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The primary function of the thyroid gland is to metabolize iodide by synthesizing thyroid hormones, which are critical regulators of growth, development and metabolism in almost all tissues. So far, research on thyroid morphogenesis has been missing an efficient stem-cell model system that allows for the in vitro recapitulation of the molecular and morphogenic events regulating thyroid follicular-cell differentiation and subsequent assembly into functional thyroid follicles. Here we report that a transient overexpression of the transcription factors NKX2-1 and PAX8 is sufficient to direct mouse embryonic stem-cell differentiation into thyroid follicular cells that organize into three-dimensional follicular structures when treated with thyrotropin. These in vitro -derived follicles showed appreciable iodide organification activity. Importantly, when grafted in vivo into athyroid mice, these follicles rescued thyroid hormone plasma levels and promoted subsequent symptomatic recovery. Thus, mouse embryonic stem cells can be induced to differentiate into thyroid follicular cells in vitro and generate functional thyroid tissue. Transient overexpression of the transcription factors NKX2-1 and PAX8 in a murine cell model is shown to direct the differentiation of embryonic stem cells towards a thyroid follicular cell lineage; the resulting three-dimensional thyroid follicles created by subsequent thyrotropin treatment show hallmarks of thyroid function in vitro and rescue thyroid function in vivo when transplanted into athyroid mice, adding to our understanding of the molecular mechanisms underlying thyroid development. Stem cells primed for thyroid production Sabine Costagliola and colleagues report a protocol that converts mouse embryonic stem cells into functional thyroid follicles in vitro . Overexpression of the transcription factors NKX2.1 and PAX8 directs differentiation towards thyroid follicular cells, which undergo self-assembly when treated with thyrotropin. The resulting three-dimensional thyroid follicles show hallmarks of thyroid function in vitro , and can rescue multiple symptoms when transplanted into athyroid mice. This work not only adds to our understanding of the molecular mechanism behind thyroid development, but also paves the way for regenerative medicine to treat congenital hypothyroidism, the most common congenital endocrine disease in humans.

It is now recognized that patient and animal models expressing genetically encoded misfolded mutant thyroglobulin (TG, the protein precursor for thyroid hormone synthesis) exhibit dramatic swelling of the endoplasmic reticulum (ER), with ER stress and cell death in thyrocytes - seen both in homozygotes (with severe hypothyroidism) and heterozygotes (with subclinical hypothyroidism). The thyrocyte death phenotype is exacerbated upon thyroidal stimulation (by thyrotropin [TSH]), as cell death is inhibited upon treatment with exogenous thyroxine. TSH stimulation might contribute to cytotoxicity by promoting ER stress or by an independent mechanism. Here we've engineered KO mice completely lacking Tg expression. Like other animals/patients with mutant TG, these animals rapidly developed severe goitrous hypothyroidism; however, thyroidal ER stress was exceedingly low - lower even than that seen in WT mice. Nevertheless, mice lacking TG exhibited abundant thyroid cell death, which depended upon renegade thyroidal iodination; cell death was completely suppressed in a genetic model lacking effective iodination or in Tg-KO mice treated with propylthiouracil (iodination inhibitor) or iodide deficiency. Thyrocytes in culture were killed not in the presence of H2O2 alone, but rather upon peroxidase-mediated iodination, with cell death blocked by propylthiouracil. Thus, in the thyroid gland bearing Tg mutation(s), TSH-stimulated iodination activity triggers thyroid cell death.

The key proteins responsible for hormone synthesis in the thyroid are glycosylated. Oligosaccharides strongly affect the function of glycosylated proteins. Both thyroid-stimulating hormone (TSH) secreted by the pituitary gland and TSH receptors on the surface of thyrocytes contain N-glycans, which are crucial to their proper activity. Thyroglobulin (Tg), the protein backbone for synthesis of thyroid hormones, is a heavily N-glycosylated protein, containing 20 putative N-glycosylated sites. N-oligosaccharides play a role in Tg transport into the follicular lumen, where thyroid hormones are produced, and into thyrocytes, where hyposialylated Tg is degraded. N-glycans of the cell membrane transporters sodium/iodide symporter and pendrin are necessary for iodide transport. Some changes in glycosylation result in abnormal activity of the thyroid and alteration of the metabolic clearance rate of hormones. Alteration of glycan structures is a pathological process related to the progression of chronic diseases such as thyroid cancers and autoimmunity. Thyroid carcinogenesis is accompanied by changes in sialylation and fucosylation, β1,6-branching of glycans, the content and structure of poly-LacNAc chains, as well as O-GlcNAcylation, while in thyroid autoimmunity the main processes affected are sialylation and fucosylation. The glycobiology of the thyroid gland is an intensively studied field of research, providing new data helpful in understanding the role of the sugar component in thyroid protein biology and disorders.

There have been conflicting reports regarding the function of miR-20a in a variety of cancer types and we previously found it to be dysregulated in sporadic versus familial papillary thyroid cancer. In this study, we studied the expression of miR-20a in normal, benign and malignant thyroid samples, and its effect on thyroid cancer cells in vitro and in vivo. The expression of miR-20a in normal, benign and malignant thyroid tissue was determined by quantitative RT-PCR. Thyroid cancer cells were transfected with miR-20a and the effect on cellular proliferation, tumor spheroid formation, and invasion was evaluated. Target genes of miR-20 were determined by genome-wide mRNA expression analysis with miR-20a overexpression in thyroid cancer cells and target prediction database. Target genes were validated by quantitative PCR and immunoblotting, and luciferase assays. MiR-20a expression was significantly higher in anaplastic thyroid cancer than in differentiated thyroid cancer, and benign and normal thyroid tissues. MiR-20a significantly inhibited thyroid cancer cell proliferation in vitro (p<0.01) and in vivo (p<0.01), tumor spheroid formation (p<0.05) and invasion (p<0.05) in multiple thyroid cancer cell lines. We found that LIMK1 was a target of miR-20a in thyroid cancer cell lines and direct knockdown of LIMK1 recapitulated the effect of miR-20a in thyroid cancer cells. To our knowledge, this is the first study to demonstrate that miR-20a plays a role as a tumor suppressor in thyroid cancer cells and targets LIMK1. Our findings suggest the upregulated expression of miR-20a in anaplastic thyroid cancer counteracts thyroid cancer progression and may have therapeutic potential.

This study investigated the therapeutic effects of Radix Scrophulariae (RS) extract on hyperthyroidism and the associated mechanisms. A hyperthyroid cell model was established using thyrotropin receptor antibody and levothyroxine sodium tablets. FRTL-5 rat thyroid cells were treated with varying concentrations of RS-containing serum. Protein expression levels of Bcl-2, Caspase-3, PCNA, Cyclin D1, MST1, LC3-II/I, and ATG5 were assessed by Western blotting to determine the optimal concentration. Subsequent experiments included RS treatment with or without MST1 overexpression or the Hippo pathway inhibitor XMU-MP-1. Transmission electron microscopy was used to visualize secretory vesicles, and immunofluorescence analysis was performed to detect thyroid-stimulating hormone receptor (TSHR) expression. Protein levels of MST1, p-LATS1, p-YAP, PCNA, Cyclin D1, Bcl-2, Caspase-3, LC3-II/I, and ATG5 were further quantified by Western blotting. The hyperthyroid model exhibited elevated expression of TSHR, Bcl-2, PCNA, and Cyclin D1 ( P  < 0.05), and reduced levels of MST1, p-LATS1, p-YAP, Caspase-3, LC3-II/I, and ATG5 ( P  < 0.05). Secretory vesicles were rarely observed. Treatment with RS-containing serum significantly downregulated TSHR, Bcl-2, PCNA, and Cyclin D1 expression ( P  < 0.05), and upregulated MST1, p-LATS1, p-YAP, Caspase-3, LC3-II/I, and ATG5 ( P  < 0.05), with abundant bilayer membrane vesicles observed. The therapeutic effects of RS were attenuated by MST1 overexpression but were restored by co-treatment with XMU-MP-1 ( P  < 0.05). RS extract may attenuate hyperthyroidism by suppressing excessive thyroid cell proliferation, enhancing apoptosis and autophagy, and activating the MST1/Hippo signaling pathway in FRTL-5 cells.

Oncocytic cell tumor of the thyroid is composed of large polygonal cells with eosinophilic cytoplasm that is rich in mitochondria. These tumors frequently have the mutations in mitochondrial DNA encoding the mitochondrial electron transport system complex I. However, the mechanism for accumulation of abnormal mitochondria is unknown. A noncanonical mitophagy system has recently been identified, and mitochondria‐eating protein (MIEAP) plays a key role in this system. We therefore hypothesized that accumulation of abnormal mitochondria could be attributed to defective MIEAP expression in these tumors. We first show that MIEAP was expressed in all the conventional thyroid follicular adenomas (FAs)/adenomatous goiters (AGs) but not in oncocytic FAs/AGs; its expression was defective not only in oncocytic thyroid cancers but also in the majority of conventional thyroid cancers. Expression of MIEAP was not correlated with methylation status of the 5′‐UTR of the gene. Our functional analysis showed that exogenously induced MIEAP, but not PARK2, reduced the amounts of abnormal mitochondria, as indicated by decreased reactive oxygen species levels, mitochondrial DNA / nuclear DNA ratios, and cytoplasmic acidification. Therefore, together with previous studies showing that impaired mitochondrial function triggers compensatory mitochondrial biogenesis that causes an increase in the amounts of mitochondria, we conclude that, in oncocytic cell tumors of the thyroid, increased abnormal mitochondria cannot be efficiently eliminated because of a loss of MIEAP expression, ie impaired MIEAP‐mediated noncanonical mitophagy. In this study, we sought to clarify the mechanisms for accumulation of abnormal mitochondria in oncocytic tumor of the thyroid and found a lack of expression of mitochondria‐eating protein (MIEAP), a molecule critical for non‐canonical mitophagy, in these tumors, and that exogenous expression of MIEAP accelerated mitochondrial turnover in oncocytic tumor cell line XTC.UC1. These data indicate that defective MIEAP expression causes accumulation of abnormal mitochondria in these tumors.

While the signaling pathways and transcription factors involved in the differentiation of thyroid follicular cells, both in embryonic and adult life, are increasingly well understood, the underlying mechanisms and potential crosstalk between the thyroid transcription factors Nkx2.1, Foxe1 and Pax8 and inductive signals remain unclear. Here, we focused on the transcription factor Sox9, which is expressed in Nkx2.1-positive embryonic thyroid precursor cells and is maintained from embryonic development to adulthood, but its function and control are unknown. We show that two of the main signals regulating thyroid differentiation, TSH and TGFβ, modulate Sox9 expression. Specifically, TSH stimulates the cAMP/PKA pathway to transcriptionally upregulate Sox9 mRNA and protein expression, a mechanism that is mediated by the binding of CREB to a CRE site within the Sox9 promoter. Contrastingly, TGFβ signals through Smad proteins to inhibit TSH-induced Sox9 transcription. Our data also reveal that Sox9 transcription is regulated by the thyroid transcription factors, particularly Pax8. Interestingly, Sox9 significantly increased the transcriptional activation of Pax8 and Foxe1 promoters and, consequently, their expression, but had no effect on Nkx2.1 . Our study establishes the involvement of Sox9 in thyroid follicular cell differentiation and broadens our understanding of transcription factor regulation of thyroid function.

Sexual dimorphism significantly influences the epidemiology of thyroid disorders, with females exhibiting higher incidence of thyroid diseases. Estrogens and their hydroxylated metabolites are key regulators of cellular redox balance and may contribute to sex-specific susceptibility through pro-oxidative mechanisms. However, the impact of individual estrogen metabolites on oxidative stress in thyroid follicular cells remains poorly defined. Here, we investigated the pro-oxidative effects of 17β-estradiol (E2) and its hydroxylated metabolites-2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2), and 16α-hydroxyestrone (16α-OHE1)-in primary porcine thyroid cell cultures from males and females. Primary follicular thyroid cells were isolated from six male and six female pigs. Cells were exposed to E2 (100 nM) or its metabolites (1 μM), with or without Fenton reaction substrates (Fe and H O ), for 24 h. Lipid peroxidation (an index of oxidative damage to lipids) was quantified using BODIPY 581/591 C11 fluorescence via flow cytometry. Basal lipid peroxidation did not differ between sexes. 2-OHE2 increased lipid peroxidation in both male and female thyroid cells, with a more pronounced effect observed in males. In contrast, 4-OHE2 selectively enhanced lipid peroxidation only in female cells. 16α-OHE1 elevated lipid peroxidation in both sexes. E2 significantly increased lipid peroxidation in both male and female cells. Among all compounds tested, E2 exhibited the most potent pro-oxidative activity, particularly in female-derived cells. These findings provide novel insights into the redox-modulating effects of estrogen metabolism in the thyroid and suggest a potential molecular basis for sex-related susceptibility to thyroid dysfunction. While based on an in vitro porcine model, the study increases our understanding of the mechanisms by which estrogenic compounds may influence thyroid pathophysiology, possibly including early events in thyroid disease development or oncogenesis.

We used atomic force microscopy (AFM) technique to measure the viscoelastic response of cancer and normal thyroid cells on different stiffness polyacrylamide gels. After applying a step in contact we recorded the stress relaxation of cells in order to measure their viscous and elastic properties. With the help of an extended version of the Hertz model, we could quantify for the first time by AFM the elastic modulus and the dynamic viscosity of cells on substrates with different stiffnesses. We have cultured anaplastic carcinoma and normal thyroid cells on three different substrates: polyacrylamide gels with elastic modulus in a range of 3–5 and 30–40 kPa and “infinitely” stiff Petri dishes. Whereas normal thyroid cells adapted their mechanical properties to different stiffness substrates, cancer cells were less affected by the surrounding stiffness. Normal cells changed the elastic modulus from 1.2 to 1.6 and to 2.6 kPa with increasing substrate stiffness; the dynamic viscosity values varied from 230 to 515 and to 470 Pa·s, accordingly. By contrast, the values for cancer cells were rather constant regardless of substrate stiffness (in average the elastic modulus was 1.3 kPa and the dynamic viscosity was 300 Pa·s). This difference in sensing and reacting to the mechanical properties of the substrate shows that normal and cancer cells interact differently with the neighboring tissue, which may be related to the ability of cancer cells to form metastases.

Despite infiltrating immune cells having an essential function in human disease and patients’ responses to treatments, mechanisms influencing variability in infiltration patterns remain unclear. Here, using bulk RNA-seq data from 46 tissues in the Genotype-Tissue Expression project, we apply cell-type deconvolution algorithms to evaluate the immune landscape across the healthy human body. We discover that 49 of 189 infiltration-related phenotypes are associated with either age or sex ( FDR  < 0.1). Genetic analyses further show that 31 infiltration-related phenotypes have genome-wide significant associations (iQTLs) ( P  < 5.0 × 10 −8 ), with a significant enrichment of same-tissue expression quantitative trait loci in suggested iQTLs ( P  < 10 −5 ). Furthermore, we find an association between helper T cell content in thyroid tissue and a COMMD3 / DNAJC1 regulatory variant ( P  = 7.5 × 10 −10 ), which is associated with thyroiditis in other cohorts. Together, our results identify key factors influencing inter-individual variability of immune infiltration, to provide insights on potential therapeutic targets. Immune infiltration provides critical information for health and disease, yet it is unclear what factors influence infiltration levels. Here, the authors analyze human tissue transcriptomes from the Genotype-Tissue Expression project to find infiltration patterns regulated by age, sex and host genetic information.