Targeting the aryl hydrocarbon receptor with FICZ regulates IL-2 and immune infiltration to alleviate Hashimoto's thyroiditis in mice.

Hashimoto's thyroiditis (HT) is the most frequent autoimmune disorder. Growing work points to the involvement of aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor, in the regulation of immune homeostasis. However, the roles of AhR and its ligands in HT remains unclear. In this study, we leveraged public human database analyses to postulate that the AhR expression was predominantly in thyroid follicular cells, correlating significantly with the thyroid infiltration levels of multiple immune cells in HT patients. Using a thyroglobulin-induced HT mouse model and in vitro thyroid follicular epithelial cell cultures, we found a significant downregulation of AhR expression in thyrocytes both in vivo and in vitro. Conversely, activating AhR by FICZ, a natural AhR ligand, mitigated inflammation and apoptosis in thyrocytes in vitro and conferred protection against HT in mice. RNA sequencing (RNA-seq) of thyroid tissues indicated that AhR activation moderated HT-associated immune or inflammatory signatures. Further, immunoinfiltration analysis indicated that AhR activation regulated immune cell infiltration in the thyroid of HT mice, such as suppressing cytotoxic CD8+ T cell infiltration and promoting anti-inflammatory M2 macrophage polarization. Concomitantly, the expression levels of interleukin-2 (IL-2), a lymphokine that downregulates immune responses, were typically decreased in HT but restored upon AhR activation. In silico validation substantiated the binding interaction between AhR and IL-2. In conclusion, targeting the AhR with FICZ regulates IL-2 and immune infiltration to alleviate experimental HT, shedding new light on the therapeutic intervention of this prevalent disease.

Technical evaluation and standardization of the human thyroid microtissue assay.

The success and sustainability of U.S. EPA efforts to reduce, refine, and replace in vivo animal testing depends on the ability to translate toxicokinetic and toxicodynamic data from in vitro and in silico new approach methods (NAMs) to human-relevant exposures and health outcomes. Organotypic culture models employing primary human cells enable consideration of human health effects and inter-individual variability but present significant challenges for test method standardization, transferability, and validation. Increasing confidence in the information provided by these in vitro NAMs requires setting appropriate performance standards and benchmarks, defined by the context of use, to consider human biology and mechanistic relevance without animal data. The human thyroid microtissue (hTMT) assay utilizes primary human thyrocytes to reproduce structural and functional features of the thyroid gland that enable testing for potential thyroid-disrupting chemicals. As a variable-donor assay platform, conventional principles for assay performance standardization need to be balanced with the ability to predict a range of human responses. The objectives of this study were to (1) define the technical parameters for optimal donor procurement, primary thyrocyte qualification, and performance in the hTMT assay, and (2) set benchmark ranges for reference chemical responses. Thyrocytes derived from a cohort of 32 demographically diverse euthyroid donors were characterized across a battery of endpoints to evaluate morphological and functional variability. Reference chemical responses were profiled to evaluate the range and chemical-specific variability of donor-dependent effects within the cohort. The data-informed minimum acceptance criteria for donor qualification and set benchmark parameters for method transfer proficiency testing and validation of assay performance.

B cells orchestrate tolerance to the neuromyelitis optica autoantigen AQP4.

Neuromyelitis optica is a paradigmatic autoimmune disease of the central nervous system, in which the water-channel protein AQP4 is the target antigen1. The immunopathology in neuromyelitis optica is largely driven by autoantibodies to AQP42. However, the T cell response that is required for the generation of these anti-AQP4 antibodies is not well understood. Here we show that B cells endogenously express AQP4 in response to activation with anti-CD40 and IL-21 and are able to present their endogenous AQP4 to T cells with an AQP4-specific T cell receptor (TCR). A population of thymic B cells emulates a CD40-stimulated B cell transcriptome, including AQP4 (in mice and humans), and efficiently purges the thymic TCR repertoire of AQP4-reactive clones. Genetic ablation of Aqp4 in B cells rescues AQP4-specific TCRs despite sufficient expression of AQP4 in medullary thymic epithelial cells, and B-cell-conditional AQP4-deficient mice are fully competent to raise AQP4-specific antibodies in productive germinal-centre responses. Thus, the negative selection of AQP4-specific thymocytes is dependent on the expression and presentation of AQP4 by thymic B cells. As AQP4 is expressed in B cells in a CD40-dependent (but not AIRE-dependent) manner, we propose that thymic B cells might tolerize against a group of germinal-centre-associated antigens, including disease-relevant autoantigens such as AQP4.

Cultured Pig Thyroid Follicular Cells: Electrical Evaluation of Epithelial Integrity.

Thyroid epithelial cells organize as enclosed follicles containing thyroid hormone precursor, iodinated thyroglobulin, with lumina bordered by the cellular apices. Transepithelial transport determines composition of compartmental milieu essential for both prohormone formation and its downstream conversion to thyroxine. Hence, not only do follicular lumina function as storage vessels but also as physiological reaction chambers into which reactive components, together with the proper salts and water, are secreted. Polarized, two-dimensional cultures of pig thyroid epithelia, prepared using established protocols, provide a convenient system for assessing transport processes subserving hormone production. This chapter details established methods for growing and evaluating integrity of primary pig thyroid cultures for downstream analysis of transport and other key physiological functions.

Myeloid cells interact with a subset of thyrocytes to promote their migration and follicle formation through NF-κB.

The pathogenesis of thyroid dysgenesis (TD) is not well understood. Here, using a combination of single-cell RNA and spatial transcriptome sequencing, we identify a subgroup of NF-κB-activated thyrocytes located at the center of thyroid tissues in postnatal mice, which maintained a partially mesenchymal phenotype. These cells actively protruded out of the thyroid primordium and generated new follicles in zebrafish embryos through continuous tracing. Suppressing NF-κB signaling affected thyrocyte migration and follicle formation, leading to a TD-like phenotype in both mice and zebrafish. Interestingly, during thyroid folliculogenesis, myeloid cells played a crucial role in promoting thyrocyte migration by maintaining close contact and secreting TNF-α. We found that cebpa mutant zebrafish, in which all myeloid cells were depleted, exhibited thyrocyte migration defects. Taken together, our results suggest that myeloid-derived TNF-α-induced NF-κB activation plays a critical role in promoting the migration of vertebrate thyrocytes for follicle generation.

Notch signaling in thyrocytes is essential for adult thyroid function and mammalian homeostasis.

The thyroid functions as an apex endocrine organ that controls growth, differentiation and metabolism1, and thyroid diseases comprise the most common endocrine disorders2. Nevertheless, high-resolution views of the cellular composition and signals that govern the thyroid have been lacking3,4. Here, we show that Notch signalling controls homeostasis and thermoregulation in adult mammals through a mitochondria-based mechanism in a subset of thyrocytes. We discover two thyrocyte subtypes in mouse and human thyroids, identified in single-cell analyses by different levels of metabolic activity and Notch signalling. Therapeutic antibody blockade of Notch in adult mice inhibits a thyrocyte-specific transcriptional program and induces thyrocyte defects due to decreased mitochondrial activity and ROS production. Thus, disrupting Notch signalling in adult mice causes hypothyroidism, characterized by reduced levels of circulating thyroid hormone and dysregulation of whole-body thermoregulation. Inducible genetic deletion of Notch1 and 2 in thyrocytes phenocopies this antibody-induced hypothyroidism, establishing a direct role for Notch in adult murine thyrocytes. We confirm that hypothyroidism is enriched in children with Alagille syndrome, a genetic disorder marked by Notch mutations, suggesting that these findings translate to humans.

Autoantibodies against type I IFNs in humans with alternative NF-κB pathway deficiency.

Patients with autoimmune polyendocrinopathy syndrome type 1 (APS-1) caused by autosomal recessive AIRE deficiency produce autoantibodies that neutralize type I interferons (IFNs)1,2, conferring a predisposition to life-threatening COVID-19 pneumonia3. Here we report that patients with autosomal recessive NIK or RELB deficiency, or a specific type of autosomal-dominant NF-κB2 deficiency, also have neutralizing autoantibodies against type I IFNs and are at higher risk of getting life-threatening COVID-19 pneumonia. In patients with autosomal-dominant NF-κB2 deficiency, these autoantibodies are found only in individuals who are heterozygous for variants associated with both transcription (p52 activity) loss of function (LOF) due to impaired p100 processing to generate p52, and regulatory (IκBδ activity) gain of function (GOF) due to the accumulation of unprocessed p100, therefore increasing the inhibitory activity of IκBδ (hereafter, p52LOF/IκBδGOF). By contrast, neutralizing autoantibodies against type I IFNs are not found in individuals who are heterozygous for NFKB2 variants causing haploinsufficiency of p100 and p52 (hereafter, p52LOF/IκBδLOF) or gain-of-function of p52 (hereafter, p52GOF/IκBδLOF). In contrast to patients with APS-1, patients with disorders of NIK, RELB or NF-κB2 have very few tissue-specific autoantibodies. However, their thymuses have an abnormal structure, with few AIRE-expressing medullary thymic epithelial cells. Human inborn errors of the alternative NF-κB pathway impair the development of AIRE-expressing medullary thymic epithelial cells, thereby underlying the production of autoantibodies against type I IFNs and predisposition to viral diseases.

Induction of epithelial-mesenchymal transition in thyroid follicular cells is associated with cell adhesion alterations and low-dose hyper-radiosensitivity.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is associated with altered cellular adhesion. We previously demonstrated that cellular adhesion influences Low-dose Hyper-Radiosensitivity (HRS) in a variety of tumor cells. However, the relationship of low-dose HRS with the phenotypic plasticity incurred by EMT during the neoplastic transformation remains to be elucidated. OBJECTIVE: To investigate whether acquisition of EMT phenotype during progressive neoplastic transformation may affect low-dose radiation sensitivity. METHODS: Primary thyroid cells obtained from a human cystic thyroid nodule were first subjected to nutritional stress. This yielded immortalized INM-Thy1 cell strain, which was further treated with either multiple γ-radiation fractions (1.5 Gy each) or repetitive cycles of 3-methylcholanthrene and phorbol-12-myristate-13-acetate, yielding two progressive transformants, viz., INM-Thy1R and INM-Thy1C. Morphological alterations, chromosomal double-minutes, cell adhesion proteins, anchorage dependency, tumorigenicity in nude mice and cellular radiosensitivity were studied in these strains. RESULTS: Both transformants (INM-Thy1R, INM-Thy1C) displayed progressive tumorigenic features, viz., soft agar colony growth and solid tumor growth in nude mice, coupled with features of epithelial-mesenchymal transition and activated Wnt pathway. Incidentally, the chemical-induced transformant (INM-Thy1C) displayed a prominent HRS (αs/αr = 29.35) which remained unaffected at high cell density. However, the parental (INM-Thy1) cell line as well as radiation-induced transformant (INM-Thy1R) failed to show this hypersensitivity. CONCLUSION: The study shows that induction of EMT in thyroid follicular cells may accompany increased susceptibility to low-dose ionizing radiation, which was attenuated by adaptive resistance acquired during radiation-induced transformation.

Antioxidant Defense Capacity Is Reduced in Thyroid Stem/Precursor Cells Compared to Differentiated Thyrocytes.

There is much evidence linking oxidative stress to thyroid cancer, and stem cells are thought to play a key role in the tumor-initiating mechanism. Their vulnerability to oxidative stress is unexplored. This study aimed to comparatively evaluate the antioxidant capacity of stem/precursor thyroid cells and mature thyrocytes. Human stem/precursor cells and mature thyrocytes were exposed to increasing concentrations of menadione, an oxidative-stress-producing agent, and reactive oxygen species (ROS) production and cell viability were measured. The expression of antioxidant and detoxification genes was measured via qPCR as well as the total antioxidant capacity and the content of glutathione. Menadione elevated ROS generation in stem/precursor thyroid cells more than in mature thyrocytes. The ROS increase was inversely correlated (p = 0.005) with cell viability, an effect that was partially prevented by the antioxidant curcumin. Most thyroid antioxidant defense genes, notably those encoding for the glutathione-generating system and phase I detoxification enzymes, were significantly less expressed in stem/precursor thyroid cells. As a result, the glutathione level and the total antioxidant capacity in stem/precursor thyroid cells were significantly decreased. This reduced antioxidant defense may have clinical implications, making stem/precursor thyroid cells critical targets for environmental conditions that are not detrimental for differentiated thyrocytes.

Pathogenesis of cancers derived from thyroid follicular cells.

The genomic simplicity of differentiated cancers derived from thyroid follicular cells offers unique insights into how oncogenic drivers impact tumour phenotype. Essentially, the main oncoproteins in thyroid cancer activate nodes in the receptor tyrosine kinase-RAS-BRAF pathway, which constitutively induces MAPK signalling to varying degrees consistent with their specific biochemical mechanisms of action. The magnitude of the flux through the MAPK signalling pathway determines key elements of thyroid cancer biology, including differentiation state, invasive properties and the cellular composition of the tumour microenvironment. Progression of disease results from genomic lesions that drive immortalization, disrupt chromatin accessibility and cause cell cycle checkpoint dysfunction, in conjunction with a tumour microenvironment characterized by progressive immunosuppression. This Review charts the genomic trajectories of these common endocrine tumours, while connecting them to the biological states that they confer.

miR-142-3p encapsulated in T lymphocyte-derived tissue small extracellular vesicles induces Treg function defect and thyrocyte destruction in Hashimoto's thyroiditis.

BACKGROUND: Hashimoto's thyroiditis (HT) is an organ-specific autoimmune disease characterized by lymphocyte infiltration that destroys thyrocyte cells. The aim of the present study was to elucidate the role and mechanisms of tissue small extracellular vesicle (sEV) microRNAs (miRNAs) in the pathogenesis of HT. METHODS: Differentially expressed tissue sEV miRNAs were identified between HT tissue and normal tissue by RNA sequencing in the testing set (n = 20). Subsequently, using quantitative real-time polymerase chain reaction (qRT‒PCR) assays and logistic regression analysis in the validation set (n = 60), the most relevant tissue sEV miRNAs to HT were verified. The parental and recipient cells of that tissue sEV miRNA were then explored. In vitro and in vivo experiments were further performed to elucidate the function and potential mechanisms of sEV miRNAs that contribute to the development of HT. RESULTS: We identified that miR-142-3p encapsulated in T lymphocyte-derived tissue sEVs can induce Treg function defect and thyrocyte destruction through an intact response loop. Inactivation of miR-142-3p can effectively protect non-obese diabetic (NOD).H-2h4 mice from HT development display reduced lymphocyte infiltration, lower antibody titers, and higher Treg cells. Looking at the mechanisms underlying sEV action on thyrocyte destruction, we found that the strong deleterious effect mediated by tissue sEV miR-142-3p is due to its ability to block the activation of the ERK1/2 signaling pathway by downregulating RAC1. CONCLUSIONS: Our findings highlight the fact that tissue sEV-mediated miR-142-3p transfer can serve as a communication mode between T lymphocytes and thyrocyte cells in HT, favoring the progression of HT.

Primary human thyrocytes maintained the function of thyroid hormone production and secretion in vitro.

PURPOSE: Thyroid cell lines are useful tools to study the physiology and pathology of the thyroid, however, they do not produce or secrete hormones in vitro. On the other hand, the detection of endogenous thyroid hormones in primary thyrocytes was often hindered by the dedifferentiation of thyrocytes ex vivo and the presence of large amounts of exogenous hormones in the culture medium. This study aimed to create a culture system that could maintain the function of thyrocytes to produce and secrete thyroid hormones in vitro. METHODS: We established a Transwell culture system of primary human thyrocytes. Thyrocytes were seeded on a porous membrane in the inner chamber of the Transwell with top and bottom surfaces exposed to different culture components, mimicking the 'lumen-capillary' structure of the thyroid follicle. Moreover, to eliminate exogenous thyroid hormones from the culture medium, two alternatives were tried: a culture recipe using hormone-reduced serum and a serum-free culture recipe. RESULTS: The results showed that primary human thyrocytes expressed thyroid-specific genes at higher levels in the Transwell system than in the monolayer culture. Hormones were detected in the Transwell system even in the absence of serum. The age of the donor was negatively related to the hormone production of thyrocytes in vitro. Intriguingly, primary human thyrocytes cultured without serum secreted higher levels of free triiodothyronine (FT3) than free thyroxine (FT4). CONCLUSION: This study confirmed that primary human thyrocytes could maintain the function of hormone production and secretion in the Transwell system, thus providing a useful tool to study thyroid function in vitro.

Thyroid-Disrupting Effects of Exposure to Fipronil and Its Metabolites from Drinking Water Based on Human Thyroid Follicular Epithelial Nthy-ori 3-1 Cell Lines.

Fipronil is a broad-spectrum insecticide used for plants and poultry. Owing to its widespread use, fipronil and its metabolites (fipronil sulfone, fipronil desulfinyl, and fipronil sulfide), termed FPM, can be frequently detected in drinking water and food. Fipronil can affect the thyroid function of animals, but the effects of FPM on the human thyroid remain unclear. We employed human thyroid follicular epithelial Nthy-ori 3-1 cells to examine combined cytotoxic responses, thyroid-related functional proteins including the sodium-iodide symporter (NIS), thyroid peroxidase (TPO), deiodinases I-III (DIO I-III), and the nuclear factor erythroid-derived factor 2-related factor 2 (NRF2) pathway induced by FPM of 1-1000-fold concentrations detected in school drinking water collected from a heavily contaminated area of the Huai River Basin. Thyroid-disrupting effects of FPM were evaluated by examining biomarkers of oxidative stress and thyroid function and tetraiodothyronine (T4) levels secreted by Nthy-ori 3-1 cells after FPM treatment. FPM activated the expression of NRF2, HO-1 (heme oxygenase 1), TPO, DIO I, and DIO II but inhibited NIS expression and increased the T4 level of thyrocytes, indicating that FPM can disrupt the function of human thyrocytes through oxidative pathways. Given the adverse impact of low FPM concentrations on human thyrocytes, supportive evidence from rodent studies, and the critical importance of thyroid hormones on development, the effects of FPM on the neurodevelopment and growth of children warrant priority attention.

Effects of Aronia melanocarpa and Hypericum perforatum aqueous extracts on hexavalent chromium induced toxicity in rat's thyrocytes.

BACKGROUND: Hexavalent chromium known as oxidizing agent is able to form reactive oxygen species. Aronia melanocarpa and Hypericum perforatum are two plants known for their antioxidant effects. Our study aimed to establish if CrVI induces apoptosis and structural changes in thyrocytes and if its effect can be counteracted by the administration of both extracts. MATERIALS AND METHODS: Wistar rats divided in five groups: C - distilled water (DW), Cr - 75 mg/L CrVI in DW for 3 months, Cr 2 - 75 mg/L CrVI in DW for 3 months followed by 1 month DW, CrA - 3 months 75 mg/L CrVI in DW and 1 month Aronia 2.5% extract, CrH - 3 months 75 mg/L CrVI in DW and 1 month Hypericum 2.5% extract. Histological assessment and qRT-PCR for evaluation of BAX and Bcl2 protein levels performed on thyroid samples. RESULTS: The Cr and Cr2 groups were those with altered cytoarchitecture: increase in the diameter of many thyroid follicles, a decrease in their number, a decrease in the height of the follicular cells. The histological examination of the CrH group revealed almost recovery of structural architecture. The BAX gene levels were higher in the Cr and Cr2 groups indicating the apoptotic activity of chromium. In extract receiving groups the BAX gene expressions were significantly lower, but the lowest level presented the CrH group. Bcl2 gene expression levels indicate antiapoptotic activity being elevated in the Cr group, followed by CrA, Cr2, and CrH groups. The BAX/Bcl2 ratio which significantly increased in the case of the Cr and Cr2 group compared to the groups that were administered the two plant extracts. CONCLUSION: The results obtained in this study confirm that CrVI has toxic effects on thyroid endocrine cells and H. perforatum has stronger antioxidant properties against the action of hexavalent chromium in thyrocytes than A. melanocarpa.

Excessive iodine induces thyroid follicular epithelial cells apoptosis by activating HIF-1α-mediated hypoxia pathway in Hashimoto thyroiditis.

BACKGROUND: Hashimoto thyroiditis (HT) is considered the most common autoimmune thyroid disease. A growing body of evidence suggests that HT incidence correlates with excessive iodine intake. We should probe the effects of excessive iodine intake in HT development and its possible mechanism. METHODS AND RESULTS: The study recruited 20 patients: 10 with HT and 10 with nodular goiter. We detected the expression of an apoptosis-related protein caspase-3 by immunohistochemistry. In vitro study, we explored the proliferation and apoptosis status in thyroid follicular cells (TFCs) stimulated with different iodine concentrations by MTT and flow cytometry. Then we performed RNA sequence analysis of Nthy-ori3-1 cells treated for 48 h with KI to probe the underlying mechanism. Finally, we used RT-PCR and siRNA interference to verify the results. We identified apoptosis in thyroid tissue obtained from HT patients coincides with the increase of caspase-3 levels. In vitro study, iodine suppressed proliferation of TFCs and promoted TFCs apoptosis in a dose-dependent manner with regulating caspase-3 activation. HIF-1α-NDRG1 mediated hypoxia pathway activation promoted the transmission of essential apoptosis signals in TFCs. CONCLUSION: Our study confirmed that excessive iodine adsorption activates the HIF-1α-mediated hypoxia pathway to promote apoptosis of TFCs, which may be an important risk factor contributing to HT development.

Defective Thyroglobulin: Cell Biology of Disease.

The primary functional units of the thyroid gland are follicles of various sizes comprised of a monolayer of epithelial cells (thyrocytes) surrounding an apical extracellular cavity known as the follicle lumen. In the normal thyroid gland, the follicle lumen is filled with secreted protein (referred to as colloid), comprised nearly exclusively of thyroglobulin with a half-life ranging from days to weeks. At the cellular boundary of the follicle lumen, secreted thyroglobulin becomes iodinated, resulting from the coordinated activities of enzymes localized to the thyrocyte apical plasma membrane. Thyroglobulin appearance in evolution is essentially synchronous with the appearance of the follicular architecture of the vertebrate thyroid gland. Thyroglobulin is the most highly expressed thyroid gene and represents the most abundantly expressed thyroid protein. Wildtype thyroglobulin protein is a large and complex glycoprotein that folds in the endoplasmic reticulum, leading to homodimerization and export via the classical secretory pathway to the follicle lumen. However, of the hundreds of human thyroglobulin genetic variants, most exhibit increased susceptibility to misfolding with defective export from the endoplasmic reticulum, triggering hypothyroidism as well as thyroidal endoplasmic reticulum stress. The human disease of hypothyroidism with defective thyroglobulin (either homozygous, or compound heterozygous) can be experimentally modeled in thyrocyte cell culture, or in whole animals, such as mice that are readily amenable to genetic manipulation. From a combination of approaches, it can be demonstrated that in the setting of thyroglobulin misfolding, thyrocytes under chronic continuous ER stress exhibit increased susceptibility to cell death, with interesting cell biological and pathophysiological consequences.

Prospects of Testing Diurnal Profiles of Expressions of TSH-R and Circadian Clock Genes in Thyrocytes for Identification of Preoperative Biomarkers for Thyroid Carcinoma.

Thyroid Nodules (TN) are frequent but mostly benign, and postoperative rate of benign TN attains the values from 70% to 90%. Therefore, there is an urgent need for identification of reliable preoperative diagnosis markers for patients with indeterminate thyroid cytology. In this study, an earlier unexplored design of research on preoperative biomarkers for thyroid malignancies was proposed. Evaluation of reported results of studies addressing the links of thyroid cancer to the circadian clockwork dysfunctions and abnormal activities of Thyroid-Stimulating Hormone (TSH) and its receptor (TSH-R) suggested diagnostic significance of such links. However, there is still a gap in studies of interrelationships between diurnal profiles of expression of circadian clock genes and TSH-R in indeterminate thyroid tissue exposed to different concentrations of TSH. These interrelationships might be investigated in future in vitro experiments on benign and malignant thyrocytes cultivated under normal and challenged TSH levels. Their design requires simultaneous measurement of diurnal profiles of expression of both circadian clock genes and TSH-R. Experimental results might help to bridge previous studies of preoperative biomarkers for thyroid carcinoma exploring diagnostic value of diurnal profiles of serum TSH levels, expression of TSH-R, and expression of circadian clock genes.

A recurrent somatic missense mutation in GNAS gene identified in familial thyroid follicular cell carcinomas in German longhaired pointer dogs.

BACKGROUND: We previously reported a familial thyroid follicular cell carcinoma (FCC) in a large number of Dutch German longhaired pointers and identified two deleterious germline mutations in the TPO gene associated with disease predisposition. However, the somatic mutation profile of the FCC in dogs has not been investigated at a genome-wide scale. RESULTS: Herein, we comprehensively investigated the somatic mutations that potentially contribute to the inherited tumor formation and progression using high depth whole-genome sequencing. A GNAS p.A204D missense mutation was identified in 4 out of 7 FCC tumors by whole-genome sequencing and in 20 out of 32 dogs' tumors by targeted sequencing. In contrast to this, in the human TC, mutations in GNAS gene have lower prevalence. Meanwhile, the homologous somatic mutation in humans has not been reported. These findings suggest a difference in the somatic mutation landscape between TC in these dogs and human TC. Moreover, tumors with the GNAS p.A204D mutation had a significantly lower somatic mutation burden in these dogs. Somatic structural variant and copy number alterations were also investigated, but no potential driver event was identified. CONCLUSION: This study provides novel insight in the molecular mechanism of thyroid carcinoma development in dogs. German longhaired pointers carrying GNAS mutations in the tumor may be used as a disease model for the development and testing of novel therapies to kill the tumor with somatic mutations in the GNAS gene.

Thyroglobulin regulates the expression and localization of the novel iodide transporter solute carrier family 26 member 7 (SLC26A7) in thyrocytes.

Solute carrier family 26 member 7 (SLC26A7), identified as a causative gene for congenital hypothyroidism, was found to be a novel iodide transporter expressed on the apical side of the follicular epithelium of the thyroid. We recently showed that TSH suppressed the expression of SLC26A7 and induces its localization to the plasma membrane, where it functions. We also showed that the ability of TSH to induce thyroid hormone synthesis is completely reversed by an autocrine negative-feedback action of thyroglobulin (Tg) stored in the follicular lumen. In the present study, we investigated the potential effect of follicular Tg on SLC26A7 expression and found that follicular Tg significantly suppressed the promoter activity, mRNA level, and protein level of SLC26A7 in rat thyroid FRTL-5 cells. In addition, follicular Tg inhibited the ability of TSH to induce the membrane localization of SLC26A7. In rat thyroid sections, the expression of SLC26A7 was weaker in follicles with a higher concentration of Tg, as evidenced by immunofluorescence staining. These results indicate that Tg stored in the follicular lumen is a feedback suppressor of the expression and membrane localization of SLC26A7, thereby downregulating the transport of iodide into the follicular lumen.

Thyroid stimulating hormone suppresses the expression and activity of cytosolic sulfotransferase 1a1 in thyrocytes.

Sulfonation is an important step in the metabolism of dopamine, estrogens, dehydroepiandrosterone, as well as thyroid hormones. However, the regulation of cytosolic sulfotransferases in the thyroid is not well understood. In a DNA microarray analysis of rat thyroid FRTL-5 cells, we found that the mRNA expression of 10 of 48 sulfotransferases was significantly altered by thyroid stimulating hormone (TSH), with that of sulfotransferase family 1A member 1 (SULT1A1) being the most significantly affected. Real-time PCR and Western blot analyses revealed that TSH, forskolin and dibutyryl cyclic AMP significantly suppressed SULT1A1 mRNA and protein levels in a time- and concentration-dependent manner. Moreover, immunofluorescence staining of FRTL-5 cells showed that SULT1A1 is localized in the perinuclear area in the absence of TSH but is spread throughout the cytoplasm with reduced fluorescence intensity in the presence of TSH. Sulfotransferase activity in FRTL-5 cells, measured using 3'-phosphoadenosine-5'-phosphosulfate as a donner and p-nitrophenol as an acceptor substrate, was significantly reduced by TSH. These findings suggest that the expression and activity of SULT1A1 are modulated by TSH in thyrocytes.