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administration to fasted mice on deiodinase, TSH, and thyroid hormone receptor (TR) gene expression in the anterior pituitary. The aim of the present study was, therefore, to evaluate fasting-induced alterations in pituitary TSHβ, D2, and TRβ2 mRNA expression, as
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– in the pituitary and gonads. We also examined the TH regulation of gene transcripts for the thyroid hormone receptors (TRs) α and β ( trα and trβ ) in the brain, liver, and gonads, as well as transcript for basic transcription element
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ABSTRACT
An aqueous two-phase partitioning study of partially purified nuclear thyroid hormone receptor from rat liver was performed. Stability of 3,5,3′-tri-iodo-l-thyronine (T3)–receptor complex and T3-binding activity in the presence of dextran or polyethylene glycol were assessed in order to determine the amount of occupied or unoccupied receptors in each phase. Partition coefficients were calculated as the ratio of receptor concentration in the upper polyethylene glycol-rich phase H2O and that in the lower dextranrich phase H2O. The partition coefficient was a sensitive function of the salt at pH above 6·1 and below 5·1. The salt had no effect on the partition coefficient at pH around 5·6. These results suggest that the isoelectric point of the thyroid hormone receptor is about 5·6, confirming previous determinations using isoelectric focusing. The partition coefficient of the receptor decreased upon T3 binding, regardless of the salt composition. In contrast, the partition coefficient of thyroxine-binding globulin increased upon T3 binding. Free T3 preferentially partitioned into the upper polyethylene glycol-rich phase and gave a partition coefficient higher than 1·0. These results strongly suggest that the decrease in the partition coefficient of the receptor upon hormone binding reflects conformational changes or changes in electrostatic properties of the receptor upon hormone binding. Such an alteration may be involved in biological activation of the receptor upon hormone binding.
J. Endocr. (1988) 119, 431–437
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liver is positively regulated by T3, primarily by binding of the liganded thyroid hormone receptor (TR)-β1 to TREs in the promoter region of the D1 gene ( Jakobs et al. 1997 , Amma et al. 2001 ). The induction of proinflammatory cytokines by
Center for Health Disparities and Molecular Medicine, School of Medicine, Loma Linda University, Loma Linda, California, USA
Division of Biochemistry, Department of Basic Sciences, School of Medicine, Loma Linda University, Loma Linda, California, USA
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Center for Health Disparities and Molecular Medicine, School of Medicine, Loma Linda University, Loma Linda, California, USA
Division of Biochemistry, Department of Basic Sciences, School of Medicine, Loma Linda University, Loma Linda, California, USA
Department of Medicine, School of Medicine, Loma Linda University, Loma Linda, California, USA
Department of Orthopedics, School of Medicine, Loma Linda University, Loma Linda, California, USA
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Contrasting developmental and tissue-specific expression of alpha and beta thyroid hormone receptor genes . EMBO Journal 9 . ( https://doi.org/10.1002/j.1460-2075.1990.tb08270.x ) Gouveia CHA Miranda-Rodrigues M Martins GM Neofiti-Papi B 2018
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ABSTRACT
The effects of histone subfractions on rat liver thyroid hormone receptor–DNA interaction were examined using an in-vitro DNA-cellulose binding assay. H1 histones bound to DNA showed reversible and potent inhibition of receptor–DNA binding without affecting receptor–hormone binding. Poly-lysine, bovine serum albumin, ovalbumin and cytochrome c did not alter receptor–DNA binding. H1 histone subfractions (calf thymus lysine-rich histone (CTL)-1, CTL-2 and CTL-3) showed potent inhibition of receptor–DNA binding indistinguishable from each other. The quantity of H1 histone subfractions bound to DNA was the same. Although each subfraction has different functional properties, inhibition of receptor–DNA binding was a common feature of all the H1 histone subfractions, which is important for the non-random distribution of the receptor in chromatin.
Binding of the receptor to core histones was investigated; it was found to bind to core histones more potently than to other proteins (H1 histone, ovalbumin and cytochrome c). Among core histone subfractions, H4 histone bound to the receptor most potently and is the candidate to be one of the acceptor sites of the receptor in chromatin.
Journal of Endocrinology (1989) 121, 337–341
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ABSTRACT
Rat liver nuclear thyroid hormone receptor lost 3,5,3′-tri-iodo-l-thyronine (T3)-binding activity with a half-life of 14 days, 4 h, 139 min, 62 min, 16 min or 6 min at 0, 36, 38, 40, 43 or 45 °C respectively, when present in crude nuclear extracts. Glycerol increased the half-life of the receptor during heat inactivation. Protection was reversible by removing the glycerol. The receptor was unstable at a pH below 6·0 or above 10·0. We also found a loss of the receptor activity during the separation of bound and free hormone using the resin test. Of several conditions tested for the separation of bound and free hormone, the addition of heated nuclear extract gave the most accurate estimation of bound hormone when using the resin test. Using these characteristics of the receptor, we purified the receptor to 1220 pmol T3-binding capacity/mg protein with a final yield of 14·6 μg/4 kg rat liver.
Journal of Endocrinology (1989) 120, 237–243
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The gene expression of thyroid hormone receptors (TR) in ECRF24 immortalized human umbilical vein endothelial cells (HUVECs) was investigated at both the mRNA and the protein level. Endothelin-1 (ET-1) and von Willebrand factor (vWF) production were measured in response to triiodothyronine (T(3)) administration. A real-time PCR technique was used to quantify the presence of mRNAs encoding for the different isoforms of the TR. The binding of T(3) to nuclear TRs was studied in isolated endothelial cell nuclei by Scatchard analysis. Expression of TR at the protein level was investigated by immunocytochemistry and Western blotting using TR-isoform-specific polyclonal rabbit antisera. ET-1 and vWF were measured in cell supernatants with a two-site immunoenzymatic assay. Scatchard analysis yielded a maximum binding capacity of 55 fmol T(3)/mg DNA (+/-200 sites/cell) with a K(d) of 125 pmol/l. Messenger RNAs encoding for the TRalpha1 and the TRalpha2 and the TRbeta1 were observed. The approximate number of mRNA molecules per cell was at least 50 molecules per cell for TRalpha1, five for TRalpha2 and two for TRbeta1. Immunocytochemistry revealed (peri)nuclear staining for TRbeta1, TRalpha1 and TRalpha2. ET-1 and vWF secretion did not increase upon addition of T(3) (10(-10)-10(-6) M). Immortalized ECRF24 HUVECs express TR, but at low levels. The number of TRs per endothelial cell is probably too low to be functional and no change in ET-1 or vWF production was found after addition of T(3). Therefore we conclude that the genomic effects of T(3) are unlikely to occur in these immortalized HUVECs.
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Many metabolic processes occur simultaneously in the liver in different locations along the porto-central axis of the liver units. These processes are often regulated by hormones, one of which is thyroid hormone which for its action depends on the presence of the different isoforms of the thyroid hormone receptor (TR). These are encoded by two genes: c-erbA-alpha encoding TRalpha1 and TRalpha2 and their respective Delta isoforms, and c-erbA-beta which encodes TRbeta1, TRbeta2 and TRbeta3. We recently found a zonal (pericentral) expression of and a diurnal variation in the TRbeta1 isoform in rat liver. We were therefore also interested to see whether TRalpha1 and TRalpha2 expression showed similar characteristics. For this reason we raised both polyclonal and monoclonal antibodies against TRalpha1 and TRalpha2 isoforms and characterised these. Antibody specificity was tested using Western blots and immunohistochemistry in liver of TR isoform-specific knockout animals. Using these antibodies we found that the TRalpha1 and TRalpha2 isoforms are zonally expressed around the central vein in rat liver. The experiments show that the portal to central gradient of TRalpha1 is broader than that of TRbeta1. Moreover, the expression of the TRalpha2 protein showed a diurnal variation with a peak in the afternoon when the animals are least active whereas no such variation was found for the TRalpha1 protein.From our data it appears that both the TRalpha1 and TRalpha2 isoforms show a zonal distribution in liver. This finding, together with the observed diurnal rhythm, has major implications for interpreting and timing experiments concerning the TR and its downstream actions in liver.
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The c-erbAalpha gene encodes two thyroid hormone receptors, TRalpha1 and TRalpha2, that arise from alternative splicing of the TRalpha pre-mRNA. TRalpha2 is not able to bind triiodothyronine (T(3)) and acts as a weak antagonist of TRs. It has been suggested that the balance of TRalpha1 to TRalpha2 is important in maintaining homeostasis. Here, we study the effect of thyroid hormone on the splicing of TRalpha under various conditions in HepG2 cells. First, T(3) was added to HepG2 cells that endogenously express TRalpha. This resulted in a decrease in the TRalpha1:TRalpha2 mRNA ratio after the addition of 10(-)(8 )M or 10(-)(7 )M T(3). Then, HepG2 cells were incubated with sera from hypothyroid or hyperthyroid patients. Sera from hyperthyroid patients (n=6) decreased the TRalpha1:TRalpha2 ratio compared with HepG2 cells incubated with sera from euthyroid patients (n=8). Sera from hypothyroid patients (n=6) had no effect on the TRalpha1:TRalpha2 ratio but supplementation with T(3) caused a decrease in the ratio. Finally, we tested sera from patients with nonthyroidal illness (NTI; n=17) which showed no effect on TRalpha splicing when compared with controls. Free thyroxine levels in sera from hypo-, eu-, and hyperthyroid patients, but not that of NTI patients, were negatively correlated (P<0.01) to the TRalpha1:TRalpha2 ratio. We next studied the expression of the splicing factors hnRNP A1 and ASF/SF2 (SF2) in relation to the splicing of the TRalpha gene. In HepG2 cells incubated with NTI sera a negative relationship was found between the ratio of hnRNP A1:SF2 and the TRalpha1:TRalpha2 ratio. A high hnRNP A1:SF2 ratio is associated with the use of the distal 5'-splice site. The splicing direction should then change towards TRalpha2, which is indeed the case. Rev-ErbA, which is partly complementary to TRalpha2 and could therefore interfere in the splicing process, did not relate to the TRalpha1:TRalpha2 ratio.In conclusion, high T(3) levels induce a low TRalpha1:TRalpha2 ratio which could protect the cell from excessive T(3)-induced gene expression. In vivo, this might be a mechanism to keep tIssues relatively euthyroid during high serum T(3) levels.
