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Graves' disease (GD), an autoimmune thyroid disease, is one of the main autoimmune diseases in the general population. It is known that the pathophysiology of this disease may be related to immunological mechanisms dysregulation. These mechanisms can be influenced by GD therapies, such as iodide or antithyroid drugs (ATD)., Objective: Verify relation between clinical, biochemical and treatment modalities used prior to surgery and histopathological characteristics observed in total thyroidectomy products from patients previously diagnosed with Graves' disease. Furthermore, these data were related to composition of lymphocytic infiltrate in terms of proportions of lymphocytes CD4 + , CD8 + , CD25 + and CD20 + . We aim to contribute to the understanding of the evolution pattern of GD, whose pathophysiology is not yet completely understood., Methods: Cross-sectional study assessing thyroidectomy products for the presence of lymphocytic infiltrate, as well as the proportion and intensity of CD4 + , CD8 + , CD25 + and CD20 + markers. We selected 50 patients who underwent total or partial thyroidectomy in a tertiary service between 1996 and 2013 due to GD with histopathological confirmation. The control group (non-autoimmune disease group) consisted of 12 patients with histopathological data compatible with normal perilesional thyroid parenchyma. The intensity of lymphocytic infiltrate and immunohistochemical expression of the markers CD4 + (helper T lymphocytes), CD8 + (cytotoxic T lymphocytes), CD25 + (regulatory T lymphocytes) and CD20 + (B lymphocytes) were retrospectively evaluated and relationship with ultrasound, laboratory and clinical data was assessed., Results: No differences were found in intensity, presence of lymphoid follicles, and expression of CD4+/CD8+/CD25+ in patients with GD who did or did not use ATD or iodide. In the group that did not use ATD, a higher proportion of CD20 + expression was found. The GD group was associated with hyperplastic epithelium and the control group was associated with simple epithelium. There was no difference in ultrasound thyroid volume between the groups. In GD patients with mild lymphocytic infiltrate, higher free thyroxin (FT4) levels were observed than those in patients with no infiltrate or moderate infiltrate., Conclusion: We found a lower proportion of intrathyroidal CD20 + B lymphocytes in patients under use of methimazole. However, no difference was observed in intrathyroidal lymphocyte subpopulations related to the short-term use of iodide. The understanding of thyroid autoimmunity, as well as identifying points of pharmacological modulation, are very important for advancement and improvement in treatments for these diseases., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Denise Engelbrecht Zantut Wittmann reports equipment, drugs, or supplies and statistical analysis were provided by State University of Campinas. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A 3D thyroid model was developed to address the limitations of 2D cultures and study the effects of compounds like 3-MNT on dehalogenase 1 (IYD) and metabolic activity. Morphology was assessed by TEM, and the expression of tissue-specific genes (TPO, TSHR, PAX8, TTF-1, NIS, IYD, TG) and metabolic features were analyzed using qRT-PCR, immunofluorescence, western blotting, ELISA, and LC-MS/MS, with and without TSH stimulus and 3-MNT treatment. Confocal and TEM analyses confirmed a follicle-like 3D structure. Expression of TPO, NIS, TG, TSH, and PAX markers was significantly higher (p < 0.05) in 3D versus 2D cultures, and ELISA showed increased TG protein production. 3-MNT treatment inhibited IYD activity, indicated by increased MIT and DIT in the media, and significantly altered (p < 0.05) NIS, TG, IYD, TSHR, and TPO expression. These findings suggest 3D thyroid cultures closely replicate tissue traits and functionality, providing a valuable tool for thyroid research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Tributyltin (TBT) is an endocrine disruptor used as a biocide in nautical paints. Even though many TBT effects in marine species are known, data in mammals are scarce, especially regarding the thyroid gland. The present study aimed to evaluate the effect of a subchronic exposure to TBT on thyroid oxidative stress of female Wistar rats. Rats received vehicle (control group), 200 or 1000 ng TBT/kg body weight/day for 40 days. After euthanasia, one part of the thyroids were collected in order to assess iodide uptake; activity and/or mRNA expression of thyroperoxidase (TPO) and dual oxidases (DUOXs); activity and/or mRNA expression of catalase, glutathione peroxidase, superoxide dismutase and NADPH oxidase 4 (CAT, GPx, SOD and NOX4); 4-hydroxynonenal (4-HNE) expression and total thiol groups levels; and mRNA expression of estrogen receptors alpha and beta (ERα and ERβ). The remaining part of the thyroid was processed for morphological analysis of estrogen receptor alpha (ERα) and for collagen deposition. Iodide uptake was not changed with treatments. TPO activity and expression were increased in the TBT1000 group (259.81% and 95.17%). The activity, but not mRNA, of CAT (17.36% TBT200; 27.10% TBT1000) and GPx (29.24% TBT200; 28.97% TBT1000) were decreased by TBT. SOD and NADPH oxidase activity, as well as thiol group and 4-HNE levels remained unchanged. Interstitial collagen deposition increased in the TBT200 group (39.54%). The mRNA expression of ERα increased in TBT-treated rats (44.87% TBT200; 36.43% TBT1000), while protein expression was increased but not reaching significance (TBT1000, p = 0.056) by TBT. Therefore, our results show that TBT increases TPO expression and reduces antioxidant enzyme activities in the thyroid gland leading to oxidative stress. Some of these effects could be mediated by the ERα pathway., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Thyroid diseases are more prevalent in women, and this difference seems to be associated with the oxidative stress found in the thyroid of females. Thyroid NADPH Oxidase 4 (NOX4) was shown to respond to estrogen, which can also modulate TGF-β1, a potent stimulator of NOX4. This study aimed to investigate the effects of TGF-β1 on redox homeostasis parameters in the rat thyroid cell PCCL3 and the interrelationship between estrogen and TGF-β1. TGF-β1 treatment increased both intra- and extracellular ROS generation along with NOX4 expression and reduced GPX and catalase activities, extracellular H2O2 scavenging capacity, and reduced thiol content. TGF-β1 mRNA and protein expression are higher in female thyroid glands of rats in comparison to males. Moreover, 17β-estradiol treatment enhanced TGF-β1 mRNA in PCCL3 cells, decreased extracellular bioavailability but did not activate Smad pathway. Our data suggest that higher levels of TGF-β1 in females are potentially related to higher ROS availability which may be associated with the sex disparity in thyroid disorders., (Copyright © 2022 Elsevier B.V. All rights reserved.)

It has long been known that thyroid hormones have implications for multiple physiological processes and can lead to serious illness when there is an imbalance in its metabolism. The connections between thyroid hormone metabolism and the immune system have been extensively described, as they can participate in inflammation, autoimmunity, or cancer progression. In addition, changes in the normal intestinal microbiota involve the activation of the immune system while triggering different pathophysiological disorders. Recent studies have linked the microbiota and certain bacterial fragments or metabolites to the regulation of thyroid hormones and the general response in the endocrine system. Even if the biology and function of the thyroid gland has attracted more attention due to its pathophysiological importance, there are essential mechanisms and issues related to it that are related to the interplay between the intestinal microbiota and the immune system and must be further investigated. Here we summarize additional information to uncover these relationships, the knowledge of which would help establish new personalized medical strategies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Thyroid cancer is the most frequent cancer of the endocrine system. Most patients are treated with thyroidectomy followed by radioiodine therapy. However, in part of the patients, a reduction of the sodium-iodide symporter (NIS) occurs, rendering radioiodine therapy ineffective. Moreover, epithelial-mesenchymal transition (EMT) may occur, leading to more aggressive and invasive features. Herein, we evaluated the effect of the flavonoid quercetin on EMT and NIS expression in BCPAP, a papillary thyroid carcinoma cell line. BCPAP was treated with 100 μM quercetin for 24 h and cell viability, apoptosis, EMT markers and NIS were evaluated. Quercetin decreased cell viability by enhancing apoptosis. The flavonoid also reduced matrix metalloproteinase 9 and increased E-cadherin mRNA levels, inhibiting BCPAP adhesion and migration. Additionally, quercetin increased NIS expression and function. Thus, our results suggest that quercetin could be useful as adjuvant in thyroid cancer therapy, inducing apoptosis, reducing invasion and increasing the efficacy of radioiodine therapy., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In rats, maternal nicotine exposure during lactation induces obesity, thyroid dysfunction, brown adipose tissue (BAT) hypofunction and liver alterations in adult offspring. Both thyroid function and lipid metabolism are influenced by gene silencing mediated by microRNAs (miRNAs). Here we investigated long-term effects of early nicotine exposure on molecular and epigenetic mechanisms closely related to thyroid and lipid metabolism, through the expression of mRNAs and miRNAs in BAT and liver of adult male and female offspring. At postnatal day 2 (PND2), lactating control (CON) or nicotine (NIC) dams were subcutaneously implanted with osmotic minipumps containing, respectively, saline or 6 mg/kg nicotine. Litters were adjusted to 3 males and 3 females. Offspring's euthanasia occurred at PND180. In the BAT, NIC females showed higher Dio2 mRNA expression, while miR-382* expression was not altered in both sexes. In the liver, NIC offspring of both sexes showed lower Dio1 mRNA expression and higher miR-224 expression, while only NIC females had higher miR-383 and miR-21 expressions. NIC offspring of both sexes showed higher mRNA expression of SCD1 in the liver; NIC males had decreased CPT1 expression, whereas NIC females had increased FASN, miR-370 and miR-122 expressions. Regardless of sex, alterations in liver Dio1, miR-224 and SCD1 expressions are involved in the disturbances caused by maternal nicotine exposure during breastfeeding. Interestingly, females had more altered miRs in the liver. Early nicotine exposure induces a sex dimorphism, particularly regarding hepatic lipid metabolism, through miRs expression., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Poly- and perfluoroalkyl substances (PFAS) are a class of endocrine disrupting chemicals (EDCs) reported to alter thyroid function. Iodide uptake by thyroid follicular cells, an early step in the synthesis of thyroid hormones, is a potential target for thyroid disruption by EDCs. The aim of the present study was to evaluate the acute effects of perfluorooctane sulfonic acid (PFOS) and perfluorooctane carboxylic acid (PFOA), two of the most abundant PFAS in the environment, on iodide transport by thyroid follicular cells in vitro. Dynamic changes in intracellular iodide concentration were monitored by live cell imaging using YFP-H148Q/I152, a genetically encoded fluorescent iodide biosensor. PFOS, but not PFOA, acutely and reversibly inhibited iodide accumulation by FRTL-5 thyrocytes, as well as by HEK-293 cells transiently expressing the Sodium Iodide Symporter (NIS). PFOS prevented NIS-mediated iodide uptake and reduced intracellular iodide concentration in iodide-containing cells, mimicking the effect of the NIS inhibitor perchlorate. PFOS did not affect iodide efflux from thyroid cells. The results of this study suggest that disruption of iodide homeostasis in thyroid cells may be a potential mechanism for anti-thyroid health effects of PFOS. The study also confirms the utility of the YFP-H148Q/I152 cell-based assay to screen environmental PFAS, and other EDCs, for anti-thyroid activity., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Organotin compounds, such as tributyltin (TBT), are common environmental contaminants and suspected endocrine-disrupting chemicals. Tributyltin is found in antifouling paints, widely used in ships and other vessels. The present study evaluated whether a 15-day treatment with TBT at a dose of 100 ng/kg/day could induce histomorphological changes in the thyroid gland of rats. TBT promoted relevant alterations in the thyroid architecture, being the most relevant histological findings the presence of increased number of small-size follicles in the treated group. In qualitative analyses, colloid vacuolization, papillary budging structures, cystic degeneration and chronic thyroiditis, were observed. Moreover, histomorphometric analysis showed statistically significant changes in the follicular architecture of TBT-treated rats, mainly a decrease in the follicle area (colloid) and an increased epithelial height that resulted in an increased epithelial height/colloid ratio. Augmented collagen deposition was also seen in the thyroids of treated groups. In immunohistochemical (IHC) analyses, the localization of NIS protein was described and a significant increased proliferation index (evaluated by Ki67 positive cells) in the treated group was reported. As an indirect measurement of oxidative stress, mitochondrial protein SDHA was also analyzed by IHC analysis. Although the cytoplasmic expression of SDHA was observed in both groups, the staining intensity score was higher in TBT-treated group. Our results suggest that besides causing histomorphological changes, environmental relevant dose of TBT treatment can also induce oxidative alterations., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Primary congenital hypothyroidism (CH) is the most common endocrine disease in children and one of the most common preventable causes of both cognitive and motor deficits. CH is a heterogeneous group of thyroid disorders in which inadequate production of thyroid hormone occurs due to defects in proteins involved in the gland organogenesis (dysembryogenesis) or in multiple steps of thyroid hormone biosynthesis (dyshormonogenesis). Dysembryogenesis is associated with genes responsible for the development or growth of thyroid cells: such as NKX2-1, FOXE1, PAX8, NKX2-5, TSHR, TBX1, CDCA8, HOXD3 and HOXB3 resulting in agenesis, hypoplasia or ectopia of thyroid gland. Nevertheless, the etiology of the dysembryogenesis remains unknown for most cases. In contrast, the majority of patients with dyshormonogenesis has been linked to mutations in the SLC5A5, SLC26A4, SLC26A7, TPO, DUOX1, DUOX2, DUOXA1, DUOXA2, IYD or TG genes, which usually originate goiter. About 800 genetic mutations have been reported to cause CH in patients so far, including missense, nonsense, in-frame deletion and splice-site variations. Many of these mutations are implicated in specific domains, cysteine residues or glycosylation sites, affecting the maturation of nascent proteins that go through the secretory pathway. Consequently, misfolded proteins are permanently entrapped in the endoplasmic reticulum (ER) and are translocated to the cytosol for proteasomal degradation by the ER-associated degradation (ERAD) machinery. Despite of all these remarkable advances in the field of the CH pathogenesis, several points on the development of this disease remain to be elucidated. The continuous study of thyroid gene mutations with the application of new technologies will be useful for the understanding of the intrinsic mechanisms related to CH. In this review we summarize the present status of knowledge on the disorders in the protein folding caused by thyroid genes mutations., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Approximately 20% of Graves' disease (GD) patients may result eventually in hypothyroidism in their natural course. Uterus globulin-associated protein 1 (UGRP1) was associated with GD in our previous study. Here we investigated the role of UGRP1 in the development of autoimmune thyroid disease (AITD). The results showed that UGRP1 was expressed in the thyrocytes of most Hashimoto's thyroiditis (HT) patients and a proportion of GD patients (293 HT and 198 GD). The pathologic features of UGRP1-positive thyrocytes resembled "Hürthle cells", and were surrounded by infiltrated leukocytes. The positivity rate of TPOAb in UGRP1-positive GD patients was much higher than that in -negative GD patients. Moreover, UGRP1 was co-expressed with Fas and HLA-DR in the thyrocytes of AITD patients. We also found IL-1β but not Th1 or Th2 cytokines was able to upregulate the expression of UGRP1. Our findings indicated that UGRP1 may be a novel marker in thyrocytes to predict GD patients who develop hypothyroidism., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In this study, we used an experimental model of congenital hypothyroidism to show that deficient thyroid hormones (TH) disrupt different neurochemical, morphological and functional aspects in the cerebral cortex of 15-day-old offspring. Our results showing decreased glutamine synthetase (GS) activity and Ca 2+ overload in the cerebral cortex of hypothyroid pups suggest misregulated glutamate metabolism associated with developmentally induced TH deficiency. The 14 C-MeAIB accumulation indicates upregulated System A activity and glutamine uptake by neurons. Energy metabolism in hypothyroid cortical slices was preserved, as demonstrated by unaltered glucose metabolism. We also found upregulated acetylcholinesterase activity, depleting acetylcholine from the synaptic cleft, pointing to disrupted cholinergic system. Increased reactive oxygen species (ROS) generation, lipid peroxidation, glutathione (GSH) depletion, which were associated with glutathione peroxidase, superoxide dismutase and gamma-glutamyltransferase downregulation suggest redox imbalance. Disrupted astrocyte cytoskeleton was evidenced by downregulated and hyperphosphorylated glial fibrillary acidic protein (GFAP). Morphological and structural characterization of the sensorimotor cerebral cortex (SCC) showed unaltered thickness of the SCC. However, decreased size of neurons on the layers II & III and IV in the right SCC and increased NeuN positive neurons in specific SCC layers, suggest that they are differently affected by the low TH levels during neurodevelopment. Hypothyroid pups presented increased number of foot-faults in the gridwalk test indicating affected motor functions. Taken together, our results show that congenital hypothyroidism disrupts glutamatergic and cholinergic neurotransmission, Ca 2+ equilibrium, redox balance, cytoskeleton integrity, morphological and functional aspects in the cerebral cortex of young rats., (Copyright © 2018. Published by Elsevier B.V.)

Graves' disease (GD) is a common autoimmune thyroid disease characterized by positive thyroid stimulating hormone receptor antibody. To better understand its molecular pathogenesis, we adopted the weighted gene co-expression network analysis to reveal co-expression modules of key genes involved in the pathogenesis of GD, protein-protein interaction network analysis to identify the hub genes related to GD development and functional analyses to explore their possible functions. Our results showed that 1) a total of 2667 differentially expressed genes in our microarray study and 16 different gene co-expression modules were associated with GD, and 2) the most significant module was associated with the percentage of macrophages, T follicular helper cells and CD4 + memory T cells and mainly enriched in immune regulation and immune response. Overall, our study reveals several key gene co-expression modules and functional pathways involved in GD, which provides some novel insights into the pathogenesis of GD., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: A single dose of potassium iodide (KI) is recommended to reduce the risk of thyroid cancer during nuclear accidents. However in case of prolonged radioiodine exposure, more than one dose of KI may be necessary. This work aims to evaluate the potential toxic effect of repeated administration of KI., Methods: Adult Wistar rats received an optimal dose of KI 1 mg/kg over a period of 1, 4 or 8 days., Results: hormonal status (TSH, FT4) of treated rats was unaffected. Contrariwise, a sequential Wolff-Chaikoff effect was observed, resulting in a prompt decrease of NIS and MCT8 mRNA expression (-58% and -26% respectively), followed by a delayed decrease of TPO mRNA expression (-33%) in conjunction with a stimulation of PDS mRNA expression (+62%)., Conclusion: we show for the first time that repeated administration of KI at 1 mg/kg/24h doesn't cause modification of thyroid hormones level, but leads to a reversible modification of the expression of genes involved in the synthesis and secretion of thyroid hormones., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction: Transforming growth factor beta (TGF-β) regulates thyroid function and growth. However, tumoral thyroid cells became resistant to this factor as they undifferentiated. Little is known about the effects of TGF-β isoforms. We compared the role of redox metabolism in the response to TGF-β isoforms between non tumoral and tumoral thyroid cells., Methodology and Results: Differentiated rat thyroid cells (FRTL-5) and human thyroid follicular carcinoma cells (WRO) were treated with the three isoforms of TGF-β. TGF-β isoforms stopped cell cycle at different steps; G1 for FRTL-5 and G2/M for WRO. The three isoforms decreased cell viability and increased ROS accumulation in both cell lines. These effects were more pronounced in FRTL-5 than in WRO, and the isoform β1 was more potent in ROS production than the other two. TGF-β isoforms decreased total glutathione, catalase expression and it activity in both cell lines. Only in FRTL-5 the lipid peroxidation was demonstrated. Moreover, TGF-β1 decreased glutathione peroxidase and mitochondrial superoxide dismutase mRNA expression and increased mitochondrial ROS in FRTL-5, but no in WRO. Pretreatment with selenium increased glutathione peroxidase activity and decreased ROS production in WRO treated with TGF-β isoforms. Furthermore, selenium partially reversed the effect of TGF-β isoforms on cell viability only in WRO cells. The knockdown of endogenous NOX4 significantly reduced the TGF-β1 effect on cell viability in WRO but no in FRTL-5., Conclusion: TGF-β disrupted the redox balance and increased ROS accumulation in both cell lines. FRTL-5 cells showed reduced antioxidant capacity and had a greater sensitivity to TGF-β isoforms, while WRO cells were more resistant. This observation provides new insights into the potential role of TGF-β in the redox regulation of thyroid cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Much research has focused on vertebrate thyroid hormone (TH) synthesis and their function in development and metabolism. While important differences in TH synthesis and signaling exist, comparative studies between vertebrates fail to explain the evolutionary origins of this important regulatory axis. For that, one needs to make sense out of the diverse TH effects which have been described in invertebrate phyla but for which a mechanistic understanding is largely missing. Almost every major group of non-vertebrate animals possesses the capability to synthesize and metabolize thyroid hormones and there is evidence for a nuclear thyroid hormone receptor mediated mechanism in the bilateria, especially in molluscs, echinoderms, cephalochordates and ascidians. Still, genomic pathways cannot fully explain many observed effects of thyroid hormones in groups such as cnidarians, molluscs, and echinoderms and it is therefore possible that TH may signal via other mechanisms, such as non-genomic signaling systems via membrane bound or cytoplasmic receptors. Here we provide a brief review of TH actions in selected invertebrate species and discuss the hypothesis that non-genomic TH action may have played a critical role in TH signaling throughout animal evolution., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Thyroxine (T4) is the major thyroid hormone in the thyroid gland and the circulation. However, it is widely accepted on the basis of abundant evidence that 3,5,3'-triiodothyronine (T3) is responsible for most, if not all, of the physiological effects of TH in extrathyroidal tissues, and T4 functions as the pro-hormone. Whether T4 has any intrinsic activity per se or is merely a pro-hormone that must be converted to T3 in order to exert any TH action has yet to be resolved. Although there are some physiological actions of T4 that are mediated by receptors at the cell membrane (non-genomic effects), the vast majority of the physiological effects of the THs identified to date involve the binding of T3 to specific nuclear receptors to regulate gene expression (genomic effects). This review examines how the role of T4 in genomic TH action has been viewed and debated during the hundred years since it was first isolated in 1914., (Copyright © 2017 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that modulate the vast majority of cellular processes. During development, the correct timing and expression of miRNAs in the tissue differentiation is essential for organogenesis and functionality. In thyroid gland, DICER and miRNAs are necessary for accurately establishing thyroid follicles and hormone synthesis. Moreover, DICER1 mutations and miRNA deregulation observed in human goiter influence thyroid tumorigenesis. The thyroid malignant transformation by MAPK oncogenes is accompanied by global miRNA changes, with a marked reduction of "tumor-suppressor" miRNAs and activation of oncogenic miRNAs. Loss of thyroid cell differentiation/function, and consequently iodine trapping impairment, is an important clinical characteristic of radioiodine-refractory thyroid cancer. However, few studies have addressed the direct role of miRNAs in thyroid gland physiology. Here, we focus on what we have learned in the thyroid follicular cell differentiation and function as revealed by cell and animal models and miRNA modulation in thyroid tumorigenesis., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Iodide Organification defects (IOD) represent 10% of cases of congenital hypothyroidism (CH) being the main genes affected that of TPO (thyroid peroxidase) and DUOX2 (dual oxidasa 2). From a patient with clinical and biochemical criteria suggestive with CH associated with IOD, TPO and DUOX2 genes were analyzed by means of PCR-Single Strand Conformation Polymorphism analysis and sequencing. A novel heterozygous compound to the mutations c.2335-1G>C (paternal mutation, intron 17) and c.3264&#95;3267delCAGC (maternal mutation, exon 24) was identified in the DUOX2 gene. Ex-vivo splicing assays and subsequent RT-PCR and sequencing analyses were performed on mRNA isolated from the HeLa cells transfected with wild-type and mutant pSPL3 expression vectors. The wild-type and c.2335-1G>C mutant alleles result in the complete inclusion or exclusion of exon 18, or in the activation of an exonic cryptic 5' ss with the consequent deletion of 169 bp at the end of this exon. However, we observed only a band of the expected size in normal thyroid tissue by RT-PCR. Additionally, the c.2335-1G>C mutation activates an unusual cryptic donor splice site in intron 17, located at position -14 of the authentic intron 17/exon 18 junction site, with an insertion of the last 14 nucleotides of the intron 17 in mutant transcripts with complete and partial inclusion of exon 18. The theoretical consequences of splice site mutation, predicted with the bioinformatics NNSplice, Fsplice, SPL, SPLM and MaxEntScan programs were investigated and evaluated in relation with the experimental evidence. These analyses confirm that c.2335-1G>C mutant allele would result in the abolition of the authentic splice acceptor site. The results suggest the coexistence in our patient of four putative truncated proteins of 786, 805, 806 and 1105 amino acids, with conservation of peroxidase-like domain and loss of gp91(phox)/NOX2-like domain. In conclusion a novel heterozygous compound was identified being responsible of IOD. Cryptic splicing sites have been characterized in DUOX2 gene for the first time. The use of molecular biology techniques is a valuable tool for understanding the molecular pathophysiology of this type of thyroid defects., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Anuran and flatfish metamorphosis are tightly regulated by thyroid hormones that are the necessary and sufficient factors that drive this developmental event. In the present study whole mount in situ hybridization (WISH) and quantitative PCR in sole are used to explore the central regulation of flatfish metamorphosis. Central regulation of the thyroid in vertebrates is mediated by the hypothalamus-pituitary-thyroid (HPT) axis. Teleosts diverge from other vertebrates as hypothalamic regulation in the HPT axis is proposed to be through hypothalamic inhibition although the regulatory factor remains enigmatic. The dynamics of the HPT axis during sole metamorphosis revealed integration between the activity of the thyrotrophes in the pituitary and the thyroid follicles. No evidence was found supporting a role for thyroid releasing hormone (trh) or corticotrophin releasing hormone (crh) in hypothalamic control of TH production during sole metamorphosis. Intriguingly the results of the present study suggest that neither hypothalamic trh nor crh expression changes during sole metamorphosis and raises questions about the role of these factors and the hypothalamus in regulation of thyrotrophs., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

We investigated thyroid state effect on capacity of rat liver mitochondria to remove exogenously produced H2O2, determining their ability to decrease fluorescence generated by an H2O2 detector system. The rate of H2O2 removal by both non respiring and respiring mitochondria was increased by hyperthyroidism and decreased by hypothyroidism. However, the rate was higher in the presence of respiratory substrates, in particular pyruvate/malate, indicating a respiration-dependent process. Generally, the changes in H2O2 removal rates mirrored those in H2O2 release rates excluding the possibility that endogenous and exogenous H2O2 competed for the removing system. Pharmacological inhibition revealed thyroid state-linked differences in antioxidant enzyme contribution to H2O2 removal which were consistent with those in antioxidant system activities. The H2O2 removal was only in part due to enzymatic systems and that imputable to non-enzymatic processes was higher in hyperthyroid and lower in hypothyroid mitochondria. The levels of cytochrome c and the light emissions, due to luminol oxidation catalyzed by cytochrome/H2O2, exhibited similar changes with thyroid state supporting the idea that non-enzymatic scavenging was mainly due to hemoprotein action, which produces hydroxyl radicals. Further support was obtained showing that the whole antioxidant capacity, which provides an evaluation of capacity of the systems, different from cytochromes, assigned to H2O2 scavenging, was lower in hyperthyroid than in hypothyroid state. In conclusion, our results show that mitochondria from hyperthyroid liver have a high capacity for H2O2 removal, which, however, leading in great part to more reactive oxygen species, results harmful for such organelles., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The transcription factor Pax8 is already known to be essential at very early stages of mouse thyroid gland development, before the onset of thyroid hormone production. In this paper we show, using a conditional inactivation strategy, that the removal of the Pax8 protein late in gland development results in severe hypothyroidism, consequent to a reduced gland size and a deranged differentiation. These results demonstrate that Pax8 is also an essential player in controlling survival and differentiation of adult thyroid follicular cells., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

TSH is the important regulator of thyroid function but detailed molecular mechanisms have not been clarified. We first generated the iodine deficient (ID) rat in which goiter is induced by accelerated endogenous TSH secretion. The result of microarray analysis demonstrated markedly increased levels of adrenomedullin 2/intermedin (AM2/IMD) expression in the ID rat thyroid. AM2/IMD is a potent vasodilator. AM2/IMD mRNA expression was induced by TSH in a rat thyroid follicular cell line FRTL-5. Immunohistochemical analysis in human normal and Graves' disease thyroid revealed that AM2/IMD immunoreactivity was detected in follicular cells and more pronounced in Graves' disease. These results indicated that TSH induced AM2/IMD expression in the rat thyroid gland and it could locally work as a potent vasodilator, resulting in the expansion of thyroid inter-follicular capillaries. AM2/IMD could also contribute to facilitate thyroid hormone synthesis possibly via vasodilation effects and/or cAMP stimulating effects in the human thyroid gland., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Thyroid hormones (TH) have a multiplicity of effects. Early in life, they mainly affect development and differentiation, while later on they have particularly important influences over metabolic processes in almost all tissues. It is now quite widely accepted that thyroid hormones have two types of effects on mitochondria. The first is a rapid stimulation of respiration, which is evident within minutes/hours after hormone treatment, and it is probable that extranuclear/non-genomic mechanisms underlie this effect. The second response occurs one to several days after hormone treatment, and leads to mitochondrial biogenesis and to a change in mitochondrial mass. The hormone signal for the second response involves both T3-responsive nuclear genes and a direct action of T3 at mitochondrial binding sites. T3, by binding to a specific mitochondrial receptor and affecting the transcription apparatus, may thus act in a coordinated manner with the T3 nuclear pathway to regulate mitochondrial biogenesis and turnover. Transcription factors, coactivators, corepressors, signaling pathways and, perhaps, all play roles in these mechanisms. This review article focuses chiefly on TH, but also looks briefly at some analogues and derivatives (on which the data is still somewhat patchy). We summarize data obtained recently and in the past to try to obtain an updated picture of the current research position concerning the metabolic effects of TH, with particular emphasis on those exerted via mitochondria., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

The extracellular-matrix protein laminin forms polymers both in vivo and in vitro. Acidification of pH leads to the formation of an artificial polymer with biomimetic properties, named polylaminin (polyLM). Follicle cells in the thyroid are in close contact with laminin, but their response to this important extracellular signal is still poorly understood. PCCL3 thyroid follicular cells cultured on glass, on regular laminin (LM) or on laminin previously polymerized in acidic pH (polyLM) showed different cell morphologies and propensities to proliferate, as well as differences in the organization of their actin cytoskeleton. On polyLM, cells displayed a typical epithelial morphology and radially organized actin fibers; whereas on LM, they spread irregularly on the substrate, lost cell contacts, and developed thick actin fibers extending through the entire cytoplasm. Iodide uptake decreased similarly in response to both laminin substrates, in comparison to glass. On both the LM and polyLM substrates, the expression of the sodium iodide symporter (NIS) decreased slightly but not significantly. NIS showed dotted immunostaining at the plasma membrane in the cells cultured on glass; on polyLM, NIS was observed mainly in the perinuclear region, and more diffusely throughout the cytoplasm on the LM substrate. Additionally, polyLM specifically favored the maintenance of cell polarity in culture. These findings indicate that PCCL3 cells can discriminate between LM and polyLM and that they respond to the latter by better preserving the phenotype observed in the thyroid tissue., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Mammalian transient receptor potential (TRP) channels are involved in many physiologically important processes. Here, we have studied the significance of the TRPC2 channel in the regulation of rat thyroid FRTL-5 cell proliferation, migration, adhesion and invasion, using stable TRPC2 (shTRPC2) knock-down cells. In the shTRPC2 cells, proliferation was decreased due to a prolonged G1/S cell cycle phase. The tumor suppressor p53 and the cyclin-dependant kinase inhibitors p27 and p21 were upregulated. Cell invasion, adhesion and migration were also attenuated in shTRPC2 cells, probably due to decreased activity of both Rac and calpain, and a decreased secretion and activity of matrix metalloproteinase 2. The attenuated proliferation, migration, invasion and ATP-evoked calcium entry was mimicked by overexpressing a non-conducting, truncated TRPC2 (TRPC2-DN) in wild type cells, and was reversed by overexpression of TRPC2-GFP in shTRPC2 cells. In conclusion, TRPC2 is an important regulator of rat thyroid cell function., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Hyperthyroidism promotes cardiac hypertrophy and the Angiotensin type 1 receptor (AT1R) has been demonstrated to mediate part of this response. Recent studies have uncovered a potentially important role for the microRNAs (miRNAs) in the control of diverse aspects of cardiac function. Then, the objective of the present study was to investigate the action promoted by hyperthyroidism on β-MHC/miR-208b expression and on α-MHC/miR-208a expression, as well as the possible contribution of the AT1R in this event. The findings of this study confirmed that AT1R is a key mediator of the cardiac hypertrophy induced by hyperthyroidism. Additionally, we demonstrated that like β-MHC, miR-208b was down-regulated in the hyperthyroid group. Similarly, like the expression of its host gene, α-MHC, miR-208a expression was up-regulated in response to hyperthyroidism. Finally, our data suggest for the first time that AT1R mediates the hyperthyroidism-induced increase on cardiac miRNA-208a/α-MHC levels, while does not influence on the reduction of miRNA-208b/β-MHC levels., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Thyroid transcription factor-1 (TTF-1) is a key regulator of thyroid development and function. In order to identify the genes whose expression depends on TTF-1 transcriptional activity within the thyrocyte we analyzed the consequence of the functional inactivation of this factor in PCCl3 cells. The expression of a fusion protein composed of the DNA binding domain of TTF-1 and of the strong repressive domain of the engrailed protein resulted in a dramatic loss of epithelial cell morphology and in proliferation arrest. These changes were reversed when the inhibition of endogenous TTF-1 was relieved. No change was observed when a similar fusion protein containing point mutations abolishing DNA binding activity was produced in the cells. Besides the expected down-regulation of expression of the main genes linked to the differentiated thyroid function, we observed a decreased expression of the transcription factors Hhex, Pax 8 and TTF-2 and of E-cadherin. By contrast, both ThOX-1 and DUOXA-1 genes were up-regulated, as well as the ones encoding vimentin and several proteins involved in cell cycle arrest. Our data thus extend the known roles of TTF-1 in thyroid development and in the expression of differentiated function in the adult organ to the control of epithelial morphology and of cell division in mature thyrocytes., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

In recent years, many studies of thyroid-disrupting effects of environmental chemicals have been published. Of special concern is the exposure of pregnant women and infants, as thyroid disruption of the developing organism may have deleterious effects on neurological outcome. Chemicals may exert thyroid effects through a variety of mechanisms of action, and some animal experiments and in vitro studies have focused on elucidating the mode of action of specific chemical compounds. Long-term human studies on effects of environmental chemicals on thyroid related outcomes such as growth and development are still lacking. The human exposure scenario with life long exposure to a vast mixture of chemicals in low doses and the large physiological variation in thyroid hormone levels between individuals render human studies very difficult. However, there is now reasonably firm evidence that PCBs have thyroid-disrupting effects, and there is emerging evidence that also phthalates, bisphenol A, brominated flame retardants and perfluorinated chemicals may have thyroid disrupting properties., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The potential autocrine role of human thyroid vascular endothelial growth factors (VEGFs) was examined using the VEGF receptor (VEGFR) inhibitor, ZM306416HCl. ZM306416HCl reduced VEGFR2 phosphorylation and inhibited endogenous, steady-state levels of p42/44 MAPK phosphorylation. It potently inhibited the secretion of plasminogen activators (PA) and increased (125)I uptake. Cell survival was compromised but rescued with insulin and TSH. Although the EGF receptor remained responsive to challenge by EGF in p42/44 MAPK assays, stimulatory effects of EGF on PA production were prevented by ZM306416HCl and those of protein kinase C stimulator, TPA reduced. In assays of (125)I uptake, ZM306416HCl prevented the inhibitory effects of EGF but not those of TPA. We conclude that autocrine VEGF may modulate thyroid function and that VEGFR inhibition increases iodide uptake and decreases PA production through regulation of p42/44 MAPK phosphorylation. VEGFR inhibition may have effects on thyroid function which may contribute to "off target" effects in clinical trials., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Iodine is a critical element involved in thyroid hormone synthesis. Its efflux into the follicular lumen is thought to occur, in part, through pendrin at the apical membrane of thyrocytes. This study attempted to investigate whether iodide administration affects SLC26A4 mRNA expression in rat thyroid and in PCCl3 cells. Rats and cells were treated or not with NaI from 30 min up to 48 h. One group was concomitantly treated with sodium perchlorate. SLC26A4 mRNA expression was also investigated in PCCl3 cells treated with actinomycin D prior to NaI treatment. Iodide administration significantly increased SLC26A4 mRNA content in both models. The simultaneous administration of NaI and perchlorate, as well as the treatment of PCCl3 cells with actinomycin D prevented this effect, indicating that intracellular iodide is essential for this event, which appears to be triggered by transcriptional mechanisms. These data show that intracellular iodide rapidly upregulates SLC26A4 mRNA expression., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Peroxisome proliferator-activated receptors (PPAR)α have been shown to exert immunomodulatory effects in autoimmune disorders; no study evaluated the effect of PPARα activation in Graves' ophthalmopathy (GO). We show the presence of PPARα, δ and γ in GO fibroblasts and preadipocytes. PPARα activators have a potent inhibitory action on the secretion of CXCL9 and CXCL11 chemokines (induced by IFNγ and TNFα) in fibroblasts and preadipocytes. The potency of the used PPARα agonists was maximum on the secretion of CXCL11 (67% inhibition by fenofibrate) in fibroblasts. The relative potency of the compounds in GO fibroblasts was different with each chemokine. PPARα agonists were stronger inhibitors of CXCL9 and CXCL11 (in GO fibroblasts and preadipocytes) than PPARγ activators. This study first shows that PPARα activators inhibit CXCL9 and CXCL11 chemokines in normal and GO fibroblasts and preadipocytes, suggesting that PPARα may be involved in the modulation of the immune response in GO., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

We used the chick embryo to study the mechanisms regulating intracellular TH availability in developing brain. TH-transporters OATP1C1 and MCT8, and deiodinases D1, D2, and D3 were expressed in a region-specific way, well before the onset of endogenous TH secretion. Between day 4 and 10 of development MCT8 and D2 mRNA levels increased, while OATP1C1 and D3 mRNA levels decreased. D2 and D3 mRNAs were translated into active protein, while no D1 activity was detectable. Injection of THs into the yolk 24h before sampling increased TH levels in the brain and resulted in decreased OATP1C1 and increased MCT8 expression in 4-day-old embryos. A compensatory response in deiodinase activity was only observed at day 8. We conclude that THs are active in the early embryonic brain and TH-transporters and deiodinases can regulate their availability. However, the absence of clear compensatory mechanisms at day 4 makes the brain more vulnerable for changes in maternal TH supply., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Thyronamines (TAM), recently described endogenous signaling molecules, exert metabolic and pharmacological actions partly opposing those of the thyromimetic hormone T(3). TAM biosynthesis from thyroid hormone (TH) precursors requires decarboxylation of the L-alanine side chain and several deiodination steps to convert e.g. L-thyroxine (T(4)) into the most potent 3-T(1)AM. Aromatic L-amino acid decarboxylase (AADC) was proposed to mediate TAM biosynthesis via decarboxylation of TH. This hypothesis was tested by incubating recombinant human AADC, which actively catalyzes dopamine production from DOPA, with several TH. Under all reaction conditions tested, AADC failed to catalyze TH decarboxylation, thus challenging the initial hypothesis. These in vitro observations are supported by detection of 3-T(1)AM in plasma of patients with AADC-deficiency at levels (46 ± 18 nM, n=4) similar to those of healthy controls. Therefore, we propose that the enzymatic decarboxylation needed to form TAM from TH is catalyzed by another unique, perhaps TH-specific, decarboxylase., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Recent strides in circadian biology over the last several decades have allowed researchers new insight into how molecular circadian clocks influence the broader physiology of mammals. Elucidation of transcriptional feedback loops at the heart of endogenous circadian clocks has allowed for a deeper analysis of how timed cellular programs exert effects on multiple endocrine axes. While the full understanding of endogenous clocks is currently incomplete, recent work has re-evaluated prior findings with a new understanding of the involvement of these cellular oscillators, and how they may play a role in constructing rhythmic hormone synthesis, secretion, reception, and metabolism. This review addresses current research into how multiple circadian clocks in the hypothalamus and pituitary receive photic information from oscillators within the hypothalamic suprachiasmatic nucleus (SCN), and how resultant hypophysiotropic and pituitary hormone release is then temporally gated to produce an optimal result at the cognate target tissue. Special emphasis is placed not only on neural communication among the SCN and other hypothalamic nuclei, but also how endogenous clocks within the endocrine hypothalamus and pituitary may modulate local hormone synthesis and secretion in response to SCN cues. Through evaluation of a larger body of research into the impact of circadian biology on endocrinology, we can develop a greater appreciation into the importance of timing in endocrine systems, and how understanding of these endogenous rhythms can aid in constructing appropriate therapeutic treatments for a variety of endocrinopathies., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Levels of numerous hormones vary across the day and night. Such fluctuations are not only attributable to changes in sleep/wakefulness and other behaviors but also to a circadian timing system governed by the suprachiasmatic nucleus of the hypothalamus. Sleep has a strong effect on levels of some hormones such as growth hormone but little effect on others which are more strongly regulated by the circadian timing system (e.g., melatonin). Whereas the exact mechanisms through which sleep affects circulating hormonal levels are poorly understood, more is known about how the circadian timing system influences the secretion of hormones. The suprachiasmatic nucleus exerts its influence on hormones via neuronal and humoral signals but it is now also apparent that peripheral tissues contain circadian clock proteins, similar to those in the suprachiasmatic nucleus, that are also involved in hormone regulation. Under normal circumstances, behaviors and the circadian timing system are synchronized with an optimal phase relationship and consequently hormonal systems are exquisitely regulated. However, many individuals (e.g., shift-workers) frequently and/or chronically undergo circadian misalignment by desynchronizing their sleep/wake and fasting/feeding cycle from the circadian timing system. Recent experiments indicate that circadian misalignment has an adverse effect on metabolic and hormonal factors such as circulating glucose and insulin. Further research is needed to determine the underlying mechanisms that cause the negative effects induced by circadian misalignment. Such research could aid the development of novel countermeasures for circadian misalignment., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

H2O2 generation by dual oxidase (DUOX) at the apex of thyroid cells is the limiting factor in the oxidation of iodide and the synthesis of thyroid hormones. Its characteristics have been investigated using different in vitro models, from the most physiological thyroid slices to the particulate fraction isolated from transfected DUOX expressing CHO cells. Comparison of the models shows that some positive controls are thyroid specific (TSH) or require the substructure of the in vivo cells (MβCD). Other controls apply to all intact cell models such as the stimulation of the PIP(2) phospholipase C pathway by ATP acting on purinergic receptors, the activation of the Gq protein downstream (NaF), or surrogates of the intracellular signals generated by this cascade (phorbol esters for protein kinase C, Ca(++) ionophore for Ca(++)). Still, other controls, exerted by intracellular Ca(++) or its substitute Mn(++), the intracellular pH, or arachidonate bear directly on the enzyme. Iodide acts at the apical membrane of the cell through an oxidized form, presumably iodohexadecanal. Cooling of the cells to 22°C blocks the activation of the PIP(2) phospholipase C cascade. All these effects are reversible. Their kinetics and concentration-effect characteristics have been defined in the four models. A general scheme of the thyroid signaling pathways regulating this metabolism is proposed. The probes characterized could be applied to other H2O2 producing cells and to pathological material., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Thyroid hormone (TH) has profound influence on metabolism that is closely linked to its effect on mitochondrial biogenesis and function. After a single injection of TH into mammals, physiological alterations (e.g. changes in oxygen consumption rates) are detectable after a lag period of ∼48h. This characteristic lag period is somewhat surprising since non-genomic responses are already detectable within minutes, and first genomic responses within some hours after administration of TH. This review provides a model to explain the characteristic lag period: TH regulates a first series of TH target genes via classical activation of gene expression by binding to thyroid hormone response elements. Some directly regulated target genes serve as intermediate factors and subsequently regulate a second series of indirect TH target genes. Intermediate factors are transcription factors (such as NRF-1, NRF-2 and PPARγ) and transcriptional coactivators (such as PGC-1α and PGC-1β). In concert with several post-translational modifications, these intermediate factors orchestrate the physiological response to thyroid hormone in vivo., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Retinoid X receptor (RXR) signaling influences thyrotrope function. Synthetic RXR agonists, rexinoids, can cause central hypothyroidism. To test the hypothesis that endogenous rexinoids contribute to the TSH 'set point', TαT1 mouse thyrotrope cells were treated with a rexinoid antagonist, LG101208. Increasing concentrations of LG101208 significantly increased TSHβ mRNA levels, indicating that the rexinoid antagonist may interfere with RXR-signaling by an endogenous rexinoid in thyrotropes. When the same experiments were repeated in the presence of charcoal-stripped serum the effect of the rexinoid antagonist was lost. Pretreatment with the transcription inhibitor DRB blocked the increase of TSHβ mRNA levels by rexinoid antagonist, indicating the primary effect is at the level of gene transcription. Mice treated with LG101208 had higher levels of serum T4, T4/TSH ratios as well as pituitary α-subunit and TSHβ mRNA compared with vehicle treated mice. Hypothalamic TRH levels were unchanged. In summary, the rexinoid antagonist, LG101208, increases TSH subunit mRNA levels in thyrotrope cells and mouse pituitaries, primarily at the level of gene transcription. These data suggest that an "endogenous rexinoid" contributes to the TSH 'set point' in thyrotropes., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The higher prevalence of thyroid disease in women suggests that estrogen (E2) might be involved in the pathophysiology of thyroid dysfunction. To approach the question of the effect of stromal cells in the modulation of thyroid epithelial cells activity, we established and characterized a homogeneous stromal cell population (TS7 cells) of rat thyroid gland. These fibroblastic cells synthesize the cytoskeleton proteins α-smooth muscle actin and vimentin, produce basement membrane components and express the cytokine transforming growth factor beta 1 (TGF-β1). Here, we hypothesized that the effects of E2 on follicular thyroid cells are mediated by TGF-β1 synthesis and secretion by stromal cells (paracrine action). Thus we investigated the effect of E2 on TGF-β1 synthesis and its signaling pathway in TS7 cells. In addition, we analyzed the role of TGF-β1 signaling pathway as mediator of TS7-PC CL3 thyroid epithelial cells interactions. We report that TS7 stromal cells expressed α and β estrogen receptors (ERα and ERβ). Further, both isoforms of TGF-β1 receptors, TGFRI and TGFRII, were also identified in TS7 cells, suggesting that these cells might be a target for this cytokine in vitro. Treatment of TS7 cells with E2 induced both synthesis and secretion of TGF-β1. This event was followed by phosphorylation of the transcription factor Smad2, a hallmark of TGF-β1 pathway activation. Co-culture of PC CL3 cells onto TS7 cells monolayers yielded round aggregates of PC CL3 cells surrounded by TS7 cells. TS7 cells induced a decrease in iodide uptake by PC CL3 cells, probably by a mechanism involving TGF-β1. Moreover, E2 affected synthesis and organization of the extracellular matrix (ECM) components, tenascin C and chondroitin sulfate, in these co-culture cells. Our results point to the TGF-β1/Smad-2 signaling pathway as a putative target of estrogen actions on thyroid stromal cells and contribute to understanding the interplay between stromal and follicular cells in thyroid physiology., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: Iodide has direct effects on thyroid function. Several iodinated lipids are biosynthesized by the thyroid and they were postulated as intermediaries in the action of iodide. Among them 6 iodo-delta-lactone (IL-delta) has been identified and proposed to play a role in thyroid autoregulation. The aim of this study was to compare the effect of iodide and IL-delta on several thyroid parameters., Methods: Thyroid bovine follicles were incubated with the different compounds during three days., Results: KI and IL-delta inhibited iodide uptake, total protein and Tg synthesis but only KI had an effect on NIS and Tg mRNAs levels. Both compounds inhibited Na+/K+ ATPase and deoxy-glucose uptake. As PAX 8, FOXE 1 and TITF1 are involved in the regulation of thyroid specific genes their mRNA levels were measured. While iodide inhibited the expression of the first two, the expression of TITF1 was stimulated by iodide and IL-delta had no effect on these parameters., Conclusion: These findings indicate that IL-delta reproduces some but not all the effects of excess iodide. These observations apply for higher micromolar concentrations of iodide while no such effects could be demonstrated at nanomolar iodide concentrations., (2010 Elsevier Ireland Ltd. All rights reserved.)

Pendred syndrome is an autosomal recessive disorder characterized by sensorineural deafness, goiter and a partial defect in iodide organification. Goiter development and hypothyroidism vary and appear to depend on nutritional iodide intake. Pendred syndrome is caused by biallelic mutations in the SLC26A4 gene, which encodes pendrin, a multifunctional anion exchanger. Pendrin is mainly expressed in the thyroid, the inner ear, and the kidney. In the thyroid, pendrin localizes to the apical membrane of thyrocytes, where it may be involved in mediating iodide efflux. Loss-of-function mutations in the SLC26A4 gene are associated with a partial iodide organification defect, presumably because of a reduced iodide efflux into the follicular lumen. In the kidney, pendrin functions as a chloride/bicarbonate exchanger. In the inner ear, pendrin is important in the maintenance of a normal anion transport and the endocochlear potential. Elucidation of the function of pendrin has provided unexpected novel insights into the pathophysiology of thyroid hormone biosynthesis, chloride retention in the kidney, and composition of the endolymph., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Thyroperoxidase-catalyzed iodination of thyroglobulin and subsequent oxidative coupling of iodinated tyrosyl residues to protein-bound iodothyronines are the key reactions in thyroid hormone biosynthesis. Under sufficient iodine supply, both synthesis steps are rate-limited by the availability of hydrogen peroxide (H(2)O(2)), which is required as final electron acceptor. The primary enzyme feeding H(2)O(2) to thyroid peroxidase is a heterodimeric NADPH oxidase complex of dual oxidase 2 (DUOX2) and DUOX maturation factor 2 (DUOXA2) at the apical plasma membrane. While the thyrotropin receptor mediates most biological effects through the Gs/adenyl cyclase/cAMP pathway, the Gq/phospholipase C-beta cascade induces H(2)O(2) generation via synergistic effects of increased intracellular calcium and protein kinase C activation on DUOX2/DUOXA2. Defects in thyroidal H(2)O(2) generation have been identified in a subset of patients with congenital hypothyroidism. These include loss-of-function mutations in DUOX2 and DUOXA2. Thyrotropin receptor mutations with preferential loss of Gq-coupling may indirectly affect H(2)O(2) production. Expressivity of the defects can be highly variable owning to the presence of genetic modifiers (e.g., the paralogs DUOX1 and DUOXA1), and environmental factors particularly nutritional iodide intake., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

The study of normal signal transduction pathways regulating the proliferation and differentiation of a cell type allows to predict and to understand the perversions of these pathways which lead to tumorigenesis. In the case of the human thyroid cell, three cascades are mostly involved in tumorigenesis: The pathways and genetic events affecting them are described. Caveats in the use of models and the interpretation of results are formulated and the still pending questions are outlined., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

Interferon alpha (IFNalpha) plays an important role in the pathogenesis of different autoimmune diseases. IFNalpha is widely used for the treatment of chronic viral infections, particularly chronic hepatitis C virus infection; however, several case reports have emerged describing autoimmune conditions, such as Graves' disease (GD), that have developed in the patients receiving IFNalpha. The mechanism by which IFNalpha is involved in GD remains poorly understood. We investigated the expression of IFNalpha and IFNalpha-inducible genes (IFIGs) in GD and found that IFIGs were overexpressed in 60% of 54 clinical diagnostic GD patients. These elevated IFIGs correlated with serological levels of autoantibody to thyroid stimulating hormone receptor (TSHR). Recombinant human IFNalpha stimulated primary cultured thyrocytes resulted in not only high level expression of IFIGs, but also, more importantly, expression of MHC-II antigens (HLA-DR3 and HLA-DR5) and TSHR in GD subjects. Furthermore, thyroid gland tissues from GD patients over express HLA-DR, TSHR and IFNalpha receptors at both messenger RNA and protein levels. Taken together, these data indicated that in GD patients, IFNalpha can function on thyroid tissue to induce a number of genes, particularly MHC class II molecules which may enhance autoantigen presentation of TSHR on thyrocytes., (Copyright 2010. Published by Elsevier Ireland Ltd.)

Arecoline is a plant alkaloid of betel nut Areca catechu. Arecoline has immunosuppressive, hepatotoxic, mutagenic and teratogenic effects, and disturbs some endocrine organs in rats. The objective is to investigate the untoward effects of arecoline on the thyroid gland in mice. Intraperitoneal injection of arecoline (10 mg/kg body weight only once) increased the serum T(3) and T(4) levels and decreased the serum TSH 20, 40 or 60 min after the treatment, with maximum effect at 40 min. Chronic arecoline treatment (10 mg/kg body weight daily for 15 days) caused light microscopic and ultrastructural degenerations of thyro-follicular cells with depletion of T(3) and T(4) levels followed by the elevation of the TSH level. Atropine (arecoline antagonist) injection prevented the changes (hyperactivity) induced by acute (40 min) arecline treatment. Arecoline initially stimulates thyroid activity, and eventually inhibits the activity; atropine prevents thyroid dysfunction induced by arecoline. Arecoline action is mediated probably via muscarinic cholinergic receptor-hypothalamic-pituitary-thyroid axis in mice., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

The THOX and DUOXA genes encode components of the oxidative machinery involved in thyroid hormone biosynthesis. Both of these genes are duplicated in mammalian genomes and are positioned in a head-to-head configuration, THOX1 facing DUOXA1 and THOX2 facing DUOXA2, respectively. The intergenic regions in both couples of genes exhibit dissimilar compositions, being highly GC-rich in the case of THOX1-DUOXA1 but not in the other case. In this study we localized precisely the transcription starts of all four genes using the RLM-RACE technique. It revealed that the distance between THOX1 and DUOXA1 transcription units is of about 70bp only, whereas THOX2 and DUOXA2 transcription starts are separated by 170bp. Analysis of these putative promoter regions revealed the presence of several potential binding sites for transcription factor Sp1 within the THOX1-DUOXA1 intergenic space, and of a TATA box and an Inr element in front of DUOXA2 and THOX2 genes, respectively. The putative promoter regions were inserted into a specifically designed vector harbouring two distinct reporter genes facing each other and their activity was investigated in transient transfection experiments in rat thyroid PCCl3 cells. Both regions exhibited bidirectional promoter activity in the assay. Gel shift experiments using extracts obtained from PCCl3 cells demonstrated the existence of at least one functional Sp1 binding site within the THOX1-DUOXA1 promoter. When Sp1 binding was abolished by mutation of the DNA sequence, a clear reduction in promoter activity in both THOX1 and DUOXA1 directions was observed in the functional assay. As these promoter sequences are well conserved in mammalian genomes, it appears very likely that the results we obtained here in the rat may be extended to the other species.

Peptide transport and expression of SoLute Carrier 15 (SLC15) peptide transporters was assessed in rat thyroid tissue and a rat thyroid cell line (PC Cl3 cells). Peptide transport was studied by monitoring the uptake of the fluorophore-conjugated dipeptide beta-Ala-Lys-N(epsilon)-7-amino-4-methyl-coumarin-3-acetic acid (Ala-Lys-AMCA). Expression of SLC15-specific mRNA transcripts was analyzed by RT-PCR. Of the two SLC15 transporters expressed in thyroid follicular cells, namely PEPT2 (SLC15A2) and PHT1 (SLC15A4), only PEPT2 was involved in peptide transport at the plasma membrane, with PHT1 most likely being intracellular. Interestingly, at the mRNA level PEPT2 was up-regulated under TSH stimulation. These findings represent the first evidence that peptide transport occurs in thyroid follicular cells. SLC15 transporters could participate to recycling of peptides derived from extracellular and lysosomal thyroglobulin proteolysis, both essential steps for thyroid hormone synthesis., (2009 Elsevier Ireland Ltd. All rights reserved.)

Despite evidence for a conserved role of thyroid-stimulating hormone (TSH) in regulating vertebrate thyroid function, molecular data on thyroid responses to TSH are mainly limited to mammalian species. In this study, we examined histological and molecular changes in the thyroid of Xenopus laevis tadpoles during a 12-day treatment with 20mg/l perchlorate (PER) and 50mg/l ethylenethiourea (ETU). Inhibition of thyroid hormone (TH) synthesis by PER and ETU was evident from developmental retardation, reduced expression of TH-regulated genes and up-regulation of tshb-A mRNA. Thyroid histopathology revealed goiters with strikingly different follicular morphologies following PER and ETU treatment. Using real-time PCR, we analyzed thyroids sampled on day 12 for differential expression of 60 candidate genes. Further temporal analyses were performed for a subset of 14 genes. Relative to the control, PER and ETU treatment modulated the expression of 51 and 49 transcripts, respectively. Particularly genes related to TH synthesis and protein metabolism were similarly affected by PER and ETU. However, several genes were differentially expressed in PER- and ETU-treated tadpoles. Specifically, goiter formation in the PER treatment was associated with low expression of genes related to DNA replication but high expression of negative growth regulators. Results from this work provide for the first time a characterization of gene expression profiles during goitrogenesis in a non-mammalian vertebrate model. Overall, our data suggest that, in addition to TSH over-stimulation, further mechanisms related to the mode of goitrogen action contribute to the regulation of thyroid gene expression.

Recent evidence derived from transgenic mouse models and findings in humans with mutations affecting thyroid hormone (TH) metabolism have convincingly supported a model of TH signalling in which regulated local adjustment of active TH concentrations is far more important than circulating plasma hormone levels. Although this theory was put forward several years ago and has been supported by significant, but inherently indirect evidence, recent insights from targeted deletion of the genes encoding deiodinase (Dio) isozymes have revived this model and greatly increased our understanding of TH metabolism. However, gene targeting proved to be a double edged sword, since the overall model was supported, but several predictions are apparently not consistent with the new experimental evidence. Human genetics further provided additional exciting data on the physiological role of Dio isozymes that need to be incorporated into any model of TH biology. The recent identification of mutations in the T3 plasma membrane transporter MCT8 has sparked new interest in the role of TH in brain function, since affected patients suffered from psychomotor retardation. Moreover, selenium (Se) and TH physiology have finally been unequivocally connected by newly identified inherited defects in a gene involved in selenoprotein biosynthesis. Finally, a link between Dio expression and energy metabolism has been delineated in mice that may hold great promise for the management of the adiposity pandemic.