Contrasting skeletal phenotypes in mice with an identical mutation targeted to thyroid hormone receptor alpha1 or beta

Thyroid hormone (T(3)) regulates bone turnover and mineralization in adults and is essential for skeletal development. Surprisingly, we identified a phenotype of skeletal thyrotoxicosis in T(3) receptor beta(PV) (TRbeta(PV)) mice in which a targeted frameshift mutation in TRbeta results in resistance to thyroid hormone. To characterize mechanisms underlying thyroid hormone action in bone, we analyzed skeletal development in TRalpha1(PV) mice in which the same PV mutation was targeted to TRalpha1. In contrast to TRbeta(PV) mice, TRalpha1(PV) mutants exhibited skeletal hypothyroidism with delayed endochondral and intramembranous ossification, severe postnatal growth retardation, diminished trabecular bone mineralization, reduced cortical bone deposition, and delayed closure of the skull sutures. Skeletal hypothyroidism in TRalpha1(PV) mutants was accompanied by impaired GH receptor and IGF-I receptor expression and signaling in the growth plate, whereas GH receptor and IGF-I receptor expression and signaling were increased in TRbeta(PV) mice. These data indicate that GH receptor and IGF-I receptor are physiological targets for T(3) action in bone in vivo. The divergent phenotypes observed in TRalpha1(PV) and TRbeta(PV) mice arise because the pituitary gland is a TRbeta-responsive tissue, whereas bone is TRalpha responsive. These studies provide a new understanding of the complex relationship between central and peripheral thyroid status.     

A point mutation (Ala229 to Thr) in the hinge domain of the c-erbA beta thyroid hormone receptor gene in a family with generalized thyroid hormone resistance.

Various point mutations in the c-erbA thyroid hormone receptor (TR) beta gene of unrelated kindreds have been reported to be responsible for different phenotypes of generalized thyroid hormone resistance. We now report a new point mutation, Td, in one of two TR beta alleles of three affected members of one family, designated family T. In contrast to the previously described point mutations, all located in the T3-binding domain of the TR beta gene, mutation Td was identified in the carboxy-terminal part of the hinge domain. Direct sequencing of the polymerase chain reaction-amplified whole coding region of the patients' fibroblast TR beta genes displayed a single guanine to adenine transition at cDNA nucleotide position 985. This altered alanine (GCC) to threonine (ACC) in codon 229. Garnier prediction of the consequence of the mutation indicated an altered secondary structure. The G----A nucleotide substitution was not present in 80 random TR beta alleles, suggesting that this point mutation is responsible for generalized thyroid hormone resistance in family T. The in vitro expressed mutant TR beta was shown to bind with high affinity to various thyroid hormone response elements. However, the affinity of the TR beta to bind to T3 was reduced 3-fold, indicating that the hinge domain of the TR beta is important for full ligand-binding activity. Moreover, it seems that multiple subdomains of the TR beta interact cooperatively to achieve optimal T3 activity.     

A thyroid hormone receptor alpha gene mutation (P398H) is associated with visceral adiposity and impaired catecholamine-stimulated lipolysis in mice

Thyroid hormone has profound effects on metabolic homeostasis, regulating both lipogenesis and lipolysis, primarily by modulating adrenergic activity. We generated mice with a point mutation in the thyroid hormone receptor alpha (TRalpha) gene producing a dominant-negative TRalpha mutant receptor with a proline to histidine substitution (P398H). The heterozygous P398H mutant mice had a 3.4-fold (p < 0.02) increase in serum thyrotropin (TSH) levels. Serum triiodothyronine (T3) and thyroxine (T4) concentrations were slightly elevated compared with wild-type mice. The P398H mice had a 4.4-fold increase in body fat (as a fraction of total body weight) (p < 0.001) and a 5-fold increase in serum leptin levels (p < 0.005) compared with wild-type mice. A 3-fold increase in serum fasting insulin levels (p < 0.002) and a 55% increase in fasting glucose levels (p < 0.01) were observed in P398H compared with wild-type mice. There was a marked reduction in norepinephrine-induced lipolysis, as reflected in reduced glycerol release from white adipose tissue isolated from P398H mice. Heart rate and cold-induced adaptive thermogenesis, mediated by thyroid hormone-catecholamine interaction, were also reduced in P398H mice. In conclusion, the TRalpha P398H mutation is associated with visceral adiposity and insulin resistance primarily due to a marked reduction in catecholamine-stimulated lipolysis. The observed phenotype in the TRalpha P398H mouse is likely due to interference with TRalpha action as well as influence on other metabolic signaling pathways. The physiologic significance of these findings will ultimately depend on understanding the full range of actions of this mutation.     

A homozygous deletion in the c-erbA beta thyroid hormone receptor gene in a patient with generalized thyroid hormone resistance: isolation and characterization of the mutant receptor.

Different point mutations have been identified in the T3-binding domain of the c-erbA beta thyroid hormone receptor gene that are associated with variant phenotypes of generalized thyroid hormone resistance (GTHR). In most cases of GTHR, heterozygotes are affected; a single mutant allele results in the inhibition of the function of normal thyroid hormone receptors. We report here a novel genetic abnormality, a 3-basepair (bp) deletion in the T3-binding domain of the beta-receptor in a kindred, S, with GTHR. One patient, S1, was the product of a consanguineous union of two heterozygotes and was homozygous for this defect. Heterozygotes from kindred S harbored a CAC deletion at nucleotides 1295-1297, which resulted in the deduced loss of amino acid residue threonine at codon 332, and they displayed elevated free T4 levels and inappropriately normal TSH levels characteristic of other kindreds with GTHR. However, patient S1, who had two mutant alleles, had markedly elevated TSH and free T4 levels and displayed profound abnormalities in brain development and linear growth. A fibroblast c-erbA beta cDNA extending from codon 175 to stop codon 457 was cloned from patient S1, sequenced, and used to create a full-length mutant cDNA. The kindred S mutant receptor was synthesized in vitro and did not bind T3. This mutant receptor did bind with similar avidity as the wild-type human beta-receptor to thyroid hormone response elements of the human TSH beta (-12 to 43 bp) and rat GH (-188 to -160 bp) genes. Kindred S showed the effect in man of heterozygous and homozygous expression of a dominant negative form of c-erbA beta.     

Severe impairment of cerebellum development in mice expressing a dominant-negative mutation inactivating thyroid hormone receptor alpha1 isoform

Thyroid hormone deficiency is known to deeply affect cerebellum post-natal development. We present here a detailed analysis of the phenotype of a recently generated mouse model, expressing a dominant-negative TRα1 mutation. Although hormonal level is not affected, the cerebellum of these mice displays profound alterations in neuronal and glial differentiation, which are reminiscent of congenital hypothyroidism, indicating a predominant function of this receptor isoform in normal cerebellum development. Some of the observed effects might result from the cell autonomous action of the mutation, while others are more likely to result from a reduction in neurotrophic factor production.     

Single base mutation in the hormone binding domain of the thyroid hormone receptor beta gene in generalised thyroid hormone resistance demonstrated by single stranded conformation polymorphism analysis.

Thyroid hormone resistance is a syndrome of considerable clinical heterogeneity. Three mutations in the c-erb A beta gene encoding the human beta thyroid hormone receptor have been described in different kindreds. We report here, in a family affected with peripheral thyroid hormone resistance, a unique point mutation in the ligand binding domain of the c-erb A beta gene resulting in histidine replacement of an arginine residue at position 438. The region in which the mutation occurred was identified by single stranded conformation polymorphism analysis and confirmed by subcloning and sequencing of the mutant alleles from each of the affected members. Binding of tri-iodothyronine to isolated nuclei from family members was normal suggesting the mechanism of thyroid hormone resistance in this family is not mediated by abnormal binding of ligand and receptor.     

Effect of fasting on thyroid hormone levels, and TR(alpha) and TR(beta) mRNA accumulation in late-stage embryo and juvenile rainbow trout, Oncorhynchus mykiss

The accumulation of mRNA encoding for hepatic and intestinal T3-receptor (TR) and body and liver masses were measured in fed and 3-week fasted juvenile and swim up stage rainbow trout embryos. Plasma and total body thyroid hormone (TH) levels were measured for juvenile and swim up stages, respectively. Fasted juveniles exhibited a lower hepatosomatic index (HSI), liver mass and plasma T4 and T3 concentrations than fed animals, but there were no changes in body mass or the accumulation of mRNA encoding for either of the TR(alpha) or TR(beta) isoforms in liver or intestine. TR(beta) mRNA accumulation was greater than TR(alpha) mRNA accumulation in both tissues. Fasted embryos had lower whole body TH levels and body, liver and intestinal tract masses, in addition to a lower intestinosomatic index. However, there was no change in HSI. Fasting did not affect whole body or hepatic TR(alpha) and TR(beta) mRNA accumulation, although intestinal tract TR(alpha) and TR(beta) mRNA accumulation was lower in the fasted embryos. The HSI and body mass changes in fasted juvenile and embryo stages, respectively, indicated that both developmental stages were impacted by fasting. Both stages also showed evidence of decreased TH production. The lower TR gene expression in the intestinal tract of fasted embryos may suggest a role for THs in the transitional stage of intestinal development during this period of development.     

Spared bone mass in rats treated with thyroid hormone receptor TR beta-selective compound GC-1

Thyrotoxicosis is frequently associated with increased bone turnover and decreased bone mass. To investigate the role of thyroid hormone receptor-beta (TR beta) in mediating the osteopenic effects of triiodothyronine (T3), female adult rats were treated daily (64 days) with GC-1 (1.5 microg/100 g body wt), a TR beta-selective thyromimetic compound. Bone mass was studied by dual-energy X-ray absorptiometry of several skeletal sites and histomorphometry of distal femur, and the results were compared with T3-treated (3 microg/100 g body wt) or control animals. As expected, treatment with T3 significantly reduced bone mineral density (BMD) in the lumbar vertebrae (L2-L5), femur, and tibia by 10-15%. In contrast, GC-1 treatment did not affect the BMD in any of the skeletal sites studied. The efficacy of GC-1 treatment was verified by a reduction in serum TSH (-52% vs. control, P < 0.05) and cholesterol (-21% vs. control, P < 0.05). The histomorphometric analysis of the distal femur indicated that T3 but not GC-1 treatment reduced the trabecular volume, thickness, and number. We conclude that chronic, selective activation of the TR beta isoform does not result in bone loss typical of T3-induced thyrotoxicosis, suggesting that the TR beta isoform is not critical in this process. In addition, our findings suggest that the development of TR-selective T3 analogs that spare bone mass represents a significant improvement toward long-term TSH-suppressive therapy.     

Discovery of a novel series of 6-azauracil-based thyroid hormone receptor ligands: potent,TR beta subtype-selective thyromimetics

In this communication, we wish to describe the discovery of a novel series of 6-azauracil-based thyromimetics that possess up to 100-fold selectivities for binding and functional activation of the beta(1)-isoform of the thyroid receptor family. Structure-activity relationship studies on the 3,5- and 3'-positions provided compounds with enhanced TR beta affinity and selectivity. Key binding interactions between the 6-azauracil moiety and the receptor have been determined through of X-ray crystallographic analysis.     

Abnormal heart rate and body temperature in mice lacking thyroid hormone receptor alpha 1.

Thyroid hormone, acting through several nuclear hormone receptors, plays important roles in thermogenesis, lipogenesis and maturation of the neonatal brain. The receptor specificity for mediating these effects is largely unknown, and to determine this we developed mice lacking the thyroid hormone receptor TR alpha 1. The mice have an average heart rate 20% lower than that of control animals, both under normal conditions and after thyroid hormone stimulation. Electrocardiograms show that the mice also have prolonged QRS- and QTend-durations. The mice have a body temperature 0.5 degrees C lower than normal and exhibit a mild hypothyroidism, whereas their overall behavior and reproduction are normal. The results identify specific and important roles for TR alpha 1 in regulation of tightly controlled physiological functions, such as cardiac pacemaking, ventricular repolarisation and control of body temperature.     

Altered behavioral rhythms and clock gene expression in mice with a targeted mutation in the Period1 gene

A group of specialized genes has been defined to govern the molecular mechanisms controlling the circadian clock in mammals. Their expression and the interactions among their products dictate circadian rhythmicity. Three genes homologous to Drosophila period exist in the mouse and are thought to be major players in the biological clock. Here we present the generation of mice in which the founding member of the family, Per1, has been inactivated by homologous recombination. These mice present rhythmicity in locomotor activity, but with a period almost 1 h shorter than wild-type littermates. Moreover, the expression of clock genes in peripheral tissues appears to be delayed in Per1 mutant animals. Importantly, light-induced phase shifting appears conserved. The oscillatory expression of clock genes and the induction of immediate-early genes in response to light in the master clock structure, the suprachiasmatic nucleus, are unaffected. Altogether, these data demonstrate that Per1 plays a distinct role within the Per family, as it may be involved predominantly in peripheral clocks and/or in the output pathways of the circadian clock.     

Thyroid hormone and DNA binding properties of a mutant c-erbA beta receptor associated with generalized thyroid hormone resistance

We have previously reported a family, Kindred A, with autosomal dominant generalized thyroid hormone resistance in which affected members were found to have a mutation in the carboxy-terminal domain of the c-erbA beta thyroid hormone receptor. In the current study, the thyroid hormone and DNA-binding properties of this mutant receptor were determined using c-erbA beta protein synthesized in vitro. Both the wild-type human placental c-erbA beta and Kindred A receptors bound [125I]-triiodothyronine, although the Kindred A receptor had decreased affinity for the hormone. The affinity for triiodothyronine was 4.5 x 10(9) M-1 and 2.3 x 10(10) M-1 for the mutant and wild-type receptors, respectively. No abnormality of DNA-binding was detected with the Kindred A receptor using a sensitive avidin-biotin DNA-binding assay with DNA fragments containing thyroid hormone response elements. The Kindred A mutant receptor which displays abnormal triiodothyronine-binding but normal DNA-binding activities in vitro acts as a dominant negative inhibitor of thyroid hormone action in man.