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To develop a cell-based in vitro thyroid-stimulating hormone (TSH) biological activity assay that can simulate in vivo pharmacodynamic mechanisms, we constructed two HEK293-TSHR cell lines based on two main cell signaling pathways (Gαs-cAMP-PKA and Gαq/11-PLC-Ca2+) that TSH depends on for its in vivo physiological function. These cell lines stably expressed the luciferase reporter driven by the cAMP response element (CRE) and nuclear factor of activated T cells (NFAT) response element, and two reporter-gene assays (RGAs) were correspondingly established and validated. The two transgenic genes could measure signals produced from the simulation of the in vivo effects of TSH from the Gαs-cAMP and Gαq/11-PLC pathways after TSH activation. TSH showed a good dose–response relationship in these two cell lines and conformed to the four-parameter model. We optimized the critical experimental parameters of these two methods and performed comprehensive methodological validation according to the International Council for Harmonization (ICH) Q2 (R1) guidelines, the Chinese Pharmacopoeia, and the United States Pharmacopoeia. The two methods showed good specificity, accuracy, precision, and linearity and can be used to aid in assessments of the biological activity of TSH drugs, product characterization, final product release, stability studies, and comparability studies for biosimilar applications.

BackgroundHormones are identified as key biological variables in tumor immunity. However, previous researches mainly focused on the immune effect of steroid hormones, while the roles that thyroid-stimulating hormone (TSH) played in the antitumor response were far from clear.MethodsThe source of TSH was determined using single-cell transcriptomic, histologic, quantitative PCR, and ELISA analysis. The influence of TSH on tumor proliferation, invasion, and immune evasion was evaluated in multiple cell lines of thyroid cancer, glioma, and breast cancer. Then transcriptomic sequencing and cellular experiments were used to identify signaling pathways. TSH receptor (TSHR) inhibitor was injected into homograft mouse tumor models with or without anti-programmed cell death protein-1 antibody.ResultsMonocyte-derived dendritic cells (moDCs) highly expressed TSHα and TSHβ2 and were the primary source of TSH in the tumor microenvironment. TSH released by moDCs promoted proliferation and invasion of tumors with high TSHR expressions, such as thyroid cancers and glioma. TSH also induced tumor programmed death-ligand 1 (PD-L1) expression through the TSHR-AC-PKA-JNK-c-JUN pathway. TSHR inhibitors reversed tumor immune evasion by inhibiting PD-L1 expression in tumor and myeloid cells and enhancing Teff activation.ConclusionsTSH–TSHR axis promotes tumor evasion in thyroid cancers and glioma. TSH suppression therapy is an effective therapeutic strategy for combination in immune checkpoint blockades.

Experimental evidence suggests that in autoimmune thyroid diseases (AITDs) the skin is a target of autoantibodies against thyroid-specific antigens; however, the role of these autoantibodies in skin alterations remains unclear. To gain insight into the function of nominally thyroid-specific genes in skin, we analyzed the expression of thyroid-stimulating hormone–receptor (TSH-R), thyroglobulin (Tg), sodium iodide symporter (NIS), and thyroperoxidase (TPO) genes in normal human skin biopsies and cultured primary keratinocytes and dermal fibroblasts. The results revealed the presence of all the transcripts in skin biopsies. However, in keratinocytes and fibroblasts, only TSH-R messenger RNA was always detected. Western blot and immunohistochemical analyses of skin specimens confirmed the presence of TSH-R protein in keratinocytes and fibroblasts. Moreover, TSH treatment induced the proliferation of cultured keratinocytes and fibroblasts and increased keratinocyte intracellular cAMP. Finally, affinity-purified IgGs from serum of patients affected by Graves’ disease, but not by chronic lymphocytic thyroiditis, stimulated cAMP accumulation in cultured keratinocytes, as well as their proliferation. In conclusion, the expression of thyroid-specific genes in cultured keratinocytes and fibroblasts and the mitogenic effects of TSH and IgGs on these cells support the concept that autoantibodies against thyroid-specific antigens may contribute to cutaneous symptoms in AITDs. JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://network.nature.com/group/jidclub

Thyroid hormones may influence risk of cancer through their role in cell differentiation, growth, and metabolism. One study of circulating thyroid hormones supports this hypothesis with respect to prostate cancer. We undertook a prospective analysis of thyroid hormones and prostate cancer risk in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study. Within the ATBC Study, a randomized controlled trial of α-tocopherol and β-carotene supplements and cancer incidence in male smokers, 402 prostate cancer cases were sampled. Controls were matched 2:1 to cases on age and date of blood collection. Odds ratios (OR) and 95% confidence intervals (CI) of prostate cancer were estimated for quintiles of serum total and free thyroxine (T4), thyroid-stimulating hormone (TSH), thyroid-binding globulin (TBG), and by categories of thyroid status. Men with serum higher TSH had a decreased risk of prostate cancer compared to men with lower TSH (Q5 vs. Q1-4: OR = 0.70, 95% CI: 0.51-0.97, p = 0.03). When the T4 and TSH measurements were combined to define men as hypothyroid, euthyroid or hyperthyroid, hypothyroid men had a lower risk of prostate cancer compared to euthyroid men (OR = 0.48, 95% CI = 0.28-0.81, p = 0.006). We observed no association between hyperthyroid status and risk, although the number of hyperthyroid men with prostate cancer was small (n = 9). In this prospective study of smokers, men with elevated TSH and those classified as being in a hypothyroid state were at decreased risk of prostate cancer. Future studies should examine the association in other populations, particularly non-smokers and other racial/ethnic groups.

Di (2-ethylhexyl) phthalate (DEHP), an environmental pollutant, is widely used as a plasticizer and causes serious pollution in the ecological environment. As previously reported, exposure to DEHP may cause thyroid dysfunction of the hypothalamic-pituitary-thyroid (HPT) axis. However, the underlying role of DEHP remains to be elucidated. The present study performed intragastrical administration of DEHP (150, 300 and 600 mg/kg) once a day for 90 consecutive days. DEHP-stimulated oxidative stress increased the thyroid follicular cavity diameter and caused thyrocyte oedema. Furthermore, DEHP exposure altered mRNA and protein levels. Thus, DEHP may perturb TH homeostasis by affecting biosynthesis, biotransformation, bio-transportation, receptor levels and metabolism through disruption of the HPT axis and activation of the thyroid-stimulating hormone (TSH)/TSH receptor signaling pathway. These results identified the formerly unappreciated endocrine-disrupting activities of phthalates and the molecular mechanisms of DEHP-induced thyrotoxicity.

Deiodinase 2 (DIO2) plays an important role in thyroid hormone metabolism and its regulation. However, molecular mechanism that regulates DIO2 activity remains unclear; only mutaions in selenocysteine insertion sequence binding protein 2 and selenocysteine tranfer RNA (tRNA [Ser]Sec ) are reported to result in decreased DIO2 activity. Two patients with clinical evidence of abnormal thyroid hormone metabolism were identified and found to have TSHR mutations as well as DIO 2 T92A single nucleotide polymorphism (SNP). Primary-cultured fibroblasts from one patient present a high level of basal DIO2 enzymatic activity, possibly due to compensation by augmented DIO2 expression. However, this high enzymatic active state yet fails to respond to accelerating TSH. Consequently, TSHR mutations along with DIO2 T92A SNP (“double hit”) may lead to a significant reduction in DIO2 activity stimulated by TSH, and thereby may have clinical relevance in a select population of hypothyroidism patients who might benefit from a T3/T4 combination therapy.

Background & Aims. Oxidative stress-related liver diseases were shown to be associated with elevated serum thyroid stimulating hormone (TSH) levels. Mitochondria are the main source of cellular reactive oxygen species. However, the relationship between TSH and hepatic mitochondrial stress/dysfunction and the underlying mechanisms are largely unknown. Here, we focused on exploring the effects and mechanism of TSH on hepatic mitochondrial stress. Methods. As the function of TSH is mediated through the TSH receptor (TSHR), Tshr-/- mice and liver-specific Tshr knockout (LKO) mice were used in our study. The thyroid-specific Tshr knockout mouse model injected with TSH (TKO+TSH) was used as a mimic for subclinical hypothyroidism (SCH) patients. Hepatic mitochondrial stress and function were analyzed in these mouse models, and the expression of key genes involved in mitochondrial stress was measured. Results. A relatively lower degree of mitochondrial stress was observed in the livers of Tshr-/- mice and LKO mice than those of their littermate counterparts. TSH caused concentration- and time-dependent effects on mitochondrial stress and cyclophilin D (CypD) acetylation in hepatocytes in vitro. Microarray and RT-PCR analyses showed that Tshr-/- mice had much higher lncRNA-AK044604 expression than their littermate counterparts. The use of the AK044604 overexpression plasmid and SIRT1 agonist proved that TSH aggravates CypD acetylation and mitochondrial stress via lncRNA-AK044604 and SIRT1. An inhibitor of CypD acetylation, cyclosporine A, suppressed TSH-induced hepatic mitochondrial stress and dysfunction. Conclusions. TSH stimulates hepatic CypD acetylation through the lncRNA-AK044604/SIRT1/SIRT3 signaling pathway, indicating an essential role for TSH in mitochondrial stress in the liver.

Deficiency in Krüppel-like zinc finger transcription factor GLI-similar 3 (GLIS3) in humans is associated with the development of congenital hypothyroidism. However, the functions of GLIS3 in the thyroid gland and the mechanism by which GLIS3 dysfunction causes hypothyroidism are unknown. In the current study, we demonstrate that GLIS3 acts downstream of thyroid-stimulating hormone (TSH) and TSH receptor (TSHR) and is indispensable for TSH/TSHR-mediated proliferation of thyroid follicular cells and biosynthesis of thyroid hormone. Using ChIP-Seq and promoter analysis, we demonstrate that GLIS3 is critical for the transcriptional activation of several genes required for thyroid hormone biosynthesis, including the iodide transporters Nis and Pds, both of which showed enhanced GLIS3 binding at their promoters. The repression of cell proliferation of GLIS3-deficient thyroid follicular cells was due to the inhibition of TSH-mediated activation of the mTOR complex 1/ribosomal protein S6 (mTORC1/RPS6) pathway as well as the reduced expression of several cell division-related genes regulated directly by GLIS3. Consequently, GLIS3 deficiency in a murine model prevented the development of goiter as well as the induction of inflammatory and fibrotic genes during chronic elevation of circulating TSH. Our study identifies GLIS3 as a key regulator of TSH/TSHR-mediated thyroid hormone biosynthesis and proliferation of thyroid follicular cells and uncovers a mechanism by which GLIS3 deficiency causes neonatal hypothyroidism and prevents goiter development.

The skin is commonly affected in thyroid diseases, but the mechanism for this association is still unclear. As the skin expresses numerous neuroendocrine elements, we tested the additional cutaneous expression of mediators operating in the hypothalamic-pituitary-thyroid axis. We found significant expression of the thyroid-stimulating hormone receptor mRNA in cultured keratinocytes, epidermal melanocytes, and melanoma cells. The presence of thyroid-stimulating hormone receptor was confirmed by northern analyses and the thyroid-stimulating hormone receptor was found to be functionally active in cyclic adenosine monophosphate signal assays. Thyroid-stimulating hormone receptor expressing cells also expressed the sodium iodide symporter and thyroglobulin genes. We also found expression of deiodinases 2 and 3 (mainly deiodinase 2) in whole skin biopsy specimens, and in the majority of epidermal and dermal cells by reverse transcription-polymerase chain reaction followed by sequencing of the amplified gene segments. There was selective expression of the gene for thyroid-stimulating hormone beta; detection of the thyroid-releasing hormone gene was minimal and thyroid-releasing hormone receptor mRNA was not detected in most of the samples. Expression of functional thyroid-stimulating hormone receptor in the skin may have significant physiologic and pathologic consequences, particularly in autoimmune conditions associated with production of stimulating antibodies against the thyroid-stimulating hormone receptor. We conclude that the expanding list of neuroendocrine elements expressed in the skin supports a strong role for this system in cutaneous biology.

The thyroid-stimulating hormone/thyrotropin (TSH) is the most relevant hormone in the control of thyroid gland physiology in adulthood. TSH effects on the thyroid gland are mediated by the interaction with a specific TSH receptor (TSHR). We studied the role of TSH/TSHR signaling on gland morphogenesis and differentiation in the mouse embryo using mouse lines deprived either of TSH (pitdw/pitdw) or of a functional TSHR (tshrhyt/tshrhytand TSHR-knockout lines). The results reported here show that in the absence of either TSH or a functional TSHR, the thyroid gland develops to a normal size, whereas the expression of thyroperoxidase and the sodium/iodide symporter are reduced greatly. Conversely, no relevant changes are detected in the amounts of thyroglobulin and the thyroid-enriched transcription factors TTF-1, TTF-2, and Pax8. These data suggest that the major role of the TSH/TSHR pathway is in controlling genes involved in iodide metabolism such as sodium/iodide symporter and thyroperoxidase. Furthermore, our data indicate that in embryonic life TSH does not play an equivalent role in controlling gland growth as in the adult thyroid.