Thyroid hormones are iodothyronines that control growth and development, as well as brain function and metabolism. Although thyroid hormone deficiency can be caused by defects of hormone synthesis and action, it has not been linked to a defect in cellular hormone transport. In fact, the physiological role of the several classes of membrane transporters remains unknown. We now report, for the first time, mutations in the monocarboxylate transporter 8 (MCT8) gene, located on the X chromosome, that encodes a 613-amino acid protein with 12 predicted transmembrane domains. The propositi of two unrelated families are males with abnormal relative concentrations of three circulating iodothyronines, as well as neurological abnormalities, including global developmental delay, central hypotonia, spastic quadriplegia, dystonic movements, rotary nystagmus, and impaired gaze and hearing. Heterozygous females had a milder thyroid phenotype and no neurological defects. These findings establish the physiological importance of MCT8 as a thyroid hormone transporter.

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The BRAF T1799A mutation is the most common genetic alteration in papillary thyroid carcinomas (PTC). It is also found in a subset of papillary microcarcinomas, consistent with a role in tumor initiation. PTCs with BRAF T1799A are often invasive and present at a more advanced stage. BRAF T1799A is found with high prevalence in tall-cell variant PTCs and in poorly differentiated and undifferentiated carcinomas arising from PTCs. To explore the role of BRAF V600E in thyroid cancer pathogenesis, we targeted its expression to thyroid cells of transgenic FVB/N mice with a bovine thyroglobulin promoter. Two Tg-BRAF V600E lines (Tg-BRAF2 and Tg-BRAF3) were propagated for detailed analysis. Tg-BRAF2 and Tg-BRAF3 mice had increased thyroid-stimulating hormone levels (>7-and f2-fold, respectively). This likely resulted from decreased expression of thyroid peroxidase, sodium iodine symporter, and thyroglobulin. All lines seemed to successfully compensate for thyroid dysfunction, as serum thyroxine/triiodothyronine and somatic growth were normal. Thyroid glands of transgenic mice were markedly enlarged by 5 weeks of age. In Tg-BRAF2 mice, PTCs were present at 12 and 22 weeks in 14 of 15 and 13 of 14 animals, respectively, with 83% exhibiting tallcell features, 83% areas of invasion, and 48% foci of poorly differentiated carcinoma. Tg-BRAF3 mice also developed PTCs, albeit with lower prevalence (3 of 12 and 4 of 9 at 12 and 22 weeks, respectively). Tg-BRAF2 mice had a 30% decrease in survival at 5 months. In summary, thyroid-specific expression of BRAF V600E induces goiter and invasive PTC, which transitions to poorly differentiated carcinomas. This closely recapitulates the phenotype of BRAF-positive PTCs in humans and supports a key role for this oncogene in its pathogenesis. (Cancer Res 2005; 65(10): 4238-45)

Dual oxidase 2 (DUOX2), an NADPH:O 2 oxidoreductase flavoprotein, is a component of the thyroid H 2 O 2 generator crucial for hormone synthesis at the apical membrane. Mutations in DUOX2 produce congenital hypothyroidism in humans. However, no functional DUOX-based NADPH oxidase has ever been reconstituted at the plasma membrane of transfected cells. It has been proposed that DUOX retention in the endoplasmatic reticulum (ER) of heterologous systems is due to the lack of an unidentified component required for functional maturation of the enzyme. By data mining of a massively parallel signature sequencing tissue expression data base, we identified an uncharacterized gene named DUOX maturation factor (DUOXA2) arranged head-to-head to and coexpressed with DUOX2. A paralog (DUOXA1) was similarly linked to DUOX1. The genomic rearrangement leading to linkage of ancient DUOX and DUOXA genes could be traced back before the divergence of echinoderms. We demonstrate that co-expression of DUOXA2, an ER-resident transmembrane protein, allows ER-to-Golgi transition, maturation, and translocation to the plasma membrane of functional DUOX2 in a heterologous system. The identification of DUOXA genes has important implications for studies of the molecular mechanisms controlling DUOX expression and the molecular genetics of congenital hypothyroidism.Generation of H 2 O 2 at the apical membrane of thyroid follicular cells is essential for iodination of thyroglobulin by thyroid peroxidase and constitutes the rate-limiting step of thyroid hormone synthesis. Dual oxidases (DUOX1 and DUOX2) 2 appear to constitute the catalytic core of the H 2 O 2 generator (1, 2). They are large homologs of the phagocyte gp91 phox /Nox2 NADPH-dependent oxidase with an N-terminal extension comprising a peroxidase-like domain. Although the crucial role of DUOX2 in thyroid hormonogenesis has been substantiated by reports of severe congenital hypothyroidism in patients with biallelic nonsense mutations (3), the understanding of structure, function, and regulation of DUOX has remained limited. The major obstacle for molecular studies of DUOX is the lack of a suitable heterologous cell system for DUOX-based functional NADPH oxidase expression. Transfected cells completely retain DUOX in the endoplasmatic reticulum (ER) (4 -8), suggesting that an unidentified component, essential for DUOX maturation, may be specifically expressed in tissues containing the functional enzyme. EXPERIMENTAL PROCEDURESData Mining and Computational Analysis-Massively parallel signature sequencing (MPSS) data (9) were obtained from the NCBI Gene Expression Omnibus repository (www.ncbi.nlm.nih.gov/geo/; records GSE1747 and GPL1443). A thyroid specificity score, as defined by Jongeneel et al. (9), was calculated for signatures with frequency Ͼ100 tags per million (ϳ30 mRNA copies/cell) in the thyroid/parathyroid library. Tags with scores ϾϪ1 were mapped to the human genome assembly using BLAST. DUOXA homologs were identified by tBLASTn searches against the NCBI nr data base and trace...

Patients with mutations in the thyroid hormone receptor ␤ (TR␤) gene manifest resistance to thyroid hormone (RTH), resulting in a constellation of variable phenotypic abnormalities. To understand the molecular basis underlying the action of mutant TR␤ in vivo, we generated mice with a targeted mutation in the TR␤ gene (TR␤PV; PV, mutant thyroid hormone receptor kindred PV) by using homologous recombination and the Cre͞loxP system. Mice expressing a single PV allele showed the typical abnormalities of thyroid function found in heterozygous humans with RTH. Homozygous PV mice exhibit severe dysfunction of the pituitary-thyroid axis, impaired weight gains, and abnormal bone development. This phenotype is distinct from that seen in mice with a null mutation in the TR␤ gene. Importantly, we identified abnormal expression patterns of several genes in tissues of TR␤PV mice, demonstrating the interference of the mutant TR with the gene regulatory functions of the wild-type TR in vivo. These results show that the actions of mutant and wild-type TR␤ in vivo are distinct. This model allows further study of the molecular action of mutant TR in vivo, which could lead to better treatment for RTH patients. The thyroid hormone 3,3Ј,5-triiodo-L-thyronine (T3) regulates growth, development, and differentiation. Its actions are mainly mediated through thyroid hormone receptors (TRs), which are ligand-dependent transcription factors (1, 2). Three ligand-activated TR isoforms have been identified, TR␣1, TR␤1, and TR␤2, which are derived from the TR␣ and TR␤ genes, respectively. Each TR isoform has a unique developmental and tissue-specific expression (1, 2). TR binds to specific DNA sequences known as thyroid hormone response elements (TRE) in the promoter regions of T3 target genes. The transcriptional activity of TR depends not only on the types of TRE but also on a host of coregulatory proteins (3).RTH is a syndrome characterized by reduced sensitivity of tissues to the action of thyroid hormone. This condition is characterized by elevated levels of circulating thyroid hormones associated with normal or high levels of serum thyroidstimulating hormone (TSH) (4). The most common form of RTH is familial with autosomal dominant inheritance (4). Patients are usually heterozygotes with only one mutant TR␤ gene, and the symptoms are mild. Moreover, clinical manifestations are variable among families with RTH and also among affected family members. Some of the clinical features that have been reported include goiter, short stature, decreased weight, tachycardia, hearing loss, attention deficit-hyperactivity disorder, decreased IQ, and dyslexia (4). One single patient homozygous for a mutant TR␤ has been reported (5). This patient displayed a complex phenotype of extreme RTH with very high levels of thyroid hormone and TSH. Most TR␤ mutants derived from RTH patients have reduced T3-binding affinities and transcriptional capacities. These TR␤ mutants exhibit a dominant-negative effect when cotransfected with wild-type TRs (6, 7). TR␤ mutan...

The primary function of thyroid gland is to metabolize iodide by synthesizing thyroid hormones that are critical regulators of growth, development and metabolism in virtually all tissues. To date, research on thyroid morphogenesis was missing an efficient stem-cell model system which allows to recapitulate in vitro the molecular and morphogenic events regulating thyroid follicular cells differentiation and subsequent assembly into functional thyroid follicles. Here we report that a transient overexpression of the transcription factors NKX2.1 and PAX8 is sufficient to direct mouse embryonic stem-cells (mESC) differentiation into thyroid follicular cells which organized into three-dimensional follicular structures when treated with thyrotropin. Those in vitro derived follicles showed significant iodide organification activity. Importantly, when grafted in vivo into athyreoid mice, these follicles rescued thyroid hormone plasma levels and promoted subsequent symptomatic recovery. Thus, mESC can be induced to differentiate into thyroid follicular cells in vitro and generate functional thyroid tissue.

Advanced human thyroid cancers, particularly those that are refractory to treatment with radioiodine (RAI), have a high prevalence of BRAF (v-raf murine sarcoma viral oncogene homolog B1) mutations. However, the degree to which these cancers are dependent on BRAF expression is still unclear. To address this question, we generated mice expressing one of the most commonly detected BRAF mutations in human papillary thyroid carcinomas (BRAF V600E ) in thyroid follicular cells in a doxycycline-inducible (dox-inducible) manner. Upon dox induction of BRAF V600E , the mice developed highly penetrant and poorly differentiated thyroid tumors. Discontinuation of dox extinguished BRAF V600E expression and reestablished thyroid follicular architecture and normal thyroid histology. Switching on BRAF V600E rapidly induced hypothyroidism and virtually abolished thyroid-specific gene expression and RAI incorporation, all of which were restored to near basal levels upon discontinuation of dox. Treatment of mice with these cancers with small molecule inhibitors of either MEK or mutant BRAF reduced their proliferative index and partially restored thyroid-specific gene expression. Strikingly, treatment with the MAPK pathway inhibitors rendered the tumor cells susceptible to a therapeutic dose of RAI. Our data show that thyroid tumors carrying BRAF V600E mutations are exquisitely dependent on the oncoprotein for viability and that genetic or pharmacological inhibition of its expression or activity is associated with tumor regression and restoration of RAI uptake in vivo in mice. These findings have potentially significant clinical ramifications.

To define the roles of circadian rhythmicity (intrinsic effects of time of day independent of the sleep or wake condition) and sleep (intrinsic effects of the sleep condition, irrespective of the time of day) on the 24-h variation in glucose tolerance, eight normal men were studied during constant glucose infusion for a total of 53 h. The period of study included 8 h of nocturnal sleep, 28 h of continuous wakefulness, and 8 h ofdaytime sleep. Blood samples for the measurement of glucose, insulin, C-peptide, cortisol, and growth hormone were collected at 20-min intervals throughout the entire study. Insulin secretion rates were derived from C-peptide levels by deconvolution. Sleep was polygraphically monitored.During nocturnal sleep, levels of glucose and insulin secretion increased by 31±5% and 60±11%, respectively, and returned to baseline in the morning. During sleep deprivation, glucose levels and insulin secretion rose again to reach a maximum at a time corresponding to the beginning of the habitual sleep period. The magnitude of the rise above morning levels averaged 17±5% for glucose and 49±8% for calculated insulin secretion. Serum insulin levels did not parallel the circadian variation in insulin secretion, indicating the existence of an approximate 40% increase in insulin clearance during the night.Daytime sleep was associated with a 16±3% rise in glucose levels, a 55±7% rise in insulin secretion, and a 39±5% rise in serum insulin.The diurnal-variation in insulin secretion was inversely related to the cortisol rhythm, with a significant correlation of the magnitudes of their morning to evening excursions. Sleep-associated rises in glucose correlated with the amount of concomitant growth hormone secreted. These studies demonstrate previously underappreciated effects of circadian rhythmicity and sleep on glucose levels, insulin secretion, and insulin clearance, and suggest that these effects could be partially mediated by cortisol and growth hormone. (J. Clin. Invest. 1991. 88:934-942.)