Dominant inhibition of thyroid hormone action selectively in the pituitary of thyroid hormone receptor-beta null mice abolishes the regulation of thyrotropin by thyroid hormone

Thyroid hormones, T4 and T3, regulate their own production by feedback inhibition of TSH and TRH synthesis in the pituitary and hypothalamus when T3 binds to thyroid hormone receptors (TRs) that interact with the promoters of the genes for the TSH subunit and TRH. All TR isoforms are believed to be involved in the regulation of this endocrine axis, as evidenced by the massive dysregulation of TSH production in mice lacking all TR isoforms. However, the relative contributions of TR isoforms in the pituitary vs. the hypothalamus remain to be completely elucidated. Thus, to determine the relative contribution of pituitary expression of TR-alpha in the regulation of the hypothalamic-pituitary-thyroid axis, we selectively impaired TR-alpha function in TR-beta null mice (TR-beta-/-) by pituitary restricted expression of a dominant negative TR-beta transgene harboring a delta337T mutation. These animals exhibited 10-fold and 32-fold increase in T4 and TSH concentrations, respectively. Moreover, the negative regulation of TSH by exogenous T3 was completely absent and a paradoxical increase in TSH concentrations and TSH-beta mRNA was observed. In contrast, prepro-TRH expression levels in T3-treated TR-beta-/- were similar to levels observed in the delta337/TR-beta-/- mice, and ligand-independent activation of TSH in hypothyroid mice was equivalently impaired. Thus, isolated TR-beta deficiency in TRH paraventricular hypothalamic nucleus neurons and impaired function of all TRs in the pituitary recapitulate the baseline hormonal disturbances that characterize mice with complete absence of all TRs.     

Effects of thyroid hormone receptor gene disruption on myosin isoform expression in mouse skeletal muscles

Skeletal muscle is known to be a target for the active metabolite of thyroid hormone, i.e., 3,5,3'-triiodothyronine (T(3)). T(3) acts by repressing or activating genes coding for different myosin heavy chain (MHC) isoforms via T(3) receptors (TRs). The diverse function of T(3) is presumed to be mediated by TR-alpha(1) and TR-beta, but the function of specific TRs in regulating MHC isoform expression has remained undefined. In this study, TR-deficient mice were used to expand our knowledge of the mechanisms by which T(3) regulates the expression of specific MHC isoforms via distinct TRs. In fast-twitch extensor digitorum longus (EDL) muscle, TR-alpha(1)-, TR-beta-, or TR-alpha(1)beta-deficient mice showed a small but statistically significant decrease (P < 0.05) of type IIB MHC content and an increased number of type I fibers. In the slow-twitch soleus, the beta/slow MHC (type I) isoform was significantly (P < 0. 001) upregulated in the TR-deficient mice, but this effect was highly dependent on the type of receptor deleted. The lack of TR-beta had no significant effect on the expression of MHC isoforms. An increase (P < 0.05) of type I MHC was observed in the TR-alpha(1)-deficient muscle. A dramatic overexpression (P < 0.001) of the slow type I MHC and a corresponding downregulation of the fast type IIA MHC (P < 0.001) was observed in TR-alpha(1)beta-deficient mice. The muscle- and fiber-specific differences in MHC isoform expression in the TR-alpha(1)beta-deficient mice resembled the MHC isoform transitions reported in hypothyroid animals, i.e., a mild MHC transition in the EDL, a dramatic but not complete upregulation of the beta/slow MHC isoform in the soleus, and a variable response to TR deficiency in different soleus muscle fibers. Thus the consequences on muscle are similar in the absence of thyroid hormone or absence of thyroid hormone receptors, indicating that TR-alpha(1) and TR-beta together mediate the known actions of T(3). However, it remains unknown how thyroid hormone exerts muscle- and muscle fiber-specific effects in its action. Finally, although developmental MHC transitions were not studied specifically in this study, the absence of embryonic and fetal MHC isoforms in the TR-deficient mice indicates that ultimately the transition to the adult MHC isoforms is not solely mediated by TRs.     

Beta 1 isoform-specific regulation of a triiodothyronine-induced gene during cerebellar development.

Although tissue-specific expression of the alpha 1 and beta 1 thyroid hormone receptors (TR-alpha 1 and TR-beta 1) suggests isoform-specific function, transfection studies to date have failed to show consistent differences in their ability to regulate gene expression. We here provide evidence that TR-beta 1 but not TR-alpha 1 regulates the expression of the gene coding for PCP-2 in cerebellar Purkinje cells during neonatal rat development and that such regulation appears to be both T3 dependent and T3 independent. Examination of neonatal rats revealed that the levels of three mRNAs expressed in cerebellar Purkinje cells (myoinositol-1,4,5-triphosphate receptor, calbindin, and PCP-2) rise from neonatal day 1 to day 15. This rise is preceded by the previously documented surge in brain T3 and TR-beta 1. Methimazole-induced hypothyroidism sharply reduces, but does not abolish, the rise in these mRNAs. Concomitant T3 administration normalizes the process. In order to establish more directly the role of TR-beta 1 and T3, cotransfection experiments were performed in CHO cells with PCP-2-lacZ construct and TR isoforms. These studies showed that TR-beta 1, even in the absence of T3, regulated the expression of the transfected PCP-2 construct. T3 augments the response to TR-beta 1 alone by 40% (P < .01). TR-alpha 1 had no effect on PCP-2-lacZ expression either in the presence or absence of T3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitation of rat tissue thyroid hormone binding receptor isoforms by immunoprecipitation of nuclear triiodothyronine binding capacity.

A panel of anti-thyroid hormone receptor (TR) antisera were generated to allow direct assay of the concentrations of the alpha 1 and beta 1 receptor isoforms in nuclear extracts from adult rat liver, kidney, brain and heart, and fetal brain. An antiserum, immunoglobulin G (IgG)-beta 1, raised against amino acid sequence 62-92 of the rat TR-beta 1 specifically precipitated only TR-beta 1 in vitro translation products. A second antiserum, IgG-alpha 1/beta, generated against a sequence that is identical in the ligand binding region of rat TR-alpha 1 and TR-beta isoforms immunoprecipitated both TR-alpha 1 and -beta 1 translation products. These IgG preparations were used to specifically immunoprecipitate thyroid hormone receptor binding activity from nuclear extracts. IgG-beta 1 cleared almost 80%, and the IgG-alpha 1/beta immunoprecipitated nearly all binding from hepatic nuclear extracts. This distribution of TR protein, 80% beta 1 and 20% alpha 1, is the same as previously reported for their respective mRNAs in liver. In heart, kidney, and brain IgG-beta 1 cleared 45, 43, and 28% of total binding, respectively, and IgG-alpha 1/beta cleared all T3 binding activity from these tissues. In agreement with an earlier study, marked variations in specific protein/mRNA ratios were noted among these tissues. Consistent with our earlier report of the presence of only very low levels of TR-beta 1 mRNA in fetal brain, IgG-beta 1 cleared just 5% of binding in this tissue. Studies using an antiserum (IgG-ch) generated against homologous segments of the hinge region in both TR-alpha 1 and -beta 1 yielded results which contrasted sharply with those of IgG-alpha 1/beta. Whereas IgG-ch could also immunoprecipitate virtually all binding from hepatic extracts it cleared only 40-50% of binding from the other tissues, including fetal brain in which TR-alpha 1 accounts for greater than 90% of binding protein. The data suggest the presence of posttranslational modification of the TR-alpha 1 protein in the hinge region, consistent with the presence in this segment of potential phosphorylation sites.     

ERβ Binds N-CoR in the Presence of Estrogens via an LXXLL-like Motif in the N-CoR C-terminus

Nuclear receptors (NRs) usually bind the corepressors N-CoR and SMRT in the absence of ligand or in the presence of antagonists. Agonist binding leads to corepressor release and recruitment of coactivators. Here, we report that estrogen receptor beta (ERbeta) binds N-CoR and SMRT in the presence of agonists, but not antagonists, in vitro and in vivo. This ligand preference differs from that of ERalpha interactions with corepressors, which are inhibited by estradiol, and resembles that of ERbeta interactions with coactivators. ERbeta /N-CoR interactions involve ERbeta AF-2, which also mediates coactivator recognition. Moreover, ERbeta recognizes a sequence (PLTIRML) in the N-CoR C-terminus that resembles coactivator LXXLL motifs. Inhibition of histone deacetylase activity specifically potentiates ERbeta LBD activity, suggesting that corepressors restrict the activity of AF-2. We conclude that the ER isoforms show completely distinct modes of interaction with a physiologically important corepressor and discuss our results in terms of ER isoform specificity in vivo.     

Modulation of testicular receptor 4 activity by mitogen-activated protein kinase-mediated phosphorylation

Testicular receptor 4 (TR4) is an orphan member of the nuclear receptor superfamily. Despite the lack of identified ligands, its functional role has been demonstrated both in animals and cell cultures. However, it remains unclear how the biological activity of TR4 is regulated without specific ligands. In this study, we showed that in the absence of specific ligands the activity of TR4 could be modulated by mitogen-activated protein kinase (MAPK)-mediated phosphorylation of its activation function 1 (AF-1) domain. A mass spectrometry-based proteome analysis of TR4 expressed in insect cells revealed three phosphorylation sites in its AF-1 domain, specifically on Ser(19), Ser(55), and Ser(68). Site-directed mutagenesis studies demonstrated the functionality of phosphorylation on Ser(19) and Ser(68) but not Ser(55). We also demonstrated that MAPK-mediated phosphorylation of the AF-1 domain rendered TR4 a repressor, mediated through the preferential recruitment of corepressor RIP140. Dephosphorylation of its AF-1 made TR4 an activator due to its selective recruitment of coactivator, P300/cyclic AMP-responsive element binding protein-binding protein-associated factor (PCAF). The biological effects were validated by using the wild type TR4 and its constitutive negative (dephosphorylated) and constitutive positive (phosphorylated) mutants in the studies of regulation of its natural target gene, apoE. This study uncovered, for the first time, a ligand-independent mechanism underlying the biological activity of TR4 that was mediated by MAPK-mediated receptor phosphorylation of AF-1 domain.     

Sick euthyroid syndrome is associated with decreased TR expression and DNA binding in mouse liver

Infection is associated with low serum thyroid hormones and thyrotropin levels. Here we demonstrate that infection also reduces thyroid hormone receptor (TR) expression. In gel shift experiments, retinoid X receptor (RXR)/TR DNA binding was reduced in mouse liver by 60 and 77%, respectively, 4 and 16 h after lipopolysaccharide (LPS) administration. Surprisingly, LPS did not decrease either TR-alpha or TR-beta protein levels at 4 h, but by 16 h TR-alpha(1), TR-alpha(2), and TR-beta levels were reduced by 55, 87, and 41%, respectively. We previously reported that LPS rapidly decreases RXR protein levels in liver. Therefore, we added RXR-beta to hepatic nuclear extracts prepared 4 h after LPS treatment, which restored RXR/TR DNA binding to a level comparable to that of controls. A similar experiment conducted on extracts prepared 16 h after LPS administration did not restore RXR/TR DNA binding. We propose that decreased RXR expression is limiting for RXR/TR DNA binding at 4 h, whereas the reduction in both TR and RXR levels results in further decreased binding at 16 h.     

Novel mode of deoxyribonucleic acid recognition by thyroid hormone receptors: thyroid hormone receptor beta-isoforms can bind as trimers to natural response elements comprised of reiterated half-sites

Thyroid hormone receptors (TRs) regulate gene expression by binding to specific DNA sequences, denoted thyroid hormone response elements (TREs). The accepted paradigm for TRs proposes that they bind as homo- or heterodimers to TREs comprised of two AGGTCA half-site sequences. In the prototypic TRE, these half-sites are arranged as direct repeats separated by a four-base spacer. This dimeric model of TR binding, derived from analysis of artificial DNA sequences, fails to explain why many natural TREs contain more than two half-sites. Therefore, we investigated the ability of different TR isoforms to bind to TREs possessing three or more half-sites. We report that the TRbeta isoforms (TRbeta0, TRbeta1, TRbeta2), but not TRalpha1, can bind to reiterated DNA elements, such as the rat GH-TRE, as complexes trimeric or greater in size. The TRbeta0 isoform, in particular, formed homo- and heterotrimers (with the retinoid X receptor) with high efficiency and cooperativity, and TRbeta0 preferentially used reporters containing these reiterated elements to drive gene expression in vivo. Our data demonstrate that TRbeta isoforms can form multimeric receptor complexes on appropriately reiterated DNA response elements, providing a functional distinction between the TR isoforms and an explanation for TREs possessing three or more half-sites.