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We report two unusual cases of resistance to thyroid hormone (RTH) in one family. The first case, a male infant, had clinical features of thyrotoxicosis in the neonatal period. In the fourth week of life weight gain was poor despite a daily intake of standard infant formula almost double the infant's estimated requirements. At this time serum free T4 (fT4) was 60.7 pmol/l (Normal range [NR] 11-25 pmol/l) and TSH was inappropriately normal at 1.8 mU/l (NR 0.3-4.0 mU/l). The infant responded clinically and biochemically to propylthiouracil (PTU) at a dose of 10 mg/kg/day. Following 27 days of treatment serum fT4 was 22.6 pmol/l and TSH had risen to 24.9 mU/l. As the infant was thriving treatment was discontinued. The infant, now aged 6 months old, remains clinically euthyroid and developmentally normal off treatment. The infant's mother, from whom he had inherited a mutation of the thyroid receptor beta (TRbeta) gene (M313T), presented earlier with secondary infertility and clinical features of thyrotoxicosis. Treatment with PTU restored her fertility and she spontaneously conceived. In the subsequent pregnancy, clinical and biochemical features of RTH improved, and she gave birth to a small but healthy female infant. In the next pregnancy, resulting in the birth of the affected male infant, clinical and biochemical features of RTH worsened, and high doses of PTU were required to maintain a clinically euthyroid state. To our knowledge, these are the first case reports of RTH associated with added features of a hypermetabolic state in infancy and secondary infertility.

The chromatin architecture of a promoter is an important determinant of its transcriptional response. For most target genes, the thyroid hormone receptor (TR) activates gene expression in response to thyroid hormone (T(3)). In contrast, the thyroid-stimulating hormone alpha-subunit (TSH alpha) gene promoter is down-regulated by TR in the presence of T(3). Here we utilize the capacity for the Xenopus oocyte to chromatinize exogenous nuclear- injected DNA to analyze the chromatin architecture of the TSH alpha promoter and how this changes upon TR-mediated regulation. Interestingly, in the oocyte, the TSH alpha promoter was positively regulated by T(3). In the inactive state, the promoter contained six loosely positioned nucleosomes. The addition of TR/retinoid X receptor together had no effect on the chromatin structure, but the inclusion of T(3) induced strong positioning of a dinucleosome in the TSH alpha proximal promoter that was bordered by regions that were hypersensitive to cleavage by methidiumpropyl EDTA. We identified a novel thyroid response element that coincided with the proximal hypersensitive region. Furthermore, we examined the consequences of mutations in TR that impaired coactivator recruitment. In a comparison with the Xenopus TR beta A promoter, we found that the effects of these mutations on transactivation and chromatin remodeling were significantly more severe on the TSH alpha promoter.

Thyroid hormones have well-documented effects on the skeleton although the mechanism of their action on bone is poorly understood. We have recently reported the presence of different thyroid hormone receptor isoforms in human bone. However, there is evidence to suggest that the expression of thyroid hormone receptor (TR) protein may not necessarily correlate with its mRNA. In this study, we used specific digoxigenin-labeled ribo probes to investigate the expression of TRalpha1, variant TRalpha2, TRbeta1, and in particular TRbeta2 mRNA in human osteophytic bone and osteoclastoma tissue in situ. The number of positive cells was expressed as the percentage of the total number of cells of the same phenotype. In osteophytes, at sites of endochondral ossification, TRalpha1, variant TRalpha2, TRbeta1, and TRbeta2 mRNA were widely distributed in undifferentiated, proliferating, mature and hypertrophic chondrocytes. At sites of bone remodeling, TRalpha1 mRNA was expressed in the majority (> 90%) of osteoblasts. TRbeta1 and the variant TR-alpha2 mRNA were moderately expressed in approximately 75% of cells with only a few osteoblasts (< 25%) expressing TRbeta2 mRNA. All the TR transcripts were highly expressed in multinucleated osteoclasts in osteoclastoma tissue. The distribution of TR mRNAs was similar to TR receptor protein expression (as we have previously reported) in both osteophytic bone and osteoclastoma tissue except TRalpha1 mRNA that was highly expressed in osteoclasts and in undifferentiated, proliferating, mature, and hypertrophic chondrocytes in contrast to its receptor protein expression. This study highlights the importance of studying both TR mRNA and receptor proteins in triiodothyronine (T3) responsive tissues. This is also the first demonstration of the presence of TRbeta2 mRNA in bone. The role of TRbeta2 in mediating the actions of thyroid hormones in bone is not known and requires further investigation.

The syndrome of resistance to thyroid hormone is associated with diverse mutations in the ligand-binding domain of the thyroid hormone beta receptor, localizing to three clusters around the hormone binding cavity. Here, we report three novel resistance to thyroid hormone mutations (S314C, S314F, and S314Y), due to different nucleotide substitutions in the same codon, occurring in six separate families. Functional characterization of these mutant receptors showed marked differences in their properties. S314F and S314Y receptor mutants exhibited significant transcriptional impairment in keeping with negligible ligand binding and were potent dominant negative inhibitors of wild-type receptor action. In contrast, the S314C mutant bound ligand with reduced affinity, such that its functional impairment and dominant negative activity manifest at low concentrations of thyroid hormone, but are more reversible at higher T3 concentrations. The degree of functional impairment of mutant receptors in vitro may correlate with the magnitude of thyroid dysfunction in vivo. Modelling these mutations using the crystal structure of thyroid hormone receptor beta shows why ligand binding is perturbed and why the phenylalanine/tyrosine mutations are more deleterious than cysteine.

Desethylamiodarone (DEA) acts as a competitive inhibitor of triiodothyronine (T3) binding to the alpha1-thyroid hormone receptor (TR alpha1) but as a non-competitive inhibitor with respect to TR beta1. To gain insight into the position of the binding site of desethylamiodarone on TR beta1 we investigated the naturally occurring mutants Y321C, R429Q, P453A, P453T and the artificial mutants L421R and E457A in the ligand binding domain of human TR beta1. The IC50 values (in microM) of DEA for P453A (50 +/- 11) and P453T (55 +/- 16) mutant TR beta1 are not different from that for the wild type TR beta1 (56 +/- 15), but the IC50 values of R429Q (32 +/- 7; P<0.001) and E457A (17 +/- 3; P<0.001) are significantly lower than of the wild type. Scatchard plots and Langmuir analyses indicate a non-competitive nature of the inhibition by DEA of T3 binding to all four mutant TR beta1s tested. Mutants P453A and P453T do not influence overall electrostatic potential, and also do not influence the affinity for DEA compared to wild type. Mutant E457A causes a change from a negatively charged amino acid to a hydrophobic amino acid, enhancing the affinity for DEA. Mutant R429Q, located in helix 11, causes an electrostatic potential change from positive to uncharged, also resulting in greater affinity for DEA. We therefore postulate that amino acids R429 and E457 are at or close to the binding site for DEA, and that DEA does not bind in the T3 binding pocket itself, in line with the non-competitive nature of the inhibition of T3 binding to TR beta1 by DEA.

Resistance to thyroid hormone (RTH) has hitherto been associated with thyroid hormone beta receptor (TRbeta) mutations which cluster in two regions (alphaalpha 310-353 and alphaalpha 429-461) of the hormone-binding domain and closely approximate the ligand-binding cavity. Here, we describe a third cluster of RTH mutations extending from alphaalpha 234-282 which constitute a third boundary of the ligand pocket. One mutant, T277A, exhibits impaired transactivation which is disproportionate to its mildly reduced ligand affinity (Ka). T3-dependent recruitment of coactivators (SRC-1, ACTR) by mutant receptor-RXR heterodimers was reduced in comparison with wild-type. Cotransfection of SRC-1 restored transactivation by T277A. In the TRbeta crystal structure this helix 3 residue is surface-exposed and is in close proximity to residues L454 and E457 in helix 12 which are known to be critical for coactivator interaction, suggesting that they all constitute part of a receptor-coactivator interface. The transcriptional function of other mutants (A234T, R243W/Q, A268D, Delta276I, A279V, R282S) in this cluster correlated with their reduced Ka and they inhibited wild-type TRbeta action in a dominant negative manner. DNA binding, heterodimerization and corepressor recruitment were preserved in all mutants, signifying the importance of these attributes for dominant negative activity and correlating with the absence of natural mutations in regions bordering the third cluster which mediate these functions.

Resistance to thyroid hormone (RTH) is characterized by elevated serum thyroid hormones, failure to suppress pituitary TSH secretion, and variable T3 responsiveness in peripheral tissues. The disorder is associated with diverse mutations that cluster within three areas of the thyroid hormone beta(TR beta) receptor. Here, we report a novel RTH mutation (R383H), which is located in a region not known to harbor naturally occurring mutations. Although the R383H mutant receptor activated positively regulated genes to an extent comparable to wild-type (WT), negative transcriptional regulation of human TSH alpha and TRH promoters was impaired in either TR beta 1 or TR beta 2 contexts, and WT receptor function was dominantly inhibited. T3-dependent changes in basal transcription with R383H were also impaired: on the TRH promoter, basal activation by unliganded R383H was not reversed by T3 to the same extent as WT; similarly transcriptional silencing by an unliganded Gal4-R383H fusion was not relieved at a T3 concentration that derepressed WT. In keeping with this, ligand-dependent corepressor release by R383H, either in a protein-protein interaction assay or as a DNA-bound heterodimer with retinoid X receptor on either positive or negative thyroid hormone response elements, was disproportionately impaired relative to its ligand-binding affinity, whereas its T3-dependent recruitment of coactivator was unimpaired. These properties were shared by another previously described RTH mutant (R429Q), and in the crystal structure of TR alpha the homologous residues interact in a polar invagination. Our data indicate a role for these residues in mediating negative transcriptional regulation and facilitating corepressor release and suggest that predominant impairment of these functions may be the minimal requirements for causation of RTH.

The mechanism of action of thyroid hormones on bone is poorly understood. Thyroid hormones may act on bone cells either indirectly by increasing secretion of growth hormone (GH) and insulin-like growth factor-1 (IGF-1), or directly by influencing target genes via specific nuclear receptors. The presence of thyroid hormone receptors (TRs) has been demonstrated in human and rodent osteoblast-like cells and cell lines and recently in osteoclasts derived from an osteoclastoma in vitro. However, their presence in human bone in situ has not been reported. We have used specific polyclonal antibodies to TR-alpha 1, -alpha 2, and -beta 1 to investigate the expression of these receptors in sections of human osteophytes and heterotopic bone. Osteoblasts and osteoclasts were identified by alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), respectively, whereas chondrocytes were identified morphologically. At sites of endochondral and intramembranous bone formation, TR-beta 1 and the splice variant -alpha 2 were widely expressed by proliferating, mature, and hypertrophic chondrocytes and also in cells within the fibrous tissue and at the bone forming surfaces, respectively. They were also detected in osteoblasts, osteoclasts, and a few osteocytes at sites of bone remodeling. In contrast, TR-alpha 1 was the least expressed and was present mainly in osteoblasts at remodeling sites and in a few mature and undifferentiated chondrocytes. Our results show, for the first time, the presence and distribution of TRs in human bone in situ and suggest that the skeletal actions of thyroid hormones may be mediated via these receptors. Further studies are required to define the role of the individual receptor isoforms in bone metabolism.

A subset of nuclear receptors, including those for thyroid hormone (TR), retinoic acid, vitamin D3, and eicosanoids, can form heterodimers with the retinoid X receptor (RXR) on DNA regulatory elements in the absence of their cognate ligands. In a mammalian two-hybrid assay, we have found that recruitment of a VP16-RXR chimera by a Gal4-TRbeta ligand-binding domain fusion is enhanced up to 50-fold by thyroid hormone (T3). This was also observed with a mutant fusion, Gal4-TR(L454A), lacking ligand-inducible activation function (AF-2) and unable to interact with putative coactivators, suggesting that the AF-2 activity of TR or intermediary cofactors is not involved in this effect. The wild-type and mutant Gal4-TR fusions also exhibited hormone-dependent recruitment of RXR in yeast. Hormone-dependent recruitment of RXR was also evident with another Gal4-TR mutant, AHTm, which does not interact with the nuclear receptor corepressor N-CoR, suggesting that ligand-enhanced dimerization is not a result of T3-induced corepressor release. Finally, we have shown that the interaction between RXR and TR is augmented by T3 in vitro, arguing against altered expression of either partner in vivo mediating this effect. We propose that ligand-dependent heterodimerization of TR and RXR in solution may provide a further level of control in nuclear receptor signaling.

Nuclear hormone receptors are hormone-regulated transcription factors that play critical roles in chordate development and homeostasis. Aberrant nuclear hormone receptors have been implicated as causal agents in a number of endocrine and neoplastic diseases. The syndrome of Resistance to Thyroid Hormone (RTH) is a human genetic disease characterized by an impaired physiological response to thyroid hormone. RTH is associated with diverse mutations in the thyroid hormone receptor beta-gene. The resulting mutant receptors function as dominant negatives, interfering with the actions of normal thyroid hormone receptors coexpressed in the same cells. We report here that RTH receptors interact aberrantly with a newly recognized family of transcriptional corepressors variously denoted as nuclear receptor corepressor (N-CoR), retinoid X receptor interacting protein-13 (RIP-13), silencing mediator for retinoid and thyroid hormone receptors (SMRT), and thyroid hormone receptor-associating cofactor (TRAC). All RTH receptors tested exhibit an impaired ability to dissociate from corepressors in the presence of thyroid hormone. Two of the RTH mutations uncouple corepressor dissociation from hormone binding; two additional RTH mutants exhibit an unusually strong interaction with corepressor under all hormone conditions tested. Finally, artificial mutants that abolish corepressor binding abrogate the dominant negative activity of RTH mutants. We suggest that an altered corepressor interaction is likely to play a critical role in the dominant negative potency of RTH mutants and may contribute to the variable phenotype in this disorder.

The syndrome of resistance to thyroid hormone is characterized by elevated serum free thyroid hormones, failure to suppress pituitary thyrotropin secretion, and variable peripheral refractoriness to hormone action. Here we describe a novel leucine to valine mutation in codon 454 (L454V) of the thyroid hormone beta receptor (TR beta) in this disorder, resulting in a mutant receptor with unusual functional properties. Although the mutant protein binds ligand comparably to wild-type receptor and forms homo- and heterodimers on direct repeat, everted repeat, or palindromic thyroid response elements, its ability to activate transcription via these elements is markedly impaired. The hydrophobic leucine residue lies within an amphipathic alpha-helix at the carboxyl terminus of TR beta and the position of the homologous residue in the crystal structure of TR alpha indicates that its side chain is solvent-exposed and might interact with other proteins. We find that two putative transcriptional mediators (RIP140 and SRC-1) exhibit hormone-dependent association with wild-type TR. In comparison, the interaction of this natural mutant (L454V) and artificial mutants (L454A, E457A) with RIP140 and SRC-1 is markedly reduced. Furthermore, coexpression of SRC-1 is able to restore the transcriptional activity of the L454V mutant receptor, indicating that the interaction of this residue with accessory proteins is critical for transcriptional activation. Finally, the occurrence of the L454V mutation in resistance to thyroid hormone, together with impaired negative regulation of the thyroid-stimulating hormone alpha promoter by this mutant, suggests that the amphipathic alpha-helix also mediates hormone-dependent transcriptional inhibition, perhaps via interaction with these or other accessory factors.

Resistance to thyroid hormone (RTH) is usually dominantly inherited and is characterized by elevated free thyroid hormones in the serum and failure to suppress pituitary thyroid stimulating hormone (TSH) secretion with variable refractoriness to hormone action in peripheral tissues. Two major forms of the disorder are recognized: asymptomatic individuals with generalized resistance (GRTH) and patients with thyrotoxic features, suggesting predominant pituitary resistance (PRTH). Molecular genetic analyses indicate that both GRTH and PRTH are associated with diverse mutations in the thyroid hormone receptor beta gene, which localize to three regions in the hormone binding domain of the receptor. In addition to being functionally impaired, the mutant receptors are also able to inhibit their wild-type counterparts in a dominant negative manner. Recognized features of RTH include failure to thrive, growth retardation and attention-deficit hyperactivity disorder in childhood, and goitre and thyrotoxic cardiac symptoms in adults. The pathogenesis of variable tissue resistance is not fully understood but may be related to the differing tissue distributions of a and b thyroid hormone receptors and variable dominant negative activity of mutant receptors on different target genes.

Hormone-dependent transcriptional activation (AF-2) by the thyroid hormone beta receptor (TR beta) localizes to its carboxyl-terminal domain. A putative transactivation sequence within this domain was analyzed by mutating individual residues to alanine. Mutant receptor carboxyl-terminal domains were tested coupled to the heterologous DNA binding domain of Gal4. A single mutant receptor (E460A) showed normal hormone binding and activation, whereas several others (P453A, F455A, L456A, F459A) exhibited impaired transactivation which correlated with their reduced ligand binding. Two mutations (L454A, E457A) were able to dissociate these properties, generating transcriptionally defective mutant proteins with preserved hormone binding. A further conservative substitution (E457D) was also nonfunctional, and these three mutations were equally deleterious when tested in the context of full-length TR beta with a natural thyroid hormone response element containing promoter. This loss of activity was not due to altered DNA binding or expression of mutant receptors in cultured cells. They also retained the ability to recruit VP16-tagged retinoid X receptor in vivo as well as bind the basal transcription factors TFIIB and TBP in vitro. Our observations indicate that conserved hydrophobic (Leu454) and charged (Glu457) residues mediate AF-2 activity of TR beta, possibly via a co-activator that has yet to be identified.

Resistance to thyroid hormone (RTH) is usually dominantly inherited and characterized by elevated thyroid hormone levels, impaired feedback inhibition of pituitary TSH production, and variable hormonal responsiveness in peripheral tissues. We have identified 20 different mutations in the thyroid hormone beta-receptor (TR beta) gene in RTH and assayed mutant receptor properties using the TSH alpha subunit gene promoter or promoters containing three different types of positive thyroid response element (TRE). Dominant negative inhibition of wild type TR beta action by mutant receptors was also tested. The mutant receptors exhibited differing transcriptional inhibitory properties and dominant negative potential with the TSH alpha promoter that correlated with their impaired hormone binding, whereas transactivation and dominant negative effects with promoters containing positive TREs varied depending on their configuration. Heterodimeric mutant receptor-retinoid X receptor (RXR) interactions, either in cultured cells or as TRE-bound complexes in gel retardation assays, were uniformly preserved, whereas homodimeric receptor interactions could not be detected in vivo, and in vitro homodimer formation on TREs was variably reduced or absent for some mutant proteins. We correlate these findings with the distribution of receptor mutations that cluster in two areas within the hormone binding domain outside putative dimerization regions and show that artificial mutations that impaired heterodimerization abrogated dominant negative activity. Therefore, we suggest that the dominant negative effect of mutant receptors in the pituitary-thyroid axis generates the characteristic biochemical abnormality of RTH and that variable resistance in other tissues may be due to response element-dependent differences in their dominant negative potential.

Objective: We wished to ascertain whether a mutation in the thyroid hormone receptor beta gene was present in a family with generalized thyroid hormone resistance syndrome and to characterize the functional properties of this mutant receptor., Design: Blood samples were obtained from family members for hormone assays and genomic DNA was isolated from leucocytes for genetic analyses., Patients: Three members (B,C,E) of a family with possible thyroid hormone resistance and two normal family members (A,D) were studied., Measurements: Basal thyroid function tests together with serum sex hormone binding globulin (SHBG) levels were measured. The thyroid hormone receptor beta gene was amplified using the polymerase chain reaction and the receptor mutation identified by sequence analysis. The ability of mutant receptor to bind T3, interact with a specific DNA sequence and to modulate target gene expression was tested. The effects of mutant receptor on co-expressed wild type receptor action were determined., Results: Patients with resistance had raised levels of T4 and T3 together with inappropriately normal serum TSH and SHBG whereas unaffected individuals had a normal hormone profile. A single nucleotide substitution corresponding to a glycine to serine mutation at codon 340 (G340S) in the hormone binding domain was identified in one of the two beta receptor gene alleles in patients with resistance, but not in the normal family members. When expressed in vitro, this receptor protein (G340S), as well as a related (G340R) mutant identified in another family, retained the ability to bind to a specific DNA sequence but were unable to bind ligand or to activate or repress target gene expression. In addition both receptor mutants were capable of inhibiting the function of wild type thyroid hormone receptor in a co-expression assay but differed in their inhibitory potential., Conclusions: We report a second type of mutation (Gly to Ser) in codon 340 of hTR beta in a family with generalized thyroid hormone resistance. Mutations at this site eliminate T3 binding, causing a loss of hormone-stimulated receptor function. However, the mutant receptors retain the ability to block normal receptor action. The occurrence of different mutations at the same site suggests that alterations in this region of the receptor may be important for generating the clinical phenotype of this disorder.

That most major physiological actions of thyroid hormones could be mediated via hormonal regulation of gene expression has been known for more than 25 years. The localization of TR in the cell nucleus, first reported almost 20 years ago, confirmed this concept. But it is only since the cloning of the TR gene and its identification as the c-erbA oncogene, accomplished 6 years ago, that we have begun to understand the details of the interaction between the hormone and its receptor and between the receptor and its target gene. Perhaps the most significant concept to emerge from the molecular studies is that the TR belongs to the superfamily of nuclear receptors for steroid hormones and morphogens such as retinoids. It highlights the evolutionary conservation of a major network of cellular signalling and intracellular regulatory pathways and which has helped bring us closer to a unified concept of the action of many growth and developmental hormones. Several important questions to be solved in the future become obvious from this brief review of the role of thyroid hormone in regulating developmental processes. Among these is the explantation for the high degree of tissue specificity of hormonal regulation of gene expression. The discovery of differential expression of the two major TR genes and the generation of multiple isoforms of the receptor by alternate splicing go some way to answering this question, but this is clearly not the sole factor determining tissue specificity. It will be most important to find out more about the interaction between the receptor and other transcription factors or nuclear proteins, some of which may be tissue specific and others expressed ubiquitously. The autoinduction of TR during amphibian metamorphosis, described above, emphasizes the intriguing question of how receptor genes are regulated during development. We know little as yet about the promoters and regulatory factors involved in this process. Finally, we have also described the recent recognition of genetic defects in TRs that underlie thyroid hormone linked diseases in humans. Future studies on the molecular genetics of receptors will enhance the importance in clinical practice of receptor linked diseases.

We have overexpressed the human beta 1 thyroid hormone receptor in insect cells using a recombinant baculovirus to a level of 5-10% of total cellular protein. The recombinant protein migrates as a 50 kDa band by SDS-PAGE and Western blot analysis. The expressed receptor binds to L-T3 with a Kd of 1.3 +/- 0.4 x 10(-10) M and to thyroid hormone analogues with an affinity hierarchy of TRIAC greater than L-T3 greater than L-T4 greater than rT3. Gel retardation assays show highly specific receptor binding to a TRE which is modified by the presence of ligand and avidin-biotin complex DNA analysis shows a Kd of 6.2 +/- 2.0 x 10(-10) M for this interaction. These results indicate high level expression of hTR beta with authentic hormone and DNA binding properties.

Thyroid hormone (T3) resistance is inherited in most cases in an autosomal dominant manner. The disorder is characterized by elevated free thyroid hormone levels and partial resistance to thyroid hormone at the cellular level. Distinct single amino acid substitutions in the ligand binding domain of the beta form of the thyroid hormone receptor have been described in two kindreds with this disorder. We used transient expression assays to characterize the functional properties of these receptor mutants, one containing a Gly to Arg change at amino acid 340 (G340R) and the other a Pro to His change at amino acid 448 (P448H). A nine amino acid carboxy terminal deletion (delta 448-456), analogous to an alteration that occurs in v-erbA, was also studied for comparison with the mutations that occur in the T3 resistance syndrome. None of the receptor mutants were able to mediate thyroid hormone dependent activation (TreTKCAT) or repression (TSH alpha CAT) of reporter genes when compared with the wild type receptor. In addition, the mutants inhibited the activity of normal alpha and beta receptor isoforms when examined in coexpression assays. This activity, referred to as dominant negative inhibition, was manifest with respect to both the positively and negatively regulated reporter genes. Although mutant receptor binding to DNA was unaffected, ligand binding studies showed that the G340R and delta 448-456 mutants failed to bind T3, whereas the P448H mutant bound hormone with reduced affinity (approximately 10% of normal) compared to the wild type receptor. Consistent with this finding, the P448H mutant receptor was partially active at higher T3 concentrations. Furthermore, the dominant negative inhibition elicited by the P448H receptor mutant at higher T3 concentrations was reversed in the presence of high doses of T3. These findings indicate that mutant beta receptors in patients with thyroid hormone resistance have reduced affinity for T3 and are functionally deficient, but impair the activity of normal receptors, thereby providing a mechanism for the dominant mode of inheritance in this disorder.

Multiple forms of human thyroid hormone (T3) receptor have been identified, including true receptors that bind T3 (alpha 1 and beta) and a splicing variant (alpha 2) that does not bind T3. The alpha 1- and beta-receptors activate transcription through interactions with positive thyroid response elements (TREs). The alpha 2 variant is unable to activate transcription and has been reported to inhibit alpha 1 or beta stimulation of positive TREs, a property referred to as dominant negative activity. In this report we have performed studies to assess the functional properties of different members of the thyroid receptor family with regard to both positive and negative transcriptional regulation. The alpha 1-, alpha 2-, and beta-receptors were each coexpressed in JEG-3 cells with either TreTKCAT (CAT = chloramphenicol acetyltransferase), a reporter gene that contains a positive TRE, or TSH alpha CAT, a negatively regulated reporter gene. The alpha 1 and beta isoforms stimulated transcription of TreTKCAT and inhibited TSH alpha CAT transcription in a T3-dependent manner, whereas the alpha 2 variant was inactive. When coexpressed with alpha 1- or beta-receptors, alpha 2 inhibited regulation of positive TREs, but the effects of alpha 2 were modest and only occurred when relatively high doses of receptor were transfected. The alpha 2-receptor variant did not affect negative regulation by alpha 1- or beta-receptors. Thus, in both positive and negative regulation, thyroid hormone receptor isoforms that bind T3 (alpha 1, beta) are functional, whereas the alpha 2 isoform, which does not bind T3, is not functional.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroid-stimulating hormone alpha and beta subunit genes are negatively regulated by thyroid hormone at the transcriptional level. Transient gene expression studies were used to demonstrate that the erbA beta form of the thyroid hormone receptor mediates negative regulation of the alpha-subunit promoter in a hormone-dependent manner. In JEG-3 choriocarcinoma cells, which are deficient in thyroid hormone receptors, coexpression of erbA beta with alpha CAT reporter genes markedly suppressed alpha CAT expression after treatment with thyroid hormone, whereas a reporter gene containing a known positive thyroid response element was induced. Thus, a single form of thyroid hormone receptor mediates both positive and negative responses to thyroid hormone in this system. Transient expression analyses of alpha gene 5' flanking sequence deletion mutants localized the negative thyroid response element to the proximal region of the promoter between -100 and +4 base pairs. The location of the negative thyroid response element in the alpha gene is therefore distinct from that of previously identified regulatory elements including the tissue-specific upstream regulatory elements, the cAMP response elements, and the glucocorticoid response elements. Overlapping segments of the alpha promoter were examined for potential thyroid hormone receptor binding sites by using gel shift assays and biotinylated DNA-binding studies. A specific thyroid hormone receptor binding site was identified between -22 and -7 base pairs, which is immediately downstream from the TATA box. This region of the alpha promoter interacts with erbA beta receptor synthesized in vitro as well as with endogenous nuclear thyroid hormone receptors, and it competes for receptor binding to a known positive thyroid response element. These studies suggest a mechanism for negative regulation in which the thyroid hormone receptor interacts with the alpha gene promoter immediately downstream of the TATA box to inhibit transcription.