Thyroid Hormone Biosynthesis Research Articles

Functional activity and tumor-specific expression of dual oxidase 2 in pancreatic cancer cells and human malignancies characterized with a novel monoclonal antibody

Dual oxidase 2 (Duox2), one of the seven members of the NADPH oxidase gene family, plays a critical role in generating H2O2 for thyroid hormone biosynthesis and as an integral part of the host defense system of the respiratory epithelium and the gastrointestinal tract. Recent evidence suggests that the regulation of Duox2 expression is under the control of pro-inflammatory cytokines and that Duox2-induced reactive oxygen species (ROS) contribute to the inflammation-related tissue injury that occurs in two pre-malignant, inflammatory conditions: chronic pancreatitis and inflammatory bowel disease. Because no reliable Duox antibodies are commercially available, we report the development of a murine monoclonal antibody (MAb) to Duox2 (clone Duox S-12) and its use for the characterization of Duox2 expression in human tumors, tumor cell lines and normal tissues. Duox S-12 specifically detected both endogenously- and ectopically-expressed Duox2 protein by immunoblotting, immunofluorescence microscopy and immunohistochemistry (where both membranous and cytoplasmic staining were present). Duox2 expression detected by Duox S-12 was functionally coupled to the generation of H2O2 in pancreatic cancer cells that expressed Duox2 and its cognate maturation factor DuoxA2. Although Duox S-12 recognizes ectopically expressed Duox1 protein because of the extensive amino acid homology between Duox1 and Duox2, the lack of substantial Duox1 mRNA expression in human tumors (except thyroid cancer) allowed us to evaluate Duox2 expression across a wide range of normal and malignant tissues by immunohistochemistry. Duox2 was expressed at elevated levels in many human cancers, most notably tumors of the prostate, lung, colon and breast while brain tumors and lymphomas demonstrated the lowest frequency of expression. The Duox-specific monoclonal antibody described here provides a promising tool for the further examination of the role of Duox-dependent reactive oxygen production in inflammation-related carcinogenesis, where alterations in oxidant tone play a critical role in cell growth and proliferation.

Recent insights into the cell biology of thyroid angiofollicular units.

In thyrocytes, cell polarity is of crucial importance for proper thyroid function. Many intrinsic mechanisms of self-regulation control how the key players involved in thyroid hormone (TH) biosynthesis interact in apical microvilli, so that hazardous biochemical processes may occur without detriment to the cell. In some pathological conditions, this enzymatic complex is disrupted, with some components abnormally activated into the cytoplasm, which can lead to further morphological and functional breakdown. When iodine intake is altered, autoregulatory mechanisms outside the thyrocytes are activated. They involve adjacent capillaries that, together with thyrocytes, form the angiofollicular units (AFUs) that can be considered as the functional and morphological units of the thyroid. In response to iodine shortage, a rapid expansion of the microvasculature occurs, which, in addition to nutrients and oxygen, optimizes iodide supply. These changes are triggered by angiogenic signals released from thyrocytes via a reactive oxygen species/hypoxia-inducible factor/vascular endothelial growth factor pathway. When intra- and extrathyrocyte autoregulation fails, other forms of adaptation arise, such as euthyroid goiters. From onset, goiters are morphologically and functionally heterogeneous due to the polyclonal nature of the cells, with nodules distributed around areas of quiescent AFUs containing globules of compact thyroglobulin (Tg) and surrounded by a hypotrophic microvasculature. Upon TSH stimulation, quiescent AFUs are activated with Tg globules undergoing fragmentation into soluble Tg, proteins involved in TH biosynthesis being expressed and the local microvascular network extending. Over time and depending on physiological needs, AFUs may undergo repetitive phases of high, moderate, or low cell and tissue activity, which may ultimately culminate in multinodular goiters.

Maintenance of iodine intake

Maintenance of iodine intake

Selenium in the thyroid: physiology and pathology

Selenium (Se) is a trace element that plays a critical role in several processes for human health. The thyroid is the organ with the highest Se content per gram of tissue; in thyroid follicular cells, Se acts as antioxidant by contrasting the production of the reactive oxygen species that are generated during thyroid hormones biosynthesis. In addition, Se is part of the active site of the deoidinases, the enzymes responsible for thyroid hormones activation and inactivation. Herein, the effects of Se supplementation in patients with thyroid related disorders have been reviewed on the basis of the studies published on this issue.

A new parameter that supports speculation on the possible mechanism of hypothyroidism induced by chemical substances in repeated-dose toxicity studies

Hypothyroidism induced by xenobiotic treatment was analyzed for possible underlying mechanism(s) on the basis of different responses of the thyroid gland and the liver, using a newly-created database of repeated-dose toxicity of 500 chemicals. Two mechanisms are proposed: direct inhibition of thyroid hormone biosynthesis in the thyroid gland, and stimulated degradation of thyroid hormone by induction of hepatic drug-metabolizing enzymes. In the database there were 10 chemicals inducing hypertrophy/hyperplasia of follicular cells in the thyroid gland and having data on thyroid glands. On the basis of the chemical structure and information available in the literature, we judged three chemicals to be typical thioamide derivatives that act directly on the thyroid gland, and the others as non-thioamide derivatives that were unlikely to have any direct action on the thyroid gland. All these chemicals were classified into two groups using the ratios of relative weight increase rate of thyroid gland versus that of the liver. These values were at least 1.7, but 3.2 or more in the most of the cases for thioamide derivatives, and 1.2 or less for non-thioamide derivatives. This background analysis suggests the feasibility of parameter-supported speculation on the possible underlying mechanism when new repeated-dose toxicity data on hypothyroidism becomes available.

Regulation of Dual Oxidase Expression and H2O2Production by Thyroglobulin

Thyroglobulin (Tg) is a macromolecular precursor in thyroid hormone synthesis to which iodine is stably bound. Tg, which is stored in the follicular space, is also a potent negative feedback regulator of follicular function, and this is achieved by suppressing mRNA levels of thyroid-specific genes such as the sodium/iodide symporter (Slc5a5), Tg, and thyroid peroxidase. Dual oxidase 1 (DUOX1) and DUOX2, originally identified in the thyroid, are nicotinamide adenine dinucleotide phosphate (NADPH) oxidases that are necessary to produce the H2O2 required for thyroid hormone biosynthesis. Since follicular Tg regulates the expression of genes that are essential for thyroid hormone synthesis, we hypothesized that Tg might also regulate DUOX expression and H2O2 production. Rat thyroid FRTL-5 cells were treated with Tg, and the mRNA expression of Duox1 and Duox2 and their corresponding maturation factors Duoxa1 and Duoxa2 were evaluated by DNA microarray and real-time PCR. Duox2 promoter activity was examined by luciferase reporter gene assay. Protein levels of DUOX2 were also examined by Western blot analysis. Intracellular H2O2 generation was quantified by a fluorescent dye, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, and acetyl ester (CM-H2DCFDA). mRNA levels of Duox2 and its activation factor Duoxa2 (but not Duox1 or Duoxa1) were significantly suppressed by Tg in a dose-dependent manner and a time-dependent fashion in rat thyroid FRTL-5 cells. DUOX2 promoter activity was significantly suppressed by Tg in a dose-dependent manner. Protein levels of DUOX2 and H2O2 generation in cells were also reduced by Tg treatment. We show that physiological concentrations of Tg suppressed the expression and function of DUOX2 in thyroid cells. These results suggest that Tg is a strong suppressor of the expression and the activity of DUOX2/DUOXA2, thereby regulating iodide organification and hormone synthesis in the thyroid. The evidence supports a reported model in which accumulated Tg in thyroid follicles plays important roles in autoregulating the function of individual follicles, which produces the basis of follicular heterogeneity.

The KCNQ1‐KCNE2 K+channel is required for adequate thyroid I−uptake

The KCNQ1 α subunit and the KCNE2 β subunit form a potassium channel in thyroid epithelial cells. Genetic disruption of KCNQ1-KCNE2 causes hypothyroidism in mice, resulting in cardiac hypertrophy, dwarfism, alopecia, and prenatal mortality. Here, we investigated the mechanistic requirement for KCNQ1-KCNE2 in thyroid hormone biosynthesis, utilizing whole-animal dynamic positron emission tomography. The KCNQ1-specific antagonist (-)-[3R,4S]-chromanol 293B (C293B) significantly impaired thyroid cell I(-) uptake, which is mediated by the Na(+)/I(-) symporter (NIS), in vivo (dSUV/dt: vehicle, 0.028 ± 0.004 min(-1); 10 mg/kg C293B, 0.009 ± 0.006 min(-1)) and in vitro (EC(50): 99 ± 10 μM C293B). Na(+)-dependent nicotinate uptake by SMCT, however, was unaffected. Kcne2 deletion did not alter the balance of free vs. thyroglobulin-bound I(-) in the thyroid (distinguished using ClO(4)(-), a competitive inhibitor of NIS), indicating that KCNQ1-KCNE2 is not required for Duox/TPO-mediated I(-) organification. However, Kcne2 deletion doubled the rate of free I(-) efflux from the thyroid following ClO(4)(-) injection, a NIS-independent process. Thus, KCNQ1-KCNE2 is necessary for adequate thyroid cell I(-) uptake, the most likely explanation being that it is prerequisite for adequate NIS activity.

PTTG-Binding Factor (PBF) Is a Novel Regulator of the Thyroid Hormone Transporter MCT8

Within the basolateral membrane of thyroid follicular epithelial cells, two transporter proteins are central to thyroid hormone (TH) biosynthesis and secretion. The sodium iodide symporter (NIS) delivers iodide from the bloodstream into the thyroid, and after TH biosynthesis, monocarboxylate transporter 8 (MCT8) mediates TH secretion from the thyroid gland. Pituitary tumor-transforming gene-binding factor (PBF; PTTG1IP) is a protooncogene that is up-regulated in thyroid cancer and that binds NIS and modulates its subcellular localization and function. We now show that PBF binds MCT8 in vitro, eliciting a marked shift in MCT8 subcellular localization and resulting in a significant reduction in the amount of MCT8 at the plasma membrane as determined by cell surface biotinylation assays. Colocalization and interaction between PBF and Mct8 was also observed in vivo in a mouse model of thyroid-specific PBF overexpression driven by a bovine thyroglobulin (Tg) promoter (PBF-Tg). Thyroidal Mct8 mRNA and protein expression levels were similar to wild-type mice. Critically, however, PBF-Tg mice demonstrated significantly enhanced thyroidal TH accumulation and reduced TH secretion upon TSH stimulation. Importantly, Mct8-knockout mice share this phenotype. These data show that PBF binds and alters the subcellular localization of MCT8 in vitro, with PBF overexpression leading to an accumulation of TH within the thyroid in vivo. Overall, these studies identify PBF as the first protein to interact with the critical TH transporter MCT8 and modulate its function in vivo. Furthermore, alongside NIS repression, PBF may thus represent a new regulator of TH biosynthesis and secretion.

PCB153 Disrupts Thyroid Hormone Homeostasis by Affecting its Biosynthesis, Biotransformation, Feedback Regulation, and Metabolism

PCB153, one of the 3 dominant congeners in the food chain, causes the disruption of the endocrine system in humans and animals. In order to elucidate the effects of PCB153 on the biosynthesis, biotransformation, regulation, metabolism, and transport of thyroid hormones (THs), Sprague-Dawley (SD) rats were dosed with PCB153 intraperitoneally (i.p.) at 0, 4, 16 and 32 mg/kg/day for 5 consecutive days and sacrificed 24 h after the last dose. Results showed that after treatment with PCB153, serum total thyroxine (TT4), total triiodothyronine (TT3), and thyrotropin releasing hormone (TRH) decreased, whereas serum thyroid stimulating hormone (TSH) concentration did not alter. The serum sodium iodide symporter (NIS), thyroid peroxidase (TPO), and thyroglobulin (Tg) levels decreased. The mRNA expressions of type 2 and 3 deiodinases (D2 and D3) reduced, but the type 1 deiodinase (D1) showed no significant change. The TSH receptor (TSHr) and TRH receptor (TRHr) levels declined. PCB153 induced hepatic enzymes, and the UDPGTs, CYP2B1, and CYP3A1 mRNA levels were significantly elevated. Taken together, the observed results from the present study indicated that PCB153 disrupted thyroid hormone homeostasis through influencing synthesis-associated proteins (NIS, TPO and Tg), deiodinases, receptors (TSHr and TRHr), and hepatic enzymes, and the decrease of D3 expression might be the compensatory response of body.

Thyroid Function Is Maintained Despite Increased Oxidative Stress in Mice Lacking Selenoprotein Biosynthesis in Thyroid Epithelial Cells

We have tested the hypothesis that selenium (Se)-containing antioxidative enzymes protect thyroid epithelial cells from oxidative damage associated with enzymatic production of hydrogen peroxide required for thyroid hormone biosynthesis. Thyroid epithelial cells therefore express antioxidative enzymes, including catalase, peroxiredoxins, thioredoxin reductases, and glutathione peroxidases (GPxs). The latter two enzyme families contain highly active peroxide-degrading enzymes that carry selenocysteine (Sec) in their active centers. Since low Se status has been associated with thyroid disorders, selenoproteins are considered essential for thyroid integrity and function. We have conditionally inactivated selenoprotein biosynthesis in thyrocytes by targeting Sec tRNA. Constitutive and inducible Cre/loxP-mediated recombination of tRNA([Ser]Sec) drastically reduced activities of selenoenzymes GPx and type I-deiodinase in thyroid extracts. Immunohistochemical staining revealed increased 4-hydroxynonenal and 3-nitro-tyrosine levels consistent with increased oxidative stress. However, gross thyroid morphology remained intact for at least 6 months after recombination. Circulating thyroid hormone levels remained normal in mutant mice, while thyrotropin (TSH) levels were moderately elevated. Challenging mutant mice with low iodine diet increased TSH, but did not lead to destruction of selenoprotein-deficient thyroids. This is the first report probing the assumed physiological roles of selenoproteins in the thyroid using a genetic loss-of-function approach. We conclude that selenoproteins protect thyrocytes from oxidative damage and modulate thyroid hormone biosynthesis, but are not essential for thyrocyte survival.

Molecular Basis of Thyroid Dyshormonogenesis: Genetic Screening in Population-Based Japanese Patients

Inborn errors of thyroid hormone biosynthesis are collectively referred to as thyroid dyshormonogenesis (DH). Seven genes have been implicated in DH, including the dual oxidase 2 gene (DUOX2), the thyroglobulin gene (TG), and the thyroid peroxidase gene (TPO). We aimed to define the prevalence and phenotypic spectrum of DH with single gene mutations. A population-based cohort of 102 patients with permanent congenital hypothyroidism was enrolled. Fourteen were diagnosed as DH and were analyzed for the seven causative genes including DUOX2, TG, and TPO. Several common mutations were screened in the remaining 88 patients. Pathogenicity of single amino acid mutations was verified in vitro. We identified four, five, and two patients with seemingly biallelic mutations in DUOX2, TG, and TPO, respectively. We also found two patients having one heterozygous DUOX2 mutation and one uncommon single-nucleotide polymorphism (SNP) p.H678R (rs57659670, allele frequency 0.035) and another two patients with homozygous p.H678R. Expression experiments and RT-PCR revealed that p.H678R is a functional SNP with theoretical 40% loss of function, supporting a role of p.H678R in the onset of DH. As for clinical phenotypes, patients with inactive DUOX2 alleles (mutations and/or p.H678R) showed characteristic time-dependent improvement of thyroid function and morphology. All three evaluated patients had a negative result in the perchlorate test. Mutations (or a functional SNP) in DUOX2, TG, or TPO were observed in 93% (95% confidence interval = 70-99%) of DH patients. Inactive DUOX2 alleles cause a broader phenotypic spectrum than currently accepted.

N-vinylpyrrolidone dimer, a novel formulation excipient, causes hepatic and thyroid hypertrophy through the induction of hepatic microsomal enzymes in rats

N-vinylpyrrolidone dimer (VPD) is a novel experimental formulation excipient intended for preclinical toxicology studies. In a previous 4-week toxicity study, VPD induced dose-dependent hepatocellular and thyroid gland hypertrophy in Sprague-Dawley (SD) rats. The objectives of the current investigation were to define the underlying molecular mechanisms of these changes. Two separate studies were conducted using male SD rats, daily doses of 300, 1000 or 3000 mg/kg of VPD, and a positive control (phenobarbital at 75 mg/kg/day): (1) a 28-day study to monitor thyroid hormone levels after 7 and 28 days of dosing; (2) a 5-day study to evaluate hepatic and thyroid gland transcriptomic changes, as well as hepatic UGT activity levels. At VPD dosages of 300 mg/kg/day and higher, 2-fold increases of serum thyroid stimulating hormone (TSH) levels were observed in male SD rats after 28 days of dosing, while serum thyroxine (T4) and triiodothyronine (T3) levels were unchanged. Liver UGT enzyme activity levels were increased in VPD-treated rats after 5 days. In addition, in the 5-day study, VPD caused increased hepatic mRNA levels of a panel of drug metabolizing enzymes (DMEs) and transporters, including Cyp3a1, Cyp2b1, Ugt 2b1, and Abcc3. Similar patterns of induction were observed in primary rat hepatocytes exposed to VPD. Transcriptomic changes in the thyroid gland were identified for genes involved in thyroid hormone biosynthesis and in the FAK, PTEN, and Wnt/β-catenin signaling pathways. Collectively, these data indicate that VPD acts as an inducer of hepatic DMEs in SD rats and that this likely leads to enhanced peripheral metabolism of T3/T4, resulting in a feedback response characterized by increased serum TSH levels, and thyroid gland hypertrophy and hyperplasia.

Mechanism of anion selectivity and stoichiometry of the Na + /I - symporter (NIS)

I(-) uptake in the thyroid, the first step in thyroid hormone biosynthesis, is mediated by the Na(+)/I(-) symporter (NIS) with an electrogenic 2Na(+):1I(-) stoichiometry. We have obtained mechanistic information on NIS by characterizing the congenital I(-) transport defect-causing NIS mutant G93R. This mutant is targeted to the plasma membrane but is inactive. Substitutions at position 93 show that the longer the side chain of the neutral residue at this position, the higher the K(m) for the anion substrates. Unlike WT NIS, which mediates symport of Na(+) and the environmental pollutant perchlorate electroneutrally, G93T/N/Q/E/D NIS, strikingly, do it electrogenically with a 21 stoichiometry. Furthermore, G93E/Q NIS discriminate between anion substrates, a discovery with potential clinical relevance. A 3D homology model of NIS based on the structure of the bacterial Na(+)/galactose transporter identifies G93 as a critical player in the mechanism of the transporter: the changes from an outwardly to an inwardly open conformation during the transport cycle use G93 as a pivot.

Identification of Classifiers for Increase or Decrease of Thyroid Peroxidase Activity in the FTC-238/hTPO Recombinant Cell Line

Thyroid peroxidase (TPO) plays an important role in thyroid hormone biosynthesis, as it catalyzes all of the essential steps in iodide organification. TPO activity can be detected using the guaiacol assay; however, this assay is complex and very time-consuming. Therefore, we focused on devising a simplified method using microarrays to detect changes in TPO activity, which is a target for disruption of the thyroid hormone axis. These experiments have systematically assessed the potential utility of transcriptomic end points as enhancements to the guaiacol assay. Previously, we demonstrated that benzophenone-2, benzophenone, perfluorooctane sulfonate, bisphenol A bis ether, and vinclozolin decreased TPO activity, and that dibutyl phthalate, carbaryl, dibenzo(a,h)anthracene, benzo(a)pyrene, and methylmercury increased TPO activity. In this work, we used human oligonucleotide chips to examine changes in the gene expression profile of FTC-238 human follicular thyroid carcinoma cells expressing human recombinant TPO, after exposure of the cells to TPO activity-disrupting agents. We identified 362 classifiers that could predict the effect of the toxicants on TPO activity with about 70% accuracy. These classifiers are potential markers for predicting the effects of chemicals on thyroid hormone production.

KCNE2 forms potassium channels with KCNA3 and KCNQ1 in the choroid plexus epithelium

Cerebrospinal fluid (CSF) is crucial for normal function and mechanical protection of the CNS. The choroid plexus epithelium (CPe) is primarily responsible for secreting CSF and regulating its composition by mechanisms currently not fully understood. Previously, the heteromeric KCNQ1-KCNE2 K(+) channel was functionally linked to epithelial processes including gastric acid secretion and thyroid hormone biosynthesis. Here, using Kcne2(-/-) tissue as a negative control, we found cerebral expression of KCNE2 to be markedly enriched in the CPe apical membrane, where we also discovered expression of KCNQ1. Targeted Kcne2 gene deletion in C57B6 mice increased CPe outward K(+) current 2-fold. The Kcne2 deletion-enhanced portion of the current was inhibited by XE991 (10 μM) and margatoxin (10 μM) but not by dendrotoxin (100 nM), indicating that it arose from augmentation of KCNQ subfamily and KCNA3 but not KCNA1 K(+) channel activity. Kcne2 deletion in C57B6 mice also altered the polarity of CPe KCNQ1 and KCNA3 trafficking, hyperpolarized the CPe membrane by 9 ± 2 mV, and increased CSF [Cl(-)] by 14% compared with wild-type mice. These findings constitute the first report of CPe dysfunction caused by cation channel gene disruption and suggest that KCNE2 influences blood-CSF anion flux by regulating KCNQ1 and KCNA3 in the CPe.

Molecules important for thyroid hormone synthesis and action - known facts and future perspectives

Thyroid hormones are of crucial importance for the functioning of nearly every organ. Remarkably, disturbances of thyroid hormone synthesis and function are among the most common endocrine disorders affecting approximately one third of the working German population. Over the last ten years our understanding of biosynthesis and functioning of these hormones has increased tremendously. This includes the identification of proteins involved in thyroid hormone biosynthesis like Thox2 and Dehal where mutations in these genes are responsible for certain degrees of hypothyroidism. One of the most important findings was the identification of a specific transporter for triiodothyronine (T3), the monocarboxylate transporter 8 (MCT8) responsible for directed transport of T3 into target cells and for export of thyroid hormones out of thyroid epithelial cells. Genetic disturbances of MCT8 in patients result in a biochemical constellation of high T3 levels in combination with low or normal TSH and thyroxine levels leading to a new syndrome of severe X-linked mental retardation. Importantly mice lacking MCT8 presented only with a mild phenotype, indicating that compensatory mechanisms exist in mice. Moreover, it has become clear that not only genomic actions of T3 exist. T3 is also capable to activate adhesion receptors and it signals via activation of PI3K and MAPK pathways. Most recently, thyroid hormone derivatives were identified, the thyronamines which are decarboxylated thyroid hormones initiating physiological actions like lowering body temperature and heart rate, thereby acting in opposite direction to the classical thyroid hormones. So far it is believed that thyronamines function via the activation of a G-protein coupled receptor, TAAR1. The objective of this review is to summarise the recent findings in thyroid hormone synthesis and action and to discuss their implications for diagnosis of thyroid disease and for treatment of patients.

Increasing Incidence of Congenital Hypothyroidism: Some Answers, More Questions

In 2007, Harris and Pass (1) reported that the incidence (birth prevalence) of congenital hypothyroidism (CH) detected by newborn screening programs in the United States had nearly doubled over the previous two decades, increasing from 1:3985 (in 1987) to 1:2273 (in 2002). In an analysis of data from their state of New York, Harris and Pass (1) found an even higher incidence, increasing from 1:3373 (in 1978) to 1:1415 (in 2005). The report of an apparent doubling was not limited to the United States. Newborn screening programs in Western Australia (1: 5747 to 1:2825) (2), Italy (1:2654 to 1:1154) (3), the northern region of the United Kingdom (1:2702 to 1:1078) (4), and Greece (1:3384 to 1:1749) (5) also reported an increasing incidence of CH. After these reports, questions naturally arose. Was this increasing incidence real, signaling an increase in the underlying etiology(ies) of CH? Or was the increasing incidence an artifact, explained by modifications of screening programs such as a change in test cutoffs? Furthermore, did the additional babies benefit from detection and early treatment? What do we know about the etiology of CH? CH is a heterogeneous disorder, with a spectrum of underlying etiologies. The most common cause is thyroid dysgenesis (ectopy, aplasia, hypoplasia, or hemiagenesis); historically, dysgenesis accounts for approximately 85% of cases. The next most common cause is thyroid dyshormonogenesis (defects in thyroid hormone biosynthesis), accounting for 10–15% of cases. Defects in thyroid hormone biosynthesis are familial, generally inherited in an autosomal recessive manner. Determination of an underlying defect is generally made by an imaging study, either ultrasonography or thyroid scintigraphy. Although imaging can determine thyroid anatomy and, if radionuclide uptake is done, some information on function, it does not elucidate the exact etiology. In fact, the underlying etiology of thyroid dysgenesis remains unknown for the vast majority of cases. Mutations in transcription factors that regulate thyroid gland development [thyroid transcription factor 2 (TTF-2), NKX2.1 (also termed TTF-1), or PAX-8] are found in only 2% of cases shown to have thyroid dysgenesis by imaging (6). Most cases of thyroid dysgenesis appear to be sporadic. It has long been known that there is a 2:1 female-to-male ratio in thyroid dysgenesis. Although this would seem to be a clue to the underlying etiology, its significance remains unknown. Previous studies from the screening program in Quebec report that the incidence of thyroid dysgenesis has remained relatively stable over the last few decades (7). Of note, with reports of increasing incidence, imaging studies now find a thyroid problem not reported in older studies—a normal-appearing thyroid gland in a eutopic location, so-called “thyroid in situ” (3, 5). Because this is associated with hypothyroidism, thyroid in situ is thought to be the result of a “functional abnormality.” Some cases of thyroid in situ have been shown to be the result of resistance to TSH binding (TSH receptor mutation), or signaling (G protein defect) (8), or to mild forms of dyshormonogenesis (hydrogen peroxide generation or maturation defects) (9). However, the exact etiology remains unknown for most cases. Hypothyroidism tends to be milder with thyroid in situ, and some cases are transient, with recovery to euthyroidism (9). Other cases of transient CH have a known etiology. Examples include babies born to mothers with Graves’ disease on antithyroid drugs, babies exposed to excess iodine (either maternal transmission or postnatal, usually from topical iodine antiseptics), and babies born to mothers with autoimmune thyroid disease

Genetic causes of congenital hypothyroidism due to dyshormonogenesis

Overview of congenital hypothyroidism caused by thyroid hormone synthesis defects, the current understanding of their pathophysiology, and clinical implications of molecular diagnoses. Genetic defects in all known thyroid-specific factors required for thyroid hormone synthesis have been described. These include defects in iodide trapping (NIS), in the facilitated iodide efflux across the apical membrane (PDS), the organification of iodide within the follicular lumen (thyroid peroxidase, DUOX2, DUOXA2), the substrate for thyroid hormone synthesis (thyroglobulin) and the ability to recover and retain intrathyroidal iodine (iodotyrosine deiodinase). Clinical and biochemical evaluation aids in selecting the most appropriate candidate gene(s). A definite molecular diagnosis of thyroid dyshormonogenesis allows genetic counseling and has prognostic value in differentiating transient from permanent congenital hypothyroidism and predicting the response of patients to iodine supplementation as adjunct or alternative treatment to L-T4 replacement. Congenital hypothyroidism due to thyroid dyshormonogenesis is a heterogenic disorder that may be caused by mutations in any of the known steps in the thyroid hormone biosynthesis pathway. An exact molecular diagnosis allows genetic counseling and the identification of asymptomatic mutation carriers at risk of recurrent hypothyroidism, and provides a rationale for adjunct iodide supplementation.

Vascular peroxidase-1 is rapidly secreted, circulates in plasma, and supports dityrosine cross-linking reactions

Members of the peroxidase–cyclooxygenase superfamily catalyze biochemical reactions essential to a broad spectrum of biological processes, including host defense, thyroid hormone biosynthesis, and modification of extracellular matrix, as well as contributing to the pathogenesis of chronic inflammatory diseases. We recently identified a novel member of this family, vascular peroxidase-1 (VPO1), that is highly expressed in the human cardiovascular system. Its biosynthesis and enzymatic properties are largely unknown. Here, we report that VPO1 was rapidly and efficiently secreted into the extracellular space when the gene was stably expressed in human embryonic kidney (HEK) cells. Secreted VPO1 is a monomer with complex N-linked oligosaccharides and exhibits peroxidase activity. Biosynthesis of endogenous VPO1 by cultured human umbilical vein endothelial cells (HUVECs) shares features exhibited by heterologous expression of recombinant VPO1 (rVPO1) in HEK cells. The proinflammatory agents lipopolysaccharide and tumor necrosis factor-α induce expression of VPO1 mRNA and protein in HUVECs. Furthermore, murine and bovine sera and human plasma contain enzymatically active VPO1. rVPO1 exhibits spectral and enzymatic properties characteristic of the peroxidase–cyclooxygenase family, except with regard to its heat stability. rVPO1 catalyzes tyrosyl radical formation and promotes dityrosine cross-linking. Taken together, these data demonstrate that VPO1 is a glycosylated heme peroxidase that is actively secreted into circulating plasma by vascular endothelial cells and shares several features with other members of the peroxidase–cyclooxygenase family, including the catalysis of dityrosine formation.

Structural stability and heme binding potential of the truncated human dual oxidase 2 (DUOX2) peroxidase domain

The essential role of human dual oxidase 2 (hDUOX2) in thyroid hormone biosynthesis defines this member of the NOX/DUOX family, whose absence due to mutation has been directly related to disease, specifically hypothyroidism. Both human DUOX isoforms, hDUOX1 and hDUOX2, are expressed in thyroid tissue; however, hDUOX1 cannot compensate for inactivation of hDUOX2, suggesting that each enzyme is differentially regulated and/or functions in a unique manner. In efforts to uncover relevant structural and functional differences we have expressed and purified the peroxidase domain of hDUOX21–599 for direct comparison with the previously studied hDUOX11–593. As was shown for hDUOX1, the truncated hDUOX2 domain purifies without a bound heme co-factor and displays no peroxidase activity. However, hDUOX21–599 displays greater stability than hDUOX11–593. Surprisingly, upon titration with heme, both isoforms bind heme with a low micromolar affinity, demonstrating that they retain a heme binding site. A conformational difference in the full-length protein and/or a protein–protein interaction may be required to increase the heme binding affinity.