To 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).
We 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.
Pituitary 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.
Using a model expressing a THRβ unable to bind T3, we showed the expression pattern of liver negative and positive regulated genes by T3.
The β isoform of thyroid hormone receptor (TR-β) has a key role in the feedback regulation of the hypothalamic-pituitary-thyroid (H-P-T) axis. The mechanism of trans-repression of the hypothalamic ...thyrotropin-releasing hormone (TRH) and pituitary thyroid-stimulating hormone (TSH) subunit genes, however, remains poorly understood. A number of distinct mechanisms for TR-β-mediated negative regulation by thyroid hormone have been proposed, including those that require and do not require DNA binding. To clarify the importance of DNA binding in negative regulation, we constructed a DNA-binding mutant of TR-β in which two amino acids within the P box were altered (GSG for EGG) to resemble that found in the glucocorticoid receptor (GR). We termed this mutant GS125, and as expected, it displayed low binding affinities for positive and negative thyroid hormone-response element (pTRE and nTRE, respectively) in gel-mobility shift assays. In transient transfection assays, the GS125 mutant abolished transactivation on three classic pTREs (DR+4, LAP, and PAL) and all negatively regulated promoters in the H-P-T axis (TRH, TSH-β, and TSH-α). However, GS125 TR-β bound to a composite TR/GR-response element and was fully functional on this hybrid TR/GR-response element. Moreover, the GS125 TR-β mutant displayed normal interactions with transcriptional cofactors in mammalian two-hybrid assays. These data do not support a DNA-binding independent mechanism for thyroid hormone negative regulation in the H-P-T axis.
Thyroid carcinoma is diagnosed in 14,000 people each year in the United States.
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Most are effectively treated by surgery, followed often by radioiodine therapy and always by thyroid hormone therapy ...to suppress the secretion of thyrotropin. These patients require monitoring for recurrence of tumor, which can occur decades later.
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In some patients, monitoring includes periodic discontinuation of thyroid hormone therapy for radioiodine scanning
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and measurement of serum thyroglobulin
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to detect residual or recurrent thyroid carcinoma. As a consequence of discontinuing thyroid hormone therapy, patients typically have symptomatic hypothyroidism, some may not have a sufficient increase in thyrotropin . . .
Congenital hypothyroidism and the thyroid hormone (T3) resistance syndrome are associated with severe central nervous system (CNS) dysfunction. Because thyroid hormones are thought to act principally ...by binding to their nuclear receptors (TRs), it is unexplained why TR knock-out animals are reported to have normal CNS structure and function. To investigate this discrepancy further, a T3binding mutation was introduced into the mouse TR-β locus by homologous recombination. Because of this T3binding defect, the mutant TR constitutively interacts with corepressor proteins and mimics the hypothyroid state, regardless of the circulating thyroid hormone concentrations. Severe abnormalities in cerebellar development and function and abnormal hippocampal gene expression and learning were found. These findings demonstrate the specific and deleterious action of unliganded TR in the brain and suggest the importance of corepressors bound to TR in the pathogenesis of hypothyroidism.
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.
Growth factors such as epidermal growth factor (EGF) and insulin regulate development and metabolism via genes containing both POU homeodomain (Pit-1) and phorbol ester (AP-1) response elements. ...Although CREB binding protein (CBP) functions as a coactivator on these elements, the mechanism of transactivation was previously unclear. We now demonstrate that CBP is recruited to these elements only after it is phosphorylated at serine 436 by growth factor–dependent signaling pathways. In contrast, p300, a protein closely related to CBP that lacks this phosphorylation site, binds only weakly to the transcription complex and in a growth factor–independent manner. A small region of CBP (amino acids 312–440), which we term GF box, contains a potent transactivation domain and mediates this effect. Direct phosphorylation represents a novel mechanism controlling coactivator recruitment to the transcription complex.
Pit-1 is a pituitary-specific transcription factor responsible for pituitary development and hormone expression in mammals. Mutations in the gene encoding Pit-1 have been found in two dwarf mouse ...strains displaying hypoplasia of growth hormone, prolactin, and thyroid-stimulating, hormone-secreting cell types in the anterior pituitary. A point mutation in this gene was identified on one allele in a patient with combined pituitary hormone deficiency. Mutant Pit-1 binds DNA normally but acts as a dominant inhibitor of Pit-1 action in the pituitary.
Hepatic gluconeogenesis is essential for maintenance of normal blood glucose concentrations and is regulated by opposing stimulatory (cyclic adenosine monophosphate, cAMP) and inhibitory (insulin) ...signaling pathways. The cAMP signaling pathway leads to phosphorylation of cAMP response element-binding (CREB) protein, resulting in recruitment of the coactivators CREB-binding protein (CBP) and p300 and subsequent activation of gluconeogenesis. Insulin signaling leads to phosphorylation of CBP at serine 436, a residue near its CREB-interacting domain, but it is unknown whether this event modulates cAMP signaling. Here, we show in vitro and in 'knock-in' mice that a mutant CBP (S436A) is aberrantly recruited to CREB protein, resulting in inappropriate activation of gluconeogenesis in the fed state and glucose intolerance resulting from increased hepatic glucose production. We propose that insulin signaling may directly regulate many cAMP signaling pathways at the transcriptional level by controlling CBP recruitment.
Cone photoreceptors in the murine retina are patterned by dorsal repression and ventral activation of S opsin. TR2, the nuclear thyroid hormone receptor isoform 2, regulates dorsal repression. To ...determine the molecular mechanism by which TR2 acts, we compared the spatiotemporal expression of TR2 and S opsin from embryonic day (E) 13 through adulthood in C57BL/6 retinae. TR2 and S opsin are expressed in cone photoreceptors only. Both are transcribed by E13, and their levels increase with cone genesis. TR2 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 TR2 suggests that repression of S opsin requires other dorsal-specific factors in addition to TR2. The mechanism by which TR2 functions was probed in transgenic animals with TR2 ablated, TR2 that is DNA binding defective, and TR2 that is ligand binding defective. These studies show that TR2 is necessary for dorsal repression, but not ventral activation of S opsin. TR2 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 TR2 is consistent with a role (direct and/or indirect) in chromatin remodeling that leads to permanent gene silencing in terminally differentiated, dorsal cone photoreceptors.
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 β isoforms of TH receptors (TRβ KO), or both (double KO). TRβ 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.
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