerted reduction in the mRNA levels of other copper-containing proteins such as metallothioneins 1 and 2 or superoxide dismutase 1. The induction of serum copper is abolished in genetically hyperthyroid mice lacking TRβ and human RTHβ patients, demonstrating an important role of TRβ for this process. Together with a previously reported TRα1 specific regulation of serum selenium, we show that the ratio of serum copper and selenium, which is largely independent of thyroid hormone levels, volume changes or sample degradation, can constitute a valuable novel biomarker for RTHβ. Moreover, it could also provide a suitable large-scale screening parameter to identify RTHα patients, which have not been identified to date.