Functional regulation of thyroid hormone receptor variant TR alpha 2 by phosphorylation.

The thyroid hormone (T3) receptor (TR) variant TR alpha 2 is abundant in brain but does not bind T3 because of its unique C terminus. The only known function of TR alpha 2, inhibition of TR-dependent transactivation, involves competition for T3 response elements. Paradoxically, in vitro-translated TR alpha 2 bound poorly to these sites. We report here that dephosphorylation of TR alpha 2 restored its DNA binding. Mutation of C-terminal serine residues to alanine (TR alpha 2-SA) was equally effective. The C terminus of TR alpha 2 was phosphorylated in a human cell line, whereas that of TR alpha 2-SA was not. Conversely, TR alpha 2-SA was a much better inhibitor of T3 action than was wild-type TR alpha 2. The dominant negative activity of TR alpha 2-SA was less than stoichiometric with TR concentration, possibly because it was unable to heterodimerize with retinoid X receptor, which enhances the binding of other TRs. Purified casein kinase II as well as a reticulocyte casein kinase II-like activity phosphorylated TR alpha 2 on serines 474 and 475. Mutation of these two residues to alanine was sufficient to restore DNA binding. Thus, DNA binding by TR alpha 2 is regulated by phosphorylation at a site distant from the DNA-binding domain. The increased dominant negative activity of a nonphosphorylatable form of TR alpha 2 suggests that phosphorylation may provide a rapid, T3-independent mechanism for cell-specific modulation of the expression of T3-responsive genes.     

SRC-p300 coactivator complex is required for thyroid hormone-induced amphibian metamorphosis

Gene activation by the thyroid hormone (T3) receptor (TR) involves the recruitment of specific coactivator complexes to T3-responsive promoters. A large number of coactivators for TR have been isolated and characterized in vitro. However, their roles and functions in vivo during development have remained largely unknown. We have utilized metamorphosis in Xenopus laevis to study the role of these coactivators during post-embryonic development. Metamorphosis is totally dependent on the thyroid hormone, and TR mediates a vast majority, if not all, of the developmental effects of the hormone. We have previously shown that TR recruits the coactivator SRC3 (steroid receptor coactivator-3) and that coactivator recruitment is essential for metamorphosis. To determine whether SRCs are indeed required, we have analyzed the in vivo role of the histone acetyltransferase p300/CREB-binding protein (CBP), which was reported to be a component of the SRC.coactivator complexes. Chromatin immunoprecipitation revealed that p300 is recruited to T3-responsive promoters, implicating a role of p300 in TR function. Further, transgenic tadpoles overexpressing a dominant negative form of p300, F-dnp300, containing only the SRC-interacting domain, displayed arrested or delayed metamorphosis. Molecular analyses of the transgenic F-dnp300 animals showed that F-dnp300 was recruited by TR (displacing endogenous p300) and inhibited the expression of T3-responsive genes. Our results thus suggest that p300 and/or its related CBP is an essential component of the TR-signaling pathway in vivo and support the notion that p300/CBP and SRC proteins are part of the same coactivator complex in vivo during post-embryonic development.     

Thyroid hormone receptor/c-erbA: control of commitment and differentiation in the neuronal/chromaffin progenitor line PC12

The c-erbA proto-oncogenes encode nuclear receptors for thyroid hormone (T3), a hormone intimately involved in mammalian brain maturation. To study thyroid hormone receptor (TR) action on neuronal cells in vitro, we expressed the chicken c-erbA/TR alpha-1 as well as its oncogenic variant v-erbA in the adrenal medulla progenitor cell line PC12. In the absence of T3, exogenous TR alpha-1 inhibits NGF-induced neuronal differentiation and represses neuron-specific gene expression. In contrast, TR alpha-1 allows normal differentiation and neuronal gene expression to occur in the presence of T3. Finally, TR alpha-1- expressing cells become NGF-responsive for proliferation when T3 is absent, but NGF-dependent for survival in presence of T3. A similar differentiation induction by NGF plus T3 was observed in a central nervous system-derived neuronal cell line (E 18) expressing exogenous TR alpha-1. Together with the finding that TR alpha-1 constitutively blocked dexamethasone-induced differentiation of PC12 cells into the chromaffin pathway, these results suggest that TR alpha-1 plays an important role in regulating commitment and maturation of neuronal progenitors. In contrast, the v-erbA oncogene, a mutated, oncogenic version of TR alpha-1, partially but constitutively inhibited NGF- induced neuronal differentiation of PC12 cells and potentiated dexamethasone-induced chromaffin differentiation, giving rise to an aberrant "interlineage" cell phenotype.     

Thyroid hormone nuclear receptors and their role in the metabolic action of the hormone

Thyroid hormone nuclear receptor molecules have been characterized as proteins of approximately 49,000 molecular weight existing in cells attached to chromatin and with 4000-8000 copies per nucleus. They bind T3 with Ka of 0.2 X 10(10) l/mol and show microheterogeneity on isoelectric focusing. Hormone responsiveness varies with receptor content in the nucleus and occupancy of receptor by T3. Recent investigations have shown that the receptors are part of the v-erbA related super family of nuclear hormone receptors. At least two types of T3 receptors (TR) exist, one coded by a gene on chromosome 3 (TR beta) and a second coded on chromosome 17 (hTR alpha). Receptors are low in the fetus and, in the adult, are dramatically reduced by starvation, illness and glucagon. Receptors function through binding of T3 or other hormone analogs to a domain in the carboxyl portion of the protein, and binding of the receptor-T3 complex through 'DNA-fingers' to specific response elements as enhancers and located in the 5'-flanking DNA of thyroid hormone responsive genes. Extensive studies on regulation of rat growth hormone have suggested binding of receptor or associated factors to several positions in the 5'-flanking DNA, and recent studies suggest that a crucial area may be a 15 bp segment between bases -179 and -164. Abnormal receptors are believed to be responsible for the syndrome of generalized resistance to thyroid hormone action, but it is yet unclear as to which form (or forms) of the receptor is abnormal in this syndrome.     

V-erba homodimers mediate the potent dominant negative activity of v-erba on everted repeats

The oncoprotein v-erbA is a mutated form of TRalpha1 that is unable to bind thyroid hormone (T3). V-erbA homodimerizes or heterodimerizes with retinoid X receptor (RXR) on core motifs arranged as direct, everted, or inverted repeats (DRs, ERs, or IRs). We created a series of v-erbA mutants in order to obtain a better understanding of the role of v-erbA homodimers versus v-erbA-RXR heterodimers in the dominant negative activity of v-erbA on ERs (the most potent v-erbA response elements). We found that one of these mutants, v-erbA mutant E325A, is able to homodimerize but unable to heterodimerize with RXR on ERs. Our data also suggest that v-erbA homodimers interact preferentially with the corepressor NCoR over SMRT and that the interaction with corepressors is stronger with v-erbA homodimers over v-erbA-RXR heterodimers. Furthermore, functional studies showed that v-erbA homodimers rather than v-erbA-RXR heterodimers mediate the dominant negative activity of v-erbA on ERs.     

Thyroid hormone responsive genes in the murine hepatocyte cell line AML 12

Thyroid hormone (T3) plays an important role in gene regulation in the liver. Previous studies have been done in complex systems such as animal models, or in transformed malignant hepatic cell lines in which thyroid hormone receptor (TR) was over-expressed by co-transfection. Therefore, the aim of this study was to characterize T3-responsive genes in a simple system, by using a non-transformed hepatic cell line that is able to express sufficient amounts of endogenous TRs. For this purpose we used the murine non-transformed hepatocyte cell line AML 12. We performed analyses using a cDNA microarray containing 15,000 murine genes. We found 12 genes to be up-regulated and 5 genes to be down-regulated in the presence of T3. For some of the genes not previously known to be regulated by T3, we confirmed the regulation by T3 using real-time PCR. Our data in AML 12 cells provide a simple and physiologically relevant system to study T3 action, without the influence of neoplastic transformation or artificial TR over-expression. Furthermore, our data describe novel T3 responsive genes and provide insight into the role of T3 in important processes such as cholesterol metabolism, bile acid secretion, oncogenesis, among others, that can be tested in future experiments in vivo.     

Differential expression of thyroid hormone receptor isoforms dictates the dominant negative activity of mutant Beta receptor

Mutations in the thyroid hormone receptor beta gene (TRbeta) cause resistance to thyroid hormone (RTH). Genetic analyses indicate that phenotypic manifestation of RTH is due to the dominant negative action of mutant TRbeta. However, the molecular mechanisms underlying the dominant negative action of mutants and how the same mutation results in marked variability of resistance in different tissues in vivo are not clear. Here we used a knock-in mouse (TRbetaPV mouse) that faithfully reproduces human RTH to address these questions. We demonstrated directly that TRbeta1 protein was approximately 3-fold higher than TRalpha1 in the liver of TRbeta(+/+) mice but was not detectable in the heart of wild-type and TRbetaPV mice. The abundance of PV in the liver of TRbeta(PV/PV) was more than TRbeta(PV/+) mice but not detectable in the heart. TRalpha1 in the liver was approximately 6-fold higher than that in the heart of wild-type and TRbetaPV mice. Using TR isoforms and PV-specific antibodies in gel shift assays, we found that in vivo, PV competed not only with TR isoforms for binding to thyroid hormone response elements (TRE) but also competed with TR for the retinoid X receptors in binding to TRE. These competitions led to the inhibition of the thyroid hormone (T(3))-positive regulated genes in the liver. In the heart, however, PV was significantly lower and thus could not effectively compete with TRalpha1 for binding to TRE, resulting in activation of the T(3)-target genes by higher levels of circulating thyroid hormones. These results indicate that in vivo, differential expression of TR isoforms in tissues dictates the dominant negative activity of mutant beta receptor, thereby resulting in variable phenotypic expression in RTH.     

Compensatory role of thyroid hormone receptor (TR) alpha 1 in resistance to thyroid hormone: study in mice with a targeted mutation in the TR beta gene and deficient in TR alpha 1

Resistance to thyroid hormone (RTH) is caused by mutations of the thyroid hormone receptor beta (TR beta) gene. Almost all RTH patients are heterozygous with an autosomal dominant pattern of inheritance. That most are clinically euthyroid suggests a compensatory role of the TR alpha1 isoform in maintaining the normal functions of thyroid hormone (T3) in these patients. To understand the role of TR alpha1 in the manifestation of RTH, we compared the phenotypes of mice with a targeted dominantly negative mutant TR beta (TR betaPV) with or without TR alpha1. TR betaPV mice faithfully recapitulate RTH in humans in that these mice demonstrate abnormalities in the pituitary-thyroid axis and impairment in growth. Here we show that the dysregulation of the pituitary-thyroid axis was worsened by the lack of TR alpha1 in TR betaPV mice, and severe impairment of postnatal growth was manifested in TR betaPV mice deficient in TR alpha1. Furthermore, abnormal expression patterns of T3-target genes in TR betaPV mice were altered by the lack of TR alpha1. These results demonstrate that the lack of TR alpha1 exacerbates the manifestation of RTH in TR betaPV mice. Therefore, TR alpha1 could play a compensatory role in mediating the functions of T3 in heterozygous patients with RTH. This compensatory role may be especially crucial for postnatal growth.