Human Thyroid Hormone Receptor Beta Research Articles

Benzotriazole ultraviolet stabilizers show potent activities as human aryl hydrocarbon receptor ligands.

Benzotriazole ultraviolet stabilizers (BUVSs) used in consumer products are raising concerns as new pollutants in the aquatic environment. We determined the agonistic activities of eight BUVSs and a chemically distinct UV absorber (4-methylbenzylidinecamphor) toward the human aryl hydrocarbon receptor (AhR) and thyroid hormone receptors alpha and beta. Although none of the BUVSs showed ligand activity against the thyroid hormone receptors, four of them (UV-P, UV-9, UV-326, and UV-090) showed significant AhR ligand activity. Their half-maximal effective concentrations (EC50) were 130 nM for UV-P, 460 nM for UV-9, and 5.1 μM for UV-090 (a value for UV-326 could not be determined). Of the numerous AhR ligands, it is well-known that those considered nontoxic are quickly metabolized by enzymes such as CYP1A1, which destroys their ability to function as ligands. Accordingly, we established a new yeast assay for simultaneous monitoring of both the strength of AhR ligand activity and ligand degradation by CYP1A1. We found the AhR ligand activities of the above four BUVSs to be stable in the presence of CYP1A1; therefore, they have the potential to accumulate and exert potent physiological effects in humans, analogous to polycyclic aromatic hydrocarbons and dioxins, which are known stable and toxic ligands.

Novel selective inhibitors of the interaction of individual nuclear hormone receptors with a mutually shared steroid receptor coactivator 2.

Nuclear hormone receptor (NR) signaling, currently a therapeutic target in multiple diseases, involves an ordered series of protein interactions to regulate transcription in response to changing hormone levels. Later steps in the process of ligand-dependent signaling are driven by a highly conserved interaction between the NRs and the steroid receptor coactivators (SRCs) that is effected by a conserved interaction motif (L1XXL2L3), known as an NR box. Using computational design and combinatorial chemistry, we have produced novel alpha-helical proteomimetics of the second NR box of SRC2 that exploit structural differences between human estrogen receptor alpha (hERalpha), human estrogen receptor beta (hERbeta), and human thyroid hormone receptor beta (hTRbeta). The resulting library sequentially replaced each leucine with non-natural side chains. Screening this library using a quantitative competition assay revealed compounds that selectively inhibit the interaction of SRC2-2 with each individual NR in preference to its interaction with the other NR. This approach generated highly selective compounds from one that had no specificity for a particular family member. These compounds represent the first family-member-selective competitive inhibitors of the protein interactions of transcription factors.

Thyroid receptor ligands. 1. Agonist ligands selective for the thyroid receptor beta1.

Endogenous thyroid receptor hormones 3,5,3',5'-tetraiodo-l-thyronine (T(4), 1) and 3,5,3'-triiodo-l-thyronine (T(3), 2) exert a significant effects on growth, development, and homeostasis in mammals. They regulate important genes in intestinal, skeletal, and cardiac muscles, the liver, and the central nervous system, influence overall metabolic rate, cholesterol and triglyceride levels, and heart rate, and affect mood and overall sense of well being. The literature suggests many or most effects of thyroid hormones on the heart, in particular on the heart rate and rhythm, are mediated through the TRalpha(1) isoform, while most actions of the hormones on the liver and other tissues are mediated more through the TRbeta(1) isoform of the receptor. Some effects of thyroid hormones may be therapeutically useful in nonthyroid disorders if adverse effects can be minimized or eliminated. These potentially useful features include weight reduction for the treatment of obesity, cholesterol lowering for treating hyperlipidemia, amelioration of depression, and stimulation of bone formation in osteoporosis. Prior attempts to utilize thyroid hormones pharmacologically to treat these disorders have been limited by manifestations of hyperthyroidism and, in particular, cardiovascular toxicity. Consequently, development of thyroid hormone receptor agonists that are selective for the beta-isoform could lead to safe therapies for these common disorders while avoiding cardiotoxicity. We describe here the synthesis and evaluation of a series of novel TR ligands, which are selective for TRbeta(1) over TRalpha(1). These ligands could potentially be useful for treatment of various disorders as outlined above. From a series of homologous R(1)-substituted carboxylic acid derivatives, increasing chain length was found to have a profound effect on affinity and selectivity in a radioreceptor binding assay for the human thyroid hormone receptors alpha(1) and beta(1) (TRalpha(1) and TRbeta(2)) as well as a reporter cell assay employing CHOK1-cells (Chinese hamster ovary cells) stably transfected with hTRalpha(1) or hTRbeta(1) and an alkaline phosphatase reporter-gene downstream thyroid response element (TRAFalpha(1) and TRAFbeta(1)). Affinity increases in the order formic, acetic, and propionic acid, while beta-selectivity is highest when the R(1) position is substituted with acetic acid. Within this series 3,5-dibromo-4-[(4-hydroxy-3-isopropylphenoxy)phenyl]acetic acid (11a) and 3,5-dichloro-4-[(4-hydroxy-3-isopropylphenoxy)phenyl]acetic acid (15) were found to reveal the most promising in vitro data based on isoform selectivity and were selected for further in vivo studies. The effect of 2, 11a, and 15 in a cholesterol-fed rat model was monitored including potencies for heart rate (ED(15)), cholesterol (ED(50)), and TSH (ED(50)). Potency for tachycardia was significantly reduced for the TRbeta selective compounds 11a and 15 compared with 2, while both 11a and 15 retained the cholesterol-lowering potency of 2. This left an approximately 10-fold therapeutic window between heart rate and cholesterol, which is consistent with the action of ligands that are approximately 10-fold more selective for TRbeta(1). We also report the X-ray crystallographic structures of the ligand binding domains of TRalpha and TRbeta in complex with 15. These structures reveal that the single amino acid difference in the ligand binding pocket (Ser277 in TRalpha or Asn331 in TRbeta) results in a slightly different hydrogen bonding pattern that may explain the increased beta-selectivity of 15.

Thyroid hormone receptor-beta mutations conferring hormone resistance and reduced corepressor release exhibit decreased stability in the N-terminal ligand-binding domain.

Resistance to thyroid hormone (RTH) syndrome is associated with mutations in the human thyroid hormone receptor-beta (hTRbeta), many of which show marked reduction in hormone binding. Here, we investigated the structural consequences of two RTH mutants (A234T and R243Q), residing in the flexible N-terminal portion of the ligand binding domain (LBD), which exhibit modestly reduced hormone binding with impaired release of corepressor. X-ray crystallography analyses revealed that these two RTH mutants modulate the position of this flexible region by either altering the movement of helix 1 (A234T) or disrupting a salt bridge (R243Q). The subsequent increased flexibility and mobility in regions after the two sites of mutation coincided with a disorganized LBD. Consistent with this finding, the ability of these mutant N-terminal regions (234-260) to recruit the remaining LBD was decreased in a ligand-dependent helix assembly assay. Collectively, these data suggest that structural information imparted by the flexible segment in the N-terminal LBD is critical for overall stability of the LBD. Thus, these structural analyses provide mechanistic insight into the etiology of RTH disease in human TRbeta mutants that exhibit hormone binding with decreased ligand-dependent corepressor release.

Synthesis and Preliminary Characterization of a Novel Antiarrhythmic Compound (KB130015) with an Improved Toxicity Profile Compared with Amiodarone

Recent developments in antiarrhythmic therapy have indicated that the best approach to pharmacologically controlling supraventricular arrhythmias and life-threatening ventricular tachyarrhythmias is by prolonging cardiac repolarization rather than by blocking conduction. In this context, amiodarone has emerged as the most potent compound, but its universal use has been limited by its toxicity profile. There are data to suggest that an important component of amiodarones antiarrhythmic action might be mediated via inhibition of thyroid hormone action in the heart. Therefore, a new series of carboxymethoxybenzoyl and benzyl derivatives of benzofuran has been prepared and evaluated as thyroid hormone receptor antagonists. Within this series, 2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran KB130015 (7) was found to reveal the most promising in vitro data. It inhibits the binding of (125)I-T(3) to the human thyroid hormone receptors (hThR) alpha(1) and beta(1). T(3)-Antagonism was confirmed in reporter cell assays employing CHOK1 cells (Chinese hamster ovary cells) stably transfected with hThR alpha(1) or hThR beta(1) and an alkaline phosphatase reporter gene downstream a thyroid response element. The derived IC(50) values were 2.2 microM for hThR alpha(1) and 4.1 microM for hThR beta(1). Compound 7 was selected for further characterization of chronic effects on ventricular papillary muscle by transmembrane electrophysiology after daily intraperitoneal injection of the ligand (40 mg/kg body weight) in guinea pigs. Compound 7 was found to prolong the action potential duration at 90% (APD(90)) repolarization time (219 +/- 22 ms, control: 186 +/- 9 ms, p < 0.01) without exhibiting any reverse rate dependency of action in a manner similar to that of amiodarone. In general, preliminary tolerance experiments with 7 demonstrated an improved safety profile compared to that of amiodarone. In summary, 7 appears to be less toxic than amiodarone while maintaining its electrophysiologic properties consistent with antiarrhythmic activity. Its potential antiarrhythmic actions warrant further investigations.

Proteasome-dependent degradation of the human estrogen receptor.

In eukaryotic cells, the ubiquitin-proteasome pathway is the major mechanism for the targeted degradation of proteins with short half-lives. The covalent attachment of ubiquitin to lysine residues of targeted proteins is a signal for the recognition and rapid degradation by the proteasome, a large multi-subunit protease. In this report, we demonstrate that the human estrogen receptor (ER) protein is rapidly degraded in mammalian cells in an estradiol-dependent manner. The treatment of mammalian cells with the proteasome inhibitor MG132 inhibits activity of the proteasome and blocks ER degradation, suggesting that ER protein is turned over through the ubiquitin-proteasome pathway. In addition, we show that in vitro ER degradation depends on ubiquitin-activating E1 enzyme (UBA) and ubiquitin-conjugating E2 enzymes (UBCs), and the proteasome inhibitors MG132 and lactacystin block ER protein degradation in vitro. Furthermore, the UBA/UBCs and proteasome inhibitors promote the accumulation of higher molecular weight forms of ER. The UBA and UBCs, which promote ER degradation in vitro, have no significant effect on human progesterone receptor and human thyroid hormone receptor beta proteins.

Molecular and structural biology of thyroid hormone receptors.

1. Thyroid hormone receptors (TR) are expressed from two separate genes (alpha and beta) and belong to the nuclear receptor superfamily, which also contains receptors for steroids, vitamins and prostaglandins. 2. Unliganded TR are bound to DNA thyroid hormone response elements (TRE) predominantly as homodimers, or as heterodimers with retinoid X-receptors (RXR), and are associated with a complex of proteins containing corepressor proteins. Ligand binding promotes corepressor dissociation and binding of a coactivator. 3. Recent studies from our group have focused on the acquisition and use of X-ray crystallographic structures of ligand-binding domains (LBD) of both the rat (r) TR alpha and the human (h) TR beta bound to several different ligands. We have also developed ligands that bind selectively to the TR beta, which may provide ways to explore the differential functions of TR alpha compared with TR beta isoforms. 4. The LBD is comprised mostly of alpha-helices. The ligand is completely buried in the receptor and forms part of its hydrophobic core. Kinetic studies suggest that the limiting step in formation of high-affinity ligand-receptor complexes is the rate of folding of the receptor around the ligand. Ligands can be fitted tightly in the ligand-binding pocket and small differences in this fitting may explain many structure-activity relationships. Interestingly, analysis of the structures of antagonists suggests that they have chemical groups, 'extensions', that could impair receptor folding around them and, thus, prevent the agonist-induced conformation changes in the receptor. 5. The TR structures allowed us to see that the mutations that occur in the syndrome of generalized resistance to thyroid hormone are located in the vicinity of the ligand-binding pocket. 6. X-ray structure of the TR has also been used to guide construction of mutations in the TR surface that block binding of various proteins important for receptor function. Studies with these TR mutants reveal that the interfaces for homo- and heterodimerization map to similar residues in helix 10 and 11 and also allow the definition of the surface for binding of coactivators, which appears to be general for nuclear receptors. Formation of this surface, which involves packing of helix 12 of the TR into a scaffold formed by helices 3 and 5, appears to be the major change in the receptor structure induced by hormone occupancy.

β-Trace Gene Expression Is Regulated by a Core Promoter and a Distal Thyroid Hormone Response Element

We isolated and characterized the human beta-Trace protein (betaTP) gene promoter. betaTP, also known as prostaglandin D2 synthase, is a lipocalin secreted from the choroid plexus and meninges into cerebrospinal fluid. Basal transcription of the betaTP gene is directed from a core promoter found within the first 325 bases of the 5'-flanking sequence. The betaTP gene promoter is responsive to thyroid hormone (3,3',5-triiodothyronine, T3) and efficiently repressed by unliganded human thyroid hormone receptor beta (TRbeta). Functional analysis of the betaTP promoter in TE671 cells revealed that responsiveness to T3 occurs in sequences 2.5 kilobase pairs 5' of the start site. Within the hormone-responsive region we identified a thyroid hormone response element (TRE) located from -2576 to -2562 base pairs relative to the transcription start site. The betaTP TRE is composed of two directly repeated consensus half-sites separated by a 3-base pair space (DR3). The betaTP TRE forms specific complexes with TRbeta. We have shown that a gene active in the choroid plexus and meninges is responsive to T3. T3 may play a role in the regulated transport of substances into the cerebrospinal fluid and ultimately the brain.

A TATA binding protein-associated factor functions as a coactivator for thyroid hormone receptors.

Transcriptional regulation by thyroid hormone receptors (TRs) requires the TR to interact with various proteins. The TATA binding protein-associated factors (TAFs) are cofactors for several transcription factors and, therefore, are candidate cofactors for the TR. To determine whether one or more of the TAFs are cofactors for TRs, direct protein interactions between human TR beta and several Drosophila TAFs were quantitated in vitro. The human (h) TR beta bound specifically to dTAFII110 and weakly to dTAFII60, but did not bind to dTAFII30 alpha, dTAFII30 beta, dTAFII40, dTAFII80, or dTAFII150. The dTAFII110:hTR beta interaction required the carboxyl-terminals of both proteins. The dTAFII110 also interacted with the hTR alpha 1 carboxyl-terminus in a yeast two-hybrid system. Thyroid hormone destabilized the dTAFII110:TR interaction in vitro, but had no effect on the interaction in the two-hybrid system. The dTAFII110 did not bind to human retinoid X receptor alpha in vitro, indicating that this TAF interacts differentially with nuclear receptors. The transcriptional function of hTR beta was enhanced by dTAFII110 in transfection assays, indicating that this TAF can function in the thyroid hormone signalling pathway. Thus, TAFII110 functions as a cofactor for TRs, and the interactions between specific TAFs and nuclear receptors may provide another level of selectivity for transcriptional responses to hormones.

Functional analysis of a proline to serine mutation in codon 453 of the thyroid hormone receptor beta 1 gene.

Mutations in the gene encoding human thyroid hormone receptor beta (hTR beta) have been associated with generalized resistance to thyroid hormone (GRTH). This disorder is associated with significant behavioral abnormalities. We examined the hTR beta gene in a family with members who manifest inappropriately normal TSH, elevated free T4, and free and total T3. Sequence analysis showed a cytosine to thymine transition at nucleotide 1642 in one allele of the index patient's genomic DNA. This altered proline to serine at codon 453. The resulting mutant receptor when expressed in vitro bound DNA with high affinity, but the T3 affinity of the receptor was impaired. The mutant TR demonstrated a dominant negative effect when cotransfected with two isoforms of wild-type receptor and also in the presence of TR variant alpha 2 in COS-1 cells. Mutations of codon 453 occur more frequently than at other sites, and four different amino acid substitutions have been reported. Significant differences in phenotype occur among affected individuals, varying from normality to moderately severe GRTH. There is no clear correlation between Ka or in vitro function of the mutant receptor, and phenotype. This study extends the association between GRTH and illness, and indicates that early diagnosis and counseling are needed in families with TR beta 1 abnormalities.

Quantitation of beta 1 triiodothyronine receptor mRNA in human tissues by competitive reverse transcription polymerase chain reaction.

Thyroid hormones act by binding to nuclear receptor proteins, the thyroid hormone receptors (TR) alpha and beta. Data from cell culture and animal studies indicate that TR expression may be regulated to modulate target organ responsiveness to thyroid hormone. To investigate whether such adaptive changes in TR expression occur in humans, we determined the mRNA levels of the hTR beta 1 in various thyroid states. Patients with overt hypo- or hyperthyroidism were enrolled in the study. Total RNA was isolated from peripheral blood mononuclear cells and hTR beta 1 mRNA levels determined by quantitative competitive reverse transcription PCR. For comparison, hTR beta 1 mRNA levels were determined in lymphocytes and normal thyroid tissue of euthyroid patients. Human TR beta 1 mRNA levels in lymphocytes were 1.8 +/- 0.4, 1.9 +/- 0.5, 1.1 +/- 0.4 10(-18) mol/microgram RNA in hypo-, eu- and hyperthyroid patients, respectively, corresponding to an estimated 0.5 - 2 molecules per cell. Although the mean hTR beta 1 mRNA levels were 40% lower in hyperthyroid than in euthyroid subjects, this difference did not reach statistical significance. Similar levels of hTR beta 1 mRNA levels were detected in thyroid gland from euthyroid patients. In summary, we developed an assay for the quantitative determination of hTR beta 1 mRNA levels in small human tissue samples, containing as little as 50 ng of total RNA. Absolute hTR beta 1 mRNA levels are very low with an estimated one molecule of mRNA being present in a mononuclear blood cell or thyrocyte. No up-regulation of hTR beta 1 was seen in hypothyroid relative to euthyroid patients. However, there is a non-significant trend towards a down-regulation of hTR beta 1 mRNA levels in hyperthyroid patients.

Importance of the most proximal GC box for activity of the promoter of human thyroid hormone receptor beta 1.

We wish to localize the sequences required for transcriptional expression of the thyroid hormone receptor beta 1 (TR beta 1). Constitutive activity of the promoter of human thyroid hormone receptor beta 1 was assessed by transient transfection of deletion constructs attached to luciferase as reporter, into P19, GH3, HepG2, H19-7, and COS1 cells. A 40-base pair fragment of the beta 1 promoter including the TATA box induced minimal luciferase activity, which was considered basal activity. The activities of various lengths of the beta 1 promoter were estimated relative to the minimal promoter in five cell lines. The region between -130 and -40 was crucial for constitutive activity in all cell lines. Further deletion analysis in HepG2 cells showed that two regions mainly augmented the transcriptional activity of the minimal 40 base pair fragment. One region located at -115 to -93, which is highly GC-rich, included the most proximal of five putative GC boxes present in the whole 1325-base pair promoter. A second region contributing to expression of TR beta 1 in HepG2 cells is at -70 to -40. Mutation of the most proximal GC box strongly suppressed transactivity of the whole promoter in P19 and HepG2 cells. In contrast, mutations in the other GC boxes did not suppress transactivation in P19 cells and slightly suppress activation in HepG2 cells. In Schneider cells, which do not express Sp1, transactivity of the region distal to -40 is positively regulated by cotransfection with a vector expressing Sp1.(ABSTRACT TRUNCATED AT 250 WORDS)

Importance of the most proximal GC box for activity of the promoter of human thyroid hormone receptor beta 1

Importance of the most proximal GC box for activity of the promoter of human thyroid hormone receptor beta 1

Factors that enhance Escherichia coli-expressed TR beta binding to T3 and DNA.

Thyroid hormone receptors (TRs) recently have been produced in E. coli by several laboratories. We produced E. coli-expressed human TR beta using the histidine/fusion protein system. Surprisingly, we observed that reticulocyte lysate, nonspecific proteins, and 1% Triton X dramatically increased both the T3- and DNA-binding activities of human TR beta. These studies demonstrate that there are a number of factors that will enhance ligand and DNA binding of E. coli-expressed TR beta. Addition of these factors to reaction samples containing E. coli-expressed TRs will help to optimize measurement conditions. These findings also suggest that experiments in which cellular proteins are added to highly purified TR preparations may require controls to eliminate contributions by nonspecific proteins.

Studies on the repression of basal transcription (silencing) by artificial and natural human thyroid hormone receptor-beta mutants.

Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate target gene transcription. Interestingly, in the absence of ligand, TRs also can repress basal transcription of positively regulated target genes, suggesting that unliganded TR may have a distinct role in gene regulation. In this paper, DNA binding, truncation, and natural human TR beta mutants were used in cotransfection and electrophoretic mobility shift assays to study various aspects of TR-mediated basal repression. Presently, little is known about the role(s) of natural human TR beta mutants on basal repression. These results show that: 1) TR binding to DNA likely is required for basal repression; 2) the amino-terminal region of TR is not required for basal repression; 3) TR homodimer binding is not absolutely required for basal repression, as TR mutants that selectively form TR-retinoid X receptor heterodimers can mediate basal repression; and 4) TR mutants with poor T3-binding affinity likely have constitutive basal repression, even in the presence of ligand. These findings provide new insight on the mechanism of basal repression by unliganded TRs.

Studies on the repression of basal transcription (silencing) by artificial and natural human thyroid hormone receptor-beta mutants

Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate target gene transcription. Interestingly, in the absence of ligand, TRs also can repress basal transcription of positively regulated target genes, suggesting that unliganded TR may have a distinct role in gene regulation. In this paper, DNA binding, truncation, and natural human TR beta mutants were used in cotransfection and electrophoretic mobility shift assays to study various aspects of TR-mediated basal repression. Presently, little is known about the role(s) of natural human TR beta mutants on basal repression. These results show that: 1) TR binding to DNA likely is required for basal repression; 2) the amino-terminal region of TR is not required for basal repression; 3) TR homodimer binding is not absolutely required for basal repression, as TR mutants that selectively form TR-retinoid X receptor heterodimers can mediate basal repression; and 4) TR mutants with poor T3-binding affinity likely have constitutive basal repression, even in the presence of ligand. These findings provide new insight on the mechanism of basal repression by unliganded TRs.

Expression of thyroid hormone receptors in human pituitary tumor cells.

To understand the expression of thyroid hormone receptors (TRs) in various types of pituitary tumor cells, we performed the reverse transcription coupled to polymerase chain reaction (RT-PCR). Specimens of pituitary adenomas were obtained by transsphenoidal adenomectomies from 2 patients with acromegaly, 6 patients with prolactinoma, 3 patients with Cushing's disease, 2 patients with gonadotropin-secreting tumors, one patient with a TSH-secreting tumor, and 3 patients with non-functioning adenoma with no clinical evidence of pituitary hormone overproduction. Human thyroid hormone receptors beta 1 and alpha 1, alpha 2 (h-TR beta 1, alpha 1, alpha 2) are expressed in nonfunctioning adenomas; h-TR alpha 1 and beta 1 are specific for prolactinomas; h-TR beta 1 is involved in acromegalies and FSH-secreting tumors, while h-TR alpha 1, alpha 2, and beta 1 are not expressed in TSH-secreting and ACTH-secreting tumors. The results suggest that human thyroid hormone receptors are differentially expressed in human pituitary tumor cells, and our studies have shed new light on the understanding of the role of TRs in pituitary tumors.

Allele-specific associated polymorphism analysis: Novel modification of SSCP for mutation detection in heterozygous alleles using the paradigm of resistance to thyroid hormone

Allele-specific polymorphism (ASAP) analysis is a modification of single-strand conformation polymorphism (SSCP) mutation screening under optimized temperature conditions in a minigel format with ethidium bromide detection. ASAP analysis was used to screen for and identify mutations within the human thyroid hormone receptor-beta (hTR-beta) gene. These mutations are the underlying cause of resistance to thyroid hormone (RTH). Eleven dissimilar known hTR-beta mutations and six previously uncharacterized mutations were accurately identified. ASAP screening extends to unique ASAP-DNA fingerprinting as an identifying signature for each novel hTR-beta mutation detected thus far. Gel-plugs from the SSCP gels containing polymorphic single-stranded DNA alleles were used without elution to prepare solid-phase sequencing templates for mutant allele PCR and sequencing (MAPS). The coupling of ASAP analysis with MAPS has eliminated many of the interpretative and technical problems associated with the sequencing of heterozygous alleles. Together, this convenient screening and sequencing methodology offers accuracy, reproducibility, speed, and the potential elimination of all radioactivity, providing a general strategy for future automated detection and characterization of genetic mutations.

Thyroid hormone receptors bind to the promoter of the mouse histone H10 gene and modulate its transcription.

It has been shown that the mouse histone H10 promoter contains a DNA element, composed of a direct repeat of the sequence GGTGACC separated by 7 nt, which is able to bind retinoic acid receptors and to modulate transcription of reporter genes following treatment with retinoic acid. We have now investigated whether this DNA motif is also responsive to thyroid hormone. We co-transfected CV-1 monkey kidney cells with chloramphenicol acetyltransferase (CAT) expression plasmids containing either 740 bp of the H10 wild-type promoter or five copies of the repeat element cloned in front of the thymidine kinase promoter and expression vectors for human thyroid hormone receptors (TRs) alpha or beta and retinoid X receptor alpha (RXR alpha). Treatment of transfected cells with triiodothyronine led to a dose-dependent increase in CAT activity. Transfection experiments with increasing amounts of expression vectors for either TR alpha or RXR alpha resulted in up to 6-fold enhancement of CAT transcription. Furthermore, point mutations within the half-sites of the response element of the H10 promoter, as well as deletions within the interspace region, lowered CAT activity to 60-80% of that of the wild-type control. Electrophoretic mobility shift assays showed that the repeat element was able to form retarded complexes with TR alpha homodimers, as well as with TR alpha-RXR alpha heterodimers. Our results suggest that thyroid hormone receptors are involved in the regulation of mouse histone H10 expression.

Three new mutations of thyroid hormone receptor-beta associated with resistance to thyroid hormone.

Three novel point mutations at nucleotides 1249, 1282, and 1614 (exons 9 and 10) of the human thyroid hormone receptor-beta gene were observed in six individuals affected by the syndrome of resistance to thyroid hormone. All three mutations occurred in a heterozygous pattern and caused the following changes in the mature form of the receptor protein: Asp322 to Asn, Glu333 to Gln, and Lys443 to Asn, respectively. The first and third point mutations arose in two unrelated families from eastern Sicily, whereas the second concerned an individual from southern Calabria, apparently presenting a sporadic form of the resistance syndrome. The clinical and biochemical features of resistance to thyroid hormone, both before and after the administration of thyroid hormones, highlight the striking intrafamilial heterogeneity in the phenotypical presentation of the syndrome.