Homozygosity for a dominant negative thyroid hormone receptor gene responsible for generalized resistance to thyroid hormone.

Generalized resistance to thyroid hormones (GRTH) commonly results from mutations in the T3-binding domain of the c-erbA beta thyroid hormone receptor gene. We have reported on a novel deletion mutation in c-erbA beta in a kindred, S, with GRTH. One patient from this kindred was the product of a consanguineous union from two affected members and was homozygous for the beta-receptor defect. This patient at 3.5 weeks of age had unprecedented elevations of TSH, free T4, and free T3 (TSH, 389 mU/L; free T4, 330.8 pmol/L; free T3, 82,719 fmol/L). He displayed a complex mixture of tissue-specific hyperthyroidism and hypothyroidism. He had delayed growth (height age, 1 3/12 yr at chronological age 2 9/12 yr) and skeletal maturation (bone age, 4 months), and developmental delay (developmental age, 8 months), but he was quite tachycardic. The homozygous patient of kindred S is markedly different from a recently reported patient with no c-erbA beta-receptor. This difference indicates that a dominant negative form of c-erbA beta in man can inhibit at least some thyroid hormone action mediated by the c-erbA alpha-receptors.     

Basal and thyroid hormone receptor auxiliary protein-enhanced binding of thyroid hormone receptor isoforms to native thyroid hormone response elements

There are three known isoforms of the rat thyroid hormone receptor, TR alpha-1, TR beta-1, and TR beta-2. The first two are expressed in all tissues, whereas TR beta-2 appears to be expressed only in the pituitary. The differences in the roles of the three receptor isoforms are unknown, but may involve preferential interaction with different subsets of thyroid hormone-regulated genes in different tissues. We tested the binding of the three TR isoforms to putative thyroid hormone response elements (TREs) from genes that are expressed in the pituitary or other tissues and are regulated by thyroid hormone. In vitro translated 35S-labeled rat TR alpha-1, rat TR beta-2, and human TR beta-1 receptors were bound to a battery of biotinylated synthetic deoxyribonucleotides containing naturally occurring putative TREs from genes expressed either in only pituitary (rat glycoprotein hormone alpha-subunit, TSH beta-subunit, and GH) or in nonpituitary (rat alpha-myosin heavy chain, malic enzyme, and Moloney murine leukemia virus promoter) tissues. All three receptor forms bound to each of the TREs. TR beta-2 did not show preferential binding to TREs of pituitary-specific genes compared to TR beta-1. Additionally, TR alpha-1 had a similar TRE-binding pattern as the TR beta s, except for possibly less binding to rat glycoprotein hormone alpha-subunit TRE. Finally, rat pituitary and liver nuclear extracts enhanced TR binding to TREs, with the greatest enhancement seen with the alpha-subunit TRE. These studies suggest that all TR isoforms bind similarly to native TREs. Also, TR binding to TREs can be differentially enhanced by interactions with nuclear proteins.     

Dominant-negative mutant thyroid hormone receptors prevent transcription from Xenopus thyroid hormone receptor beta gene promoter in response to thyroid hormone in Xenopus tadpoles in vivo.

We describe a dominant-negative approach in vivo to assess the strong, early upregulation of thyroid hormone receptor beta (TR beta) gene in response to thyroid hormone, characteristic of the onset of natural and thyroid hormone-induced amphibian metamorphosis, 3,3',5-Triiodo-thyronine (T3) treatment of organ cultures of premetamorphic Xenopus tadpole tails coinjected in vivo with the wild-type Xenopus TR beta (wt-xTR beta) and three different thyroid responsive element chloramphenicol acetyltransferase (TRE-CAT) reporter constructs, including a direct repeat +4 (DR +4) element in the -200/+87 fragment of the xTR beta promoter, resulted in a 4- to 8-fold enhancement of CAT activity. Two human C-terminal TR beta 1 mutants (delta-hTR beta 1 and Ts-hTR beta 1), an artificial Xenopus C-terminal deletion mutant (mt-xTR beta), and the oncogenic viral homology v-erbA, none of which binds T3, inhibited this T3 response of the endogenous wt-xTR in Xenopus XTC-2 cells cotransfected with the -1600/+87 xTR beta promoter-CAT construct, the potency of the dominant-negative effect of these mutant TRs being a function of the strength of their heterodimerization with Xenopus retinoid X receptor gamma. Coinjection of the dominant-negative Xenopus and human mutant TR beta s into Xenopus tadpole tails totally abolished the T3 responsiveness of the wt-xTR beta with different TREs, including the natural DR +4 TRE of the xTR beta promoter.     

The oligomeric state of thyroid receptor regulates hormone binding kinetics

We previously reported that mutations in the thyroid hormone receptor (TR) surface that mediates dimer and heterodimer formation do not alter affinity for cognate hormone (triiodothyronine (T(3))) yet dramatically enhance T(3) association and dissociation rates. This study aimed to show that TR oligomeric state influences binding and dissociation kinetics. We performed binding assays using marked hormone ((125)I-T(3)) and TRs expressed and purified by different methods. We find that T(3) associates with TRs with biphasic kinetics in solution; a rapid step (half-life ±0.1 h) followed by a slower second step (half-life ±5 h) and that purification of monomers suggests that biphasic kinetics are due to the presence of monomers and dimers in our preparations. In support of this idea, incubation of TR ligand binding domain monomers with corepressor peptide induces dimer formation and decreases association rates and T(3) binds to, and dissociates from, a TRβ mutant that only forms dimers (TRβD355R) with slow single-phase kinetics. In addition, heterodimer formation with retinoid X receptors also influences ligand binding kinetics. Together, these results suggest that the dimer/heterodimer surface is allosterically coupled to the hormone binding pocket and that different interactions at this surface exert different effects on ligand binding that may be relevant for TR actions in the cell.     

The effect of endocrine disrupting chemicals on thyroid hormone binding to Japanese quail transthyretin and thyroid hormone receptor

We investigated the effect of endocrine disrupting chemicals (EDCs), including medical, industrial, and agricultural chemicals, on 3,3',5-L-[125I]triiodothyronine ([125I]T3) binding to purified Japanese quail transthyretin (qTTR), a major thyroid hormone-binding protein in plasma, and to the ligand-binding domain of thyroid hormone receptor beta (qTR LBD). Scatchard plots of T3 binding to qTTR and qTR LBD revealed two classes of binding sites, with Kd values of 6.9 and 185 nM, and a single class of binding sites, with Kd value of 0.31 nM, respectively. Among the test chemicals, diethylstilbestrol was the most powerful inhibitor of [125I]T3 binding to qTTR (IC50 < 0.4 nM). Diethylstilbestrol, ioxynil (IC50 =1.1+/-0.5 nM) and pentachlorophenol (IC50 = 6.3+/-3.8 nM) displaced [125I]T3 from qTTR more effectively than unlabeled T3 (IC50 = 9.7+/-0.9 nM) did. Although malathion, 4-nonylphenol, bisphenol A and n-butylbenzyl phthalate were effective inhibitors of [125I]T3 binding to qTTR, their potency was two orders of magnitude less than that of T3. All test chemicals except for diethylstilbestrol had either a weak or no effect on [125I]T3 binding to qTR LBD. These results show that several EDCs tested in this study target qTTR rather than qTR LBD.     

The roles of three forms of human thyroid hormone receptor in gene regulation

We have expressed three forms of human thyroid hormone receptor (hTR alpha 1, alpha 2, and beta) in cultured cells by transient transfection. hTR alpha 1 and beta transfected cells showed increased triiodothyronine (T3) binding capacity, but hTR alpha 2 transfected cells did not. When hTR alpha 1 or beta was cotransfected with pUrGH(S), in which a portion of the rat GH 5' flanking region (-236/-147) was ligated into the CAT reporter plasmid (pUTKAT1), T3 increased CAT gene expression. When hTR alpha 2 was cotransfected with pUrGH(S), T3 did not alter CAT gene expression. When hTR alpha 1 or beta was cotransfected with pUrGH(O), in which a synthetic oligonucleotide representing the TRE from the rat GH 5' flanking region (-189/-160) was substituted for the natural enhancer in pUTKAT1, T3 increased CAT gene expression. When hTR alpha 1 and beta were cotransfected with pUrGH(O), induction by T3 was increased. When hTR alpha 2 was cotransfected with hTR alpha 1 or beta, induction by T3 was decreased. These results indicate that hTR alpha 1 and beta function as native TR, that hTR alpha 1 and beta can recognize the same TRE, that hTR alpha 1 and beta can function additively, and that hTR alpha 2 can inhibit the action of hTR alpha 1 and beta.     

Tissue-specific differential repression of gene expression by a dominant negative mutant of thyroid hormone beta1 receptor.

Resistance to thyroid hormone (RTH) is a genetic disease caused by the mutations of the thyroid hormone beta receptor (TRbeta) gene, producing receptors with a dominant negative action. The present study addressed the question as to whether tissue-specific factors modulate the dominant negative function in different tissues. We prepared stably transfected pituitary GH3 (GH3-PV) and liver SK-Hep-1 (SK-Hep-1-PV) cell lines with a potent dominant negative mutant, PV. The growth hormone (GH) and the malic enzyme genes (ME) in GH3 and SK-Hep-1, respectively, are directly regulated by the thyroid hormone, 3,3,'5-triiodo-L-thyronine (T3). The ratio of the expressed PV/endogenous TRbeta1 proteins was approximately 20 and 5 for GH3-PV and SK-Hep-1-PV cells, respectively. However, the T3-activated expression of the GH gene in GH3-PV and ME gene in SK-Hep-1-PV was repressed by approximately 30% and 90%, respectively, indicating the lack of correlation of PV/TRpbeta1 protein ratio with the dominant negative potency of mutant PV. Furthermore, the synergistic effect of the pituitary-specific factor 1 on the TR-mediated GH promoter activity was not repressed by mutant PV. Taken together, these results suggest that the dominant negative effect of mutant TR is variable in the tissues studied.     

Discrimination of DNA response elements for thyroid hormone and estrogen is dependent on dimerization of receptor DNA binding domains.

We and others have previously shown that a two-amino acid substitution in the base of the first zinc finger of the glucocorticoid receptor DNA binding domain (DBD) is sufficient to alter the receptor's target DNA from a glucocorticoid response element (GRE) to an estrogen response element (ERE). Activation of a thyroid hormone response element (TRE) has been shown to require an additional five-amino acid change in the second zinc finger of the thyroid hormone receptor (TR). Using closely related TRE and ERE sequences, we report that a receptor containing the TR DBD activates the ERE poorly, and receptors containing essential amino acids of the estrogen receptor (ER) DBD activate the TRE poorly. The ER DBD (expressed in Escherichia coli) selectively bound to a 32P-labeled ERE (32P-ERE) as a dimer and a 32P-TRE as a monomer, whereas the TR DBD bound 32P-TRE as a dimer and 32P-ERE as a monomer. When hybrid receptor DBDs were examined, we found that the five amino acids in the second zinc finger of the TR necessary for TRE activation were also essential for dimer formation on a TRE. Dimer formation of ER on an ERE was localized to the second half of the second zinc finger. These results suggest that the ability of ER and TR to functionally discriminate between an ERE and a TRE is a result of dimerization of their DBDs.     

Thyroid hormone resistance in the heart: role of the thyroid hormone receptor beta isoform

Several cardiac genes possess thyroid hormone (TH) response elements regulated by TH receptors. Mutation in TR-beta gene causes the human syndrome of resistance to TH, which is characterized by elevated serum concentration of T(4) and T(3) and variable degrees of insensitivity to TH. It is unclear, however, whether a mutant TR-beta could function as a dominant negative in the heart when expressed from the endogenous locus. A well-described resistance to TH (Delta337T) was either introduced into germline of mice (KI-mut) or expressed as a transgene in the heart using a cardiac-specific promoter (KS-mut). Mice were studied at baseline, after 5-propyl-2-thiouracil (PTU) or after PTU and T(3) treatment (PTU + T(3)). PTU + T(3) treatment significantly increased left ventricular mass in all groups compared with baseline measurements, although the increase in left ventricular mass was significantly less in KI-mut animals. Baseline heart rates (HRs) were similar in wild-type (WT) and KI-mut but were lower in KS-mut animals. After TH deprivation (PTU), HR decreased in WT and KI-mut animals; similarly, HR increased in WT and KI-mut after PTU + T(3). In contrast, HR in KS-mut animals did not change after either treatment. Except for cardiac hypertrophy, the presence of a germline TR-beta mutation had surprisingly little effect on cardiac function.     

The role of serum binding proteins in determining free thyroid hormone concentrations during development in quail

Japanese quail were used as a model for studying the role of binding proteins in determining free T4 (FT4) and free T3 (FT3) concentrations during development. Adults were used to characterize thyroid hormone binding; developmental stages studied were late embryonic, perinatal, hatchling, and juvenile. Total and free hormones were determined directly by RIA, and free hormones were determined indirectly by equilibrium dialysis. Binding proteins were identified by electrophoresis of serum preincubated with labeled hormones. Serum FT4 and FT3 concentrations in adult quail were equivalent to those in humans. T4 bound principally to albumin and secondarily to prealbumin; T3 bound principally to alpha-globulin and secondarily to albumin and gamma-globulin. A specific T4-binding globulin, as in mammals, was not present. The relative affinity of stripped serum was greater for T4 than for T3. In late embryos, FT4 concentrations rise as a result of a marked increase in total T4 (TT4) and modest increases in binding proteins. The perinatal peak in FT4 reflects the perinatal surge of TT4 without a change in binding proteins. From days 1-6 posthatching, FT4 decreases as a consequence of TT4 decreasing faster than the decrease in binding. In juveniles, FT4 concentrations stabilize as increases in TT4 are paralleled by increases in serum binding. T3 binding shows few significant differences from adult values during development, so FT3 concentrations follow closely the pattern of TT3 changes. These results demonstrate that developmental changes in serum binding proteins play a significant role in determining the pattern of free thyroid hormones, especially for FT4, by modulating the total hormone concentrations controlled by the hypothalamic-pituitary axis.     

Lacking thyroid hormone receptor beta gene does not influence alterations in peripheral thyroid hormone metabolism during acute illness

The downregulation of liver deiodinase type 1 (D1) is supposed to be one of the mechanisms behind the decrease in serum tri-iodothyronine (T3) observed during non-thyroidal illness (NTI). Liver D1 mRNA expression is positively regulated by T3, mainly via the thyroid hormone receptor (TR)beta1. One might thus expect that lacking the TRbeta gene would result in diminished downregulation of liver D1 expression and a smaller decrease in serum T3 during illness. In this study, we used TRbeta-/- mice to evaluate the role of TRbeta in lipopolysaccharide (LPS, a bacterial endotoxin)-induced changes in thyroid hormone metabolism. Our results show that the LPS-induced serum T3 and thyroxine and liver D1 decrease takes place despite the absence of TRbeta. Furthermore, we observed basal differences in liver D1 mRNA and activity between TRbeta-/- and wild-type mice and TRbeta-/- males and females, which did not result in differences in serum T3. Serum T3 decreased rapidly after LPS administration, followed by decreased liver D1, indicating that the contribution of liver D1 during NTI may be limited with respect to decreased serum T3 levels. Muscle D2 mRNA did not compensate for the low basal liver D1 observed in TRbeta-/- mice and increased in response to LPS in TRbeta-/- and WT mice. Other (TRbeta independent) mechanisms like decreased thyroidal secretion and decreased binding to thyroid hormone-binding proteins probably play a role in the early decrease in serum T3 observed in this study.