As the genome of experimental animals has become easier to manipulate, a number of mouse models have been developed to understand in vivo thyroid hormone action. A major site of thyroid hormone action is the HPT axis. While several methods are available that provide a detailed understanding of the HPT axis in mice, many authors choose to include only cursory data about this axis, which can lead to erroneous conclusions about in vivo thyroid hormone action. A standard protocol is proposed to evaluate the HPT axis in mice.

AimsTo understand how thyroid hormone (TH) regulates tissue-specific gene expression in patients with the syndrome of resistance to TH (RTHβ), we used a mouse model that replicates the human RTHβ, specifically the ∆337T mutation in the thyroid hormone receptor β (THRβ).Main methodsWe investigated the expression of key TH target genes in the pituitary and liver of TRβ∆337T and wild type THRβ mice by qPCR before and after a T3 suppression test consisting of the administration of increasing concentrations of T3 to hypothyroid mice.Key findingsPituitary Tshb and Cga expression decreased and Gh expression increased in TRβ∆337T mice after T3 suppression. The stimulation of positively regulated TH genes was heterogeneous in the liver. Levels of liver Me1 and Thsrp were elevated in TRβ∆337T mice after T3 administration. Slc16a2 and Gpd2 did not respond to T3 stimulation in the liver of TRβ∆337T mice whereas Dio1 response was lower than that observed in WT mice. Moreover, although Chdh and Upd1 genes were negatively regulated in the liver, the expression of these genes was elevated after T3 suppression. We did not observe significant changes in THRα expression in the liver and pituitary, while THRβ levels were diminished in the pituitary and increased in the liver.SignificanceUsing a model expressing a THRβ unable to bind T3, we showed the expression pattern of liver negative and positive regulated genes by T3.

The gene encoding human preproTRH was isolated from a human lung fibroblast genomic DNA library with a rat preproTRH cDNA fragment. The transcriptional unit is 3.3 kilobases in size and contains three exons interrupted by two introns of approximately 1050 and 650 base pairs, respectively. Exon 1 encodes the 5′untranslated region of the mRNA, exon 2 the putative signal sequence and the initial portion of propeptide, and exon 3 encodes the remainder of the propeptide, which contains six copies of the TRH sequence in contrast to five copies in the rat preproTRH gene. The predicted human preproTRH peptide structure has 242 amino acids compared to 255 amino acids in the rat. Homology with rat preproTRH is 73.3% and 59.5% at the nucleic acid and amino acid levels, respectively. Intron-exon splicing sites and 5′ and 3′ mRNA borders were confirmed rigorously by sequencing a human preproTRH cDNA using the polymerase chain reaction and human hypothalamic cDNA.

Mice bearing the genomic mutation Δ337T on the thyroid hormone receptor β (TRβ) gene present the classical signs of resistance to thyroid hormone (TH), with high serum TH and TSH. This mutant TR is unable to bind TH, remains constitutively bound to co-repressors, and has a dominant negative effect on normal TRs. In this study, we show that homozygous (TRβΔ337T) mice for this mutation have reduced body weight, length, and body fat content, despite augmented relative food intake and relative increase in serum leptin. TRβΔ337T mice exhibited normal glycemia and were more tolerant to an i.p. glucose load accompanied by reduced insulin secretion. Higher insulin sensitivity was observed after single insulin injection, when the TRβΔ337T mice developed a profound hypoglycemia. Impaired hepatic glucose production was confirmed by the reduction in glucose generation after pyruvate administration. In addition, hepatic glycogen content was lower in homozygous TRβΔ337T mice than in wild type. Collectively, the data suggest that TRβΔ337T mice have deficient hepatic glucose production, by reduced gluconeogenesis and lower glycogen deposits. Analysis of liver gluconeogenic gene expression showed a reduction in the mRNA of phosphoenolpyruvate carboxykinase, a rate-limiting enzyme, and of peroxisome proliferator-activated receptor-γ coactivator 1α, a key transcriptional factor essential to gluconeogenesis. Reduction in both gene expressions is consistent with resistance to TH action via TRβ, reproducing a hypothyroid phenotype. In conclusion, mice carrying the Δ337T-dominant negative mutation on the TRβ are leaner, exhibit impaired hepatic glucose production, and are more sensitive to hypoglycemic effects of insulin.

Cone photoreceptors in the murine retina are patterned by dorsal repression and ventral activation of S opsin. TR beta 2, the nuclear thyroid hormone receptor beta isoform 2, regulates dorsal repression. To determine the molecular mechanism by which TR beta 2 acts, we compared the spatiotemporal expression of TR beta 2 and S opsin from embryonic day (E) 13 through adulthood in C57BL/6 retinae. TR beta 2 and S opsin are expressed in cone photoreceptors only. Both are transcribed by E13, and their levels increase with cone genesis. TR beta 2 is expressed uniformly, but transiently, across the retina. mRNA levels are maximal by E17 at completion of cone genesis and again minimal before P5. S opsin is also transcribed by E13, but only in ventral cones. Repression in dorsal cones is established by E17, consistent with the occurrence of patterning during cone cell genesis. The uniform expression of TR beta 2 suggests that repression of S opsin requires other dorsal-specific factors in addition to TR beta 2. The mechanism by which TR beta 2 functions was probed in transgenic animals with TR beta 2 ablated, TR beta 2 that is DNA binding defective, and TR beta 2 that is ligand binding defective. These studies show that TR beta 2 is necessary for dorsal repression, but not ventral activation of S opsin. TR beta 2 must bind DNA and the ligand T3 (thyroid hormone) to repress S opsin. Once repression is established, T3 no longer regulates dorsal S opsin repression in adult animals. The transient, embryonic action of TR beta 2 is consistent with a role (direct and/or indirect) in chromatin remodeling that leads to permanent gene silencing in terminally differentiated, dorsal cone photoreceptors.

Context: Combined pituitary hormone deficiency (CPHD) in humans is caused by mutations of pituitary-specific transcription factors such as Pit-1. Although many patients with CPHD have an autosomal recessive disorder caused by a Pit-1 DNA- binding mutation, there are a number of reports of mutant Pit-1 molecules that either by prediction or through experimentation bind normally to DNA.Objective: The objective of this study was to understand the pathophysiological mechanisms of mutant Pit-1 molecules with intact DNA binding.Design: DNA- binding and functional studies were used to assess five Pit-1 mutations: F135C, R143Q, A158P, K216E, and R271W.Results: In gel-shift studies using well-characterized DNA- binding elements from the GH and prolactin genes, the K126E mutant displayed markedly enhanced Pit-1 dimer binding to either element, whereas the R271W mutant bound with high avidity, but only as a monomer. In contrast, the R143Q mutant was unable to bind these elements, and the F135C and A158P mutants displayed near-normal DNA-binding characteristics. We observed that CBP/p300 bound poorly to the A158P and K216E mutant Pit-1 molecules, but bound normally to the F135C, R143Q, and R271W mutants. In functional assays, CBP/p300 cotransfection with mutant Pit-1 expression vectors resulted in less transactivation of either the GH or prolactin reporter genes.Conclusions: From these studies, we suggest that CBP/p300 recruitment and Pit-1 dimerization are necessary for Pit-1 target gene activation and are important in the pathogenesis of CPHD.

Hypercholesterolemia is found in patients with hypothyroidism and resistance to thyroid hormone. In this study, we examined cholesterol metabolism in a thyroid hormone receptor beta ( TR-beta) mutant mouse model of resistance to thyroid hormone. Whereas studies of cholesterol metabolism have been reported in TR-beta knock-out mice, generalized expression of a non-ligand binding TR-beta protein in this knock-in model more fully recapitulates the hypothyroid state, because the hypothyroid effect of TRs is mediated by the unliganded receptor. In the hypothyroid state, a high cholesterol diet increased serum cholesterol levels in wild-type animals (WT) but either did not change or reduced levels in mutant (MUT) mice relative to hypothyroidism alone. 7 alpha-Hydroxylase (CYP7A1) is the rate-limiting enzyme in cholesterol metabolism and mRNA levels were undetectable in the hypothyroid state in all animals. triiodothyronine replacement restored CYP7A1 mRNA levels in WT mice but had minimal effect in MUT mice. In contrast, a high cholesterol diet markedly induced CYP7A1 levels in MUT but not WT mice in the hypothyroid state. Elevation of CYP7A1 mRNA levels and reduced hepatic cholesterol content in MUT animals are likely because of cross-talk between TR-beta and liver X receptor alpha(LXR-alpha), which both bind to a direct repeat +4 ( DR+4) element in the CYP7A1 promoter. In transfection studies, WT but not MUT TR-beta antagonized induction of this promoter by LXR-alpha. Electromobility shift analysis revealed that LXR/RXR heterodimers bound to the DR+4 element in the presence of MUT but not WT TR-beta. A mechanism for cross-talk, and potential antagonism, between TR-beta and LXR-alpha is proposed.

P-Lim (Lhx3a) is a LIM homeodomain transcription factor essential for pituitary development and motor neuron specification in mice. The Lhx3 gene encodes two isoforms, which differ in their amino (N) termini, Lhx3a and 3b. The P-Lim DNA binding site on the glycoprotein hormone alpha subunit (alpha-GSU) gene promoter is conserved in mammals. P-Lim plays a pivotal role in mediating thyrotropin-releasing hormone (TRH) signaling by binding CREB binding protein (CBP), as we have reported previously. Here, we demonstrate that P-Lim (Lhx3a) but not Lhx3b can mediate TRH signaling and bind to CBP. Moreover, TRH specifically induces P-Lim-CBP binding through the N-termini of P-Lim. We also found that the protein kinase C (PKC) phosphorylation site within the N-terminus of P-Lim is responsible for the P-Lim-CBP binding suggesting that the TRH signaling pathway phosphorylates P-Lim. These studies have elucidated the molecular mechanism by which TRH stimulates alpha-GSU gene expression.

Thyroid hormone (TH) action is mediated by TH receptors (TRs), which are members of the nuclear hormone receptor superfamily. In vitro studies have demonstrated that TR activity is regulated by interactions with corepressor and coactivator proteins (CoRs and CoAs, respectively). TH stimulation is thought to involve dissociation of CoRs and recruitment of CoAs to the liganded TR. In contrast, negative regulation by TH is thought to occur via recruitment of CoRs to the liganded TR. The physiological role of CoAs bound to TRs, however, has yet to be defined. In this study, we used gene-targeting techniques to mutate the TR-beta locus within its activation function-2 (AF-2) domain (E457A). This mutation was chosen because it completely abolished CoA recruitment in vitro, while preserving normal triiodothyronine (T3) binding and CoR interactions. As expected, TH-stimulated gene expression was reduced in homozygous E457A mice. However, these animals also displayed abnormal regulation of the hypothalamic-pituitary-thyroid axis. Serum thyroxine, T3, and thyroid-stimulating hormone (TSH) levels and pituitary Tshb mRNA levels were inappropriately elevated compared with those of WT animals, and L-T3 treatment failed to suppress serum TSH and pituitary Tshb mRNA levels. Therefore, the AF-2 domain of TR-beta is required for positive and, paradoxically, for negative regulation by TH in vivo.

Hypothalamic thyrotropin-releasing hormone (TRH) stimulates thyroid-stimulating hormone (TSH) secretion from the anterior pituitary. TSH then initiates thyroid hormone (TH) synthesis and release from the thyroid gland. Although opposing TRH and TH inputs regulate the hypothalamic-pituitary-thyroid axis, TH negative feedback is thought to be the primary regulator. This hypothesis, however, has yet to be proven in vivo. To elucidate the relative importance of TRH and TH in regulating the hypothalamic-pituitary-thyroid axis, we have generated mice that lack either TRH, the beta isoforms of TH receptors ( TR beta KO), or both ( double KO). TR beta knock-out ( KO) mice have significantly higher TH and TSH levels compared with wild-type mice, in contrast to double KO mice, which have reduced TH and TSH levels. Unexpectedly, hypothyroid double KO mice also failed to mount a significant rise in serum TSH levels, and pituitary TSH immunostaining was markedly reduced compared with all other hypothyroid mouse genotypes. This impaired TSH response, however, was not due to a reduced number of pituitary thyrotrophs because thyrotroph cell number, as assessed by counting TSH immunopositive cells, was restored after chronic TRH treatment. Thus, TRH is absolutely required for both TSH and TH synthesis but is not necessary for thyrotroph cell development.

Peroxisome proliferator-activated receptor gamma (PPARgamma) interacts with retinoid X receptor (RXR) on PPAR response elements (PPREs) to regulate transcription of PPAR-responsive genes. To investigate the binding of PPARgamma and RXR to PPREs, three mutations were constructed in the DNA-binding domains of PPARgamma; two of the mutants maintained the structure of zinc finger I (PPARgamma-GS and PPARgamma-AA), and a third mutation disrupted the protein structure of zinc finger I (PPARgamma-CS). Results indicated that the mutations of PPARgamma that maintained intact zinc fingers were capable of binding to a variety of PPREs in the presence of RXR and could activate transcription on several PPREs. In parallel, a mutation was created in the DNA-binding domain of RXRalpha that maintained the structure of the zinc fingers (RXR-GS) but did not bind DNA and was transcriptionally inactive. Examination of the 3' half-site of several PPREs revealed that variations from the consensus sequence reduced or abolished transcriptional activity, but conversion to consensus improved transcriptional activity with PPARgamma-GS and PPARgamma-AA. Examination of the 5' half-site indicated that the upstream three nucleotides were more important for transcriptional activity than the downstream three nucleotides. Our data demonstrated that stringent binding of RXR to the 3' half-site of a PPRE is more influential on the binding of the PPARgamma/RXR heterodimer than the ability of PPARgamma to bind DNA. Thus, unlike RXR, PPARgamma exhibits promiscuity in binding on a PPRE, suggesting that the definition of a PPRE for PPARgamma may need to be expanded.

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.

Thyroid hormone receptors (TRs) are cellular homologues of the viral erythroblastic leukemia oncogene (v-erbA). TRs (c-crbA isoforms) are derived from two separate gene loci in mammals: a and beta. Through a series of knockout experiments in mice in which one or several of the TR isoforms were deleted, it has been demonstrated that the TR-beta isoforms control central regulation of thyroid-stimulating hormone. Of these isoforms, TR-beta2 is the most important in mediating negative feedback control of the hypothalamic-pituitary-thyroid axis. Further analysis of TR knockout animals revealed, however, that they exhibited a much milder overall phenotype than hypothyroid animals, indicating that receptor loss was not equivalent to ligand loss in vivo. To understand this apparent paradox, we generated animals expressing a non-T3 binding receptor (delta337T) from the TR-beta allele. These mice displayed a complete hypothyroid phenotype, demonstrating that the unliganded TR mediates the effect of hypothyroidism. Because this mutant TR constitutively binds to nuclear coreprssors, it also suggests that this class of proteins is essential for mediating hypothyroidism in vivo.

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.