TSH Receptor Expression Research Articles

The Absence of Thyroid-Stimulating Hormone Receptor Expression on Natural Killer T Cells: Implications for the Immune–Endocrine Interaction

The expression of thyroid-stimulating hormone receptor (TSHR) has been documented on various immune cells, including B lymphocytes, T lymphocytes, Natural Killer (NK) cells, monocytes, and dendritic cells (DCs). Natural Killer T (NKT) cells serve as a crucial link between innate and adaptive immunity, playing significant roles in immunological interactions and autoimmune diseases. The aim of the present study was to evaluate the presence of TSHR on NKT cells. Our research involved patients with thyroid disease, as well as healthy controls. Peripheral blood mononuclear cells (PBMCs) and, thereafter, NKT cells were isolated from 86 patients with benign nodular thyroid disease with and without autoimmune thyroid disease (AITD) (28 and 56 cases, respectively), and TSHR expression was analyzed using fluorescence-activated cell sorting (FACS). In order to confirm the results, the reverse-transcription polymerase chain reaction (RT-PCR) method was used in cells obtained from healthy individuals. Our findings obtained with application of the FACS method revealed that TSHR is not expressed on NKT cells in either AITD or non-AITD patients, though TSHR was detected in the total PBMC population (TSHR+ cells 2.77%). The absence of TSHR on NKT cells was further confirmed with RT-PCR in healthy individuals (p < 0.0001). These results questioned the previously suggested direct influence of NKT cells on AITD development.

Zirconium- 89 Labeled Antibody K1-70 for PET Imaging of Thyroid-stimulating Hormone Receptor Expression in Thyroid Cancer.

Thyroid-stimulating hormone receptor (TSHR) is a G-protein coupled receptor that is highly expressed on benign and malignant thyroid tissues. TSHR binding and activation has long been a component of thyroid cancer molecular imaging and radiotherapy, by promoting expression of the sodium-iodide symporter (NIS) and incorporation of I-131 into thyroid hormones. Here, we report the radiosynthesis and preclinical evaluation of a Zirconium-89 (89Zr) labeled TSHR antibody to serve as a positron emission tomography (PET) diagnostic correlate for therapeutic agents targeting TSHR without reliance on NIS. TSHR human monoclonal antibody K1-70 was conjugated to chelator desferrioxamine-p-benzyl-isothiocyanate, followed by labeling with Zr-89, yielding the radiotracer 89Zr-DFO-TSHR-Ab. The in vitro cellar uptake and binding affinity of 89Zr-DFO-TSHR-Ab were analyzed in three new TSHR stable overexpressing tumor cell lines and their corresponding wild types (WT) with low or no TSHR expression. 89Zr-DFO-TSHR-Ab PET/CT imaging of TSHR expression was evaluated in tumor mouse models bearing one TSHR-positive tumor and other negative control with or without the coinjection of antibody K1-70, and then verified by radiotracer biodistribution study and tumor immunohistochemistry (IHC). The conjugate DFO-TSHR-Ab was labeled with Zr-89 at 37°C for 60min and purified by PD-10 column in radiochemical yields of 68.8 ± 9.9%, radiochemical purities of 98.7 ± 0.8%, and specific activities of 19.1 ± 2.7mCi/mg (n = 5). In vitro cell studies showed 89Zr-DFO-TSHR-Ab had significantly high uptake on TSHR expressing tumor cells with nanomolar affinity and high potency. Preclinical PET/CT imaging revealed that 89Zr-DFO-TSHR-Ab selectively detected TSHR expressing thyroid tumors and displayed improved in vivo performance with the coinjection of unlabeled TSHR antibody K1-70 leading to higher uptake in TSHR expressing tumors than parental WT tumors and physiologic tissues; this observation was confirmed by the biodistribution and immunostaining analyses. We synthesized 89Zr-labeled antibody K1-70 as a new radiopharmaceutical for PET imaging of TSHR. 89Zr-DFO-TSHR-Ab has high radioactive uptake and retention in TSHR expressing tumors and cleared quickly from most background tissues in mouse models. Our study demonstrated that 89Zr-DFO-TSHR-Ab has the potential for PET imaging of TSHR-positive thyroid cancer and monitoring TSHR-targeted therapy.

Metabolomics reveals the implication of acetoacetate and ketogenic diet therapy in radioiodine-refractory differentiated thyroid carcinoma.

Patients with radioiodine-refractory (RAIR) differentiated thyroid carcinoma (DTC; RAIR-DTC) have a poor prognosis. The aim of this study was to provide new insights and possibilities for the diagnosis and treatment of RAIR-DTC. The metabolomics of 24 RAIR-DTC and 18 non-radioiodine-refractory (NonRAIR) DTC patients samples were analyzed by liquid chromatograph-mass spectrometry. Cellular radioiodine uptake was detected with γ counter. Sodium iodide symporter (NIS) expression and thyroid stimulating hormone receptor (TSHR) were measured by Western blot analysis. CCK8 and colony formation assays were used to measure cellular proliferation. Scratch and transwell assays were performed to assess cell migration and invasion. Annexin V/PI staining was used to detect cell apoptosis. Cell growth in vivo was evaluated by a tumor xenograft model. The acetoacetate (AcAc) level was measured by ELISA. Pathological changes, Ki67, NIS, and TSHR expression were investigated by immunohistochemistry. The metabolite profiles of RAIR could be distinguished from those of NonRAIR, with AcAc significantly lower in RAIR. The significantly different metabolic pathway was ketone body metabolism. AcAc increased NIS and TSHR expression and improved radioiodine uptake. AcAc inhibited cell proliferation, migration, and invasion, and as well promoted cell apoptosis. Ketogenic diet (KD) elevated AcAc levels and significantly suppressed tumor growth, as well as improved NIS and TSHR expression. Significant metabolic differences were observed between RAIR and NonRAIR, and ketone body metabolism might play an important role in RAIR-DTC. AcAc improved cellular iodine uptake and had antitumor effects for thyroid carcinoma. KD might be a new therapeutic strategy for RAIR-DTC.

Immune checkpoints: new insights into the pathogenesis of thyroid eye disease.

Thyroid eye disease (TED) is a disfiguring autoimmune disease characterized by changes in the orbital tissues and is caused by abnormal thyroid function or thyroid-related antibodies. It is the ocular manifestation of Graves' disease. The expression of thyroid-stimulating hormone receptor (TSHR) and the insulin-like growth factor-1 receptor (IGF-1 R) on the cell membrane of orbital fibroblasts (OFs) is responsible for TED pathology. Excessive inflammation is caused when these receptors in the orbit are stimulated by autoantibodies. CD34+ fibrocytes, found in the peripheral blood and orbital tissues of patients with TED, express immune checkpoints (ICs) like MHC II, B7, and PD-L1, indicating their potential role in presenting antigens and regulating the immune response in TED pathogenesis. Immune checkpoint inhibitors (ICIs) have significantly transformed cancer treatment. However, it can also lead to the occurrence of TED in some instances, suggesting the abnormality of ICs in TED. This review will examine the overall pathogenic mechanism linked to the immune cells of TED and then discuss the latest research findings on the immunomodulatory role of ICs in the development and pathogenesis of TED. This will offer fresh perspectives on the study of pathogenesis and the identification of potential therapeutic targets.

Role of thyroid stimulating hormone in the maintenance and functioning of the human corpus luteum.

To evaluate the impact of high thyroid stimulating hormone (TSH) levels on human granulosa-luteal (hGL) cells. hGL cells were isolated from follicular aspirates derived from patients undergoing IVF treatment without any thyroid disorder (serum TSH 0.5-2mU/L). Cells were cultured at 37°C in DMEM, supplemented with 5% FBS. The cells were treated with 1nM LH and increasing concentrations of TSH. At the end of culture, conditioned medium and cells were collected to analyze progesterone production, cell viability, and mRNA levels of genes involved in the steroidogenesis process. Human ovarian tissues were analyzed for TSH receptor (TSHR) expression by IHC. The expression of TSHR was detected in human corpus luteum by IHC and in hGL by RT-PCR. In hGL cells, TSH treatment did not modulate progesterone production nor the expression of steroidogenic genes, such as p450scc and HSD3b 1/2. However, TSH induced a dose-dependent increase in cell death. Finally, TSH did not affect LH-induced p450scc and HSD3b1/2 expression while LH partially reverted TSH negative effect on cell death in hGL. Elevated TSH levels in hypothyroid women may be associated with impaired CL functioning and maintenance. These findings open a new line of research for the importance of the treatment of women with thyroid dysfunction that could contribute to the onset of infertility.

Exercise Attenuates Doxorubicin‐Induced Myocardial Injury by Inhibiting TSHR and Regulating Macrophage Polarization Through miR‐30d‐5p/GALNT7

Objective: Doxorubicin (DOX) is an extensively used chemotherapeutic agent that induces cardiotoxicity. Studies have reported that exercise (EXE) can alleviate DOX‐induced cardiotoxicity. Therefore, this study aimed to explore the mechanism by which EXE attenuates DOX‐induced myocardial injury.Methods: In this study, cell and animal models of DOX‐induced myocardial injury were constructed. The animal model was subjected to EXE intervention.Results: In this study, in vitro experiments revealed that miR‐30d‐5p negatively regulated polypeptide N‐acetylgalactosaminyltransferase 7 (GALNT7) and that GALNT7 negatively regulated the expression of thyroid stimulating hormone receptor (TSHR). miR‐30d‐5p downregulated the expression of GALNT7, promoted the expression of TSHR, and promoted macrophage M1 polarization, thus aggravating cardiomyocyte injury. In vivo experiments revealed that EXE intervention significantly downregulated miR‐30d‐5p and TSHR expression, upregulated GALNT7, reduced inflammation, and promoted M2 macrophage polarization, thereby alleviating DOX‐induced myocardial injury. In addition, overexpression of miR‐30d‐5p or knockdown of GALNT7 weakened the intervention effect of EXE, whereas overexpression of GALNT7 or knockdown of TSHR promoted the effect of EXE.Conclusion: EXE can modulate the miR‐30d‐5p/GALNT7 axis to inhibit the expression of TSHR, thereby regulating the polarization of macrophages to the M2 phenotype and ultimately alleviating DOX‐induced myocardial injury, which provides new targets and strategies for the clinical treatment of myocardial injury.

Impact of ibrutinib on inflammation in a mouse model of Graves' orbitopathy.

Bruton's tyrosine kinase (BTK) and interleukin (IL)-2 Inducible T-cell Kinase (ITK) inhibitors have anti-inflammatory properties. We investigated the therapeutic effect of ibrutinib, an orally bioavailable BTK/ITK inhibitor, in a mouse model of Graves' orbitopathy (GO). Genetic immunization was performed through intramuscular administration of the recombinant plasmid, pCMV6-hTSHR cDNA, to 8-week-old female BALB/c mice. Serum levels of T3, T4, and thyroid-stimulating hormone receptor (TSHR) antibodies (TRAbs) were quantified using enzyme-linked immunosorbent assay. Histopathological changes in orbital tissues were examined using immunohistochemistry (IHC) staining for TSHR and various inflammatory markers. Following successful genetic immunization, ibrutinib was orally administered daily for 2 weeks in the GO model mice. After treatment, the mRNA and protein expression levels of BTK, ITK, IL-1β, and IL-6 in orbital tissues were evaluated using real-time PCR and Western blotting. In total, 20 mice were sacrificed to confirm successful genetic immunization. The GO mouse group exhibited significantly increased serum T3, T4, and TRAb levels. IHC revealed increased expression of TSHR, IL-1β, IL-6, transforming growth factor-β1, interferon-γ, CD40, CD4, BTK, and ITK in the GO mouse model. The orbital inflammation was significantly attenuated in ibrutinib-treated mice. The mRNA and protein expression levels of BTK, ITK, IL-1β, and IL-6 in orbital tissue were lower in ibrutinib-treated GO mouse group compared to the phosphate-buffered saline-treated GO mouse group. The GO mouse model demonstrated enhanced BTK and ITK expression. Ibrutinib, a BTK/ITK inhibitor, suppressed the inflammatory cytokine production. These findings highlight the potential involvement of BTK/ITK in the inflammatory pathogenesis of GO, suggesting its role as a novel therapeutic target.

Perfluorooctanoic acid disrupts thyroid-specific genes expression and regulation via the TSH-TSHR signaling pathway in thyroid cells

Perfluorooctanoic acid (PFOA) is a highly persistent and widespread chemical in the environment with endocrine disruption effects. Although it has been reported that PFOA can affect multiple aspects of thyroid function, the exact mechanism by which it reduces thyroxine levels has not yet been elucidated. In this study, FRTL-5 rat thyroid follicular cells were used as a model to study the toxicity of PFOA to the genes related to thyroid hormone synthesis and their regulatory network. Our results reveal that PFOA interfered with the phosphorylation of the cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB) induced by thyroid-stimulating hormone (TSH), as well as the transcription levels of paired box 8 (PAX8), thyroid transcription factor 1 (TTF1), sodium/iodide cotransporter (NIS), thyroglobulin (TG), and thyroid peroxidase (TPO). However, the above outcomes can be alleviated by enhancing cAMP production with forskolin treatment. Further investigations showed that PFOA reduced the mRNA level of TSH receptor (TSHR) and impaired its N-glycosylation, suggesting that PFOA has disrupting effects on both transcriptional regulation and post-translational regulation. In addition, PFOA increased endoplasmic reticulum (ER) stress and decreased ER mass in FRTL-5 cells. Based on these findings, it can be inferred that PFOA disrupts the TSH-activated cAMP signaling pathway by inhibiting TSHR expression and its N-glycosylation. We propose that this mechanism may contribute to the decrease in thyroid hormone levels caused by PFOA. Our study sheds light on the molecular mechanism by which PFOA can disrupt thyroid function and provides new insights and potential targets for interventions to counteract the disruptive effects of PFOA.

FRI488 The Effect Of Thyroid Stimulating Hormone On Brown Adipose Tissue In Humans

Abstract Disclosure: L. Salej: None. S.D. Kodani: None. N. Chaves: None. K.N. Youssef: None. R. Monahan-Earley: None. H. Halle: None. H. Shaelah: None. P. Annie: None. J.A. Parker: None. A.M. Cypess: None. G. Kolodny: None. Y. Tseng: None. B.C. James: None. A. Gavrila-Filip: None. Obesity and diabetes are two main public health concerns due to their high impact on global morbidity and mortality. In an effort to find new treatment strategies, highly active metabolic tissues such as brown adipose tissue (BAT) have been studied as potential targets. Animal studies have shown that TSH may have a direct local effect on BAT mass and activity by stimulating local T3 production via increased deiodinase-2 (DIO2) expression and also by increasing the expression of uncoupling protein-1 (UCP-1), the key molecule in BAT thermogenesis. We hypothesized that high circulating TSH levels could increase BAT activity by increasing local T3 production and generating a local tissue "thyrotoxicosis". We studied 7 patients with thyroid cancer who underwent thyroid surgery and required recombinant TSH (rhTSH) administration for standard follow-up procedures. Each patient underwent 2 PET-CT scans for assessment of BAT volume and activity, first one day after the rhTSH administration (high TSH state) and then 1-3 months later on suppressive thyroid hormone (TH) treatment (low TSH state). Indirect calorimetry and serum thyroid function tests, fasting glucose, insulin, HbA1c, and lipid panel were measured. Our results show that there was a statistically significant difference in TSH levels (p = 0.0095), with stable circulating T4 and T3 between the low and high TSH states. There was no statistically significant difference in the BAT volume and activity (SUVmax) between the high TSH and low TSH states. Age was a significant predictor of BAT SUVmax (p = 0.008; coeff = -0.39, CI = -0.65-0.13), while BMI was a significant predictor for resting energy expenditure (REE) (p = 0.04; coeff = -203.95, CI = -396.12 - -10.78). Based on these findings, along with those from our previous study[1] showing that circulating TH levels may not be directly correlated with BAT function, we have extended our study to assess the intracellular thyroid hormone status in BAT. We have collected deep peri-thyroidal adipose tissue and subcutaneous white adipose tissue (WAT) as a control group from patients undergoing thyroid surgery. In these samples, we will measure mRNA expression of TSH receptors, TH receptors, β-adrenergic receptors, DIO2 and UCP-1 and evaluate for correlations with circulating TSH and TH levels as well as different metabolic parameters to get a deeper insight into BAT function in humans. Reference: 1. Gavrila A et al. Variable Cold-Induced Brown Adipose Tissue Response to Thyroid Hormone Status. Thyroid. 2017 Jan 1;27(1):1-10. Presentation: Friday, June 16, 2023

Thyrotropin receptor autoantibody (TRAb) enhance the expression of thyrotropin receptor on mouse brain vascular endothelial cells: in vivo and in vitro.

The possibility that thyrotropin receptor (TSHR) expression in non-thyroid tissue is well-documented. However, there is insufficient data on the expression of TSHR in medulla oblongata regions, particularly when focusing on the background of encephalopathy associated with hyperthyroidism. In this study, we explored the expression of the functional TSHR in Graves' disease (GD) mouse cerebral vascular endothelial cells and the effects of thyrotropin receptor autoantibody (TRAb) on its expression. A mouse model of GD was constructed with an adenovirus overexpressing TSHR289. The location and expression of the TSHR gene and protein in vivo were determined via RT-qPCR, Western blot, and immunofluorescence techniques. The effect of TRAb on the expression of functional TSHR in vitro was investigated using bEnd.3 cells. Our results show that medulla oblongata vascular endothelial cells from GD mice expressed higher levels of TSHR compared to control mice. In an in vitro experiment, novel results demonstrated that after treatment with a monoclonal TSHR-specific agonistic antibody (M22), the expression of TSHR on the bEnd.3 cells increased at both the protein and mRNA levels. Furthermore, compared with bEnd.3 cells were treated with IBMX only, those treated with M22 showed increased cAMP production. This study suggested that TSHR is expressed and functionally active in the mouse medulla oblongata and in vitro-cultured bEnd.3 cells and TRAb (M22) increased the expression of TSHR on bEnd.3 cells.

Somatostatin Receptor Type 2 and Thyroid-Stimulating Hormone Receptor Expression in Oncocytic Thyroid Neoplasms: Implications for Prognosis and Treatment

Somatostatin receptor type 2 (SSTR2) and thyroid-stimulating hormone receptor (TSHR) display variable expression in primary thyroid tumors and have been implicated as theranostic targets. This study was designed to explore the differential expression of SSTR2 and TSHR in oncocytic (Hurthle cell) carcinoma (OC) vs oncocytic adenoma (OA). We performed a retrospective review for oncocytic neoplasms treated at our institution from 2012 to 2019. Formalin-fixed paraffin-embedded tissue blocks were used for tissue microarray construction. Tissue microarray blocks were cut into 5-μm sections and stained with anti-SSTR2 and anti-TSHR antibodies. Immunostains were analyzed by 3 independent pathologists. χ2 and logistic regression analysis were used to analyze clinical and pathologic variables. Sixty-seven specimens were analyzed with 15 OA and 52 OC. The mean age was 57 years, 61.2% were women, and 70% were White. SSTR2 positivity was noted in 2 OA (13%) and 15 OC (28%; 10 primary, 4 recurrent, and 1 metastatic) (P = .22). TSHR positivity was noted in 11 OA (73%) and 32 OC (62%; 31 primary and 1 metastatic) (P = .40). Those who presented with or developed clinical recurrence/metastasis were more likely to be SSTR2-positive (50% vs 21%; P = .04) and TSHR-negative (64.3% vs 28.9%; P = .02) than primary OC patients. Widely invasive OC was more likely to be SSTR2-positive compared to all other OC subtypes (minimally invasive and angioinvasive) (P = .003). For all patients with OC, TSHR positivity was inversely correlated with SSTR2 positivity (odds ratio, 0.12; CI, 0.03-0.43; P = .006). This relationship was not seen in the patients with OA (odds ratio, 0.30; CI, 0.01-9.14; P = .440). Our results show that recurrent/metastatic OC was more likely to be SSTR2-positive and TSHR-negative than primary OC. Patients with OC displayed a significant inverse relationship between SSTR2 and TSHR expression that was not seen in patients with OA. This may be a key relationship that can be used to prognosticate and treat OCs.

Radix Scrophulariae Extracts Exert Effect on Hyperthyroidism via MST1/Hippo Signaling Pathway.

To explore the mechanism of Radix Scrophulariae (RS) extracts in the treatment of hyperthyroidism rats by regulating proliferation, apoptosis, and autophagy of thyroid cell through the mammalian sterile 20-like kinase 1 (MST1)/Hippo pathway. Twenty-four rats were randomly divided into 4 groups according to a random number table: control, model group, RS, and RS+Hippo inhibitor (XMU-MP-1) groups (n=6 per group). Rats were gavaged with levothyroxine sodium tablet suspension (LST, 8 μ g/kg) for 21 days except for the control group. Afterwards, rats in the RS group were gavaged with RS extracts at the dose of 1,350 mg/kg, and rats in the RS+XMU-MP-1 group were gavaged with 1,350 mg/kg RS extracts and 1 mg/kg XMU-MP-1. After 15 days of administration, thyroid gland was taken for gross observation, and histopathological changes were observed by hematoxylin-eosin staining. The structure of Golgi secretory vesicles in thyroid tissues was observed by transmission electron microscopy. The expression of thyrotropin receptor (TSH-R) was observed by immunohistochemistry. Terminal-deoxynucleoitidyl transferase mediated nick end labeling assay was used to detect cell apoptosis in thyroid tissues. Real-time quantity primer chain reaction and Western blot were used to detect the expressions of MST1, p-large tumor suppressor gene 1 (LATS1), p-Yes1 associated transcriptional regulator (YAP), proliferating cell nuclear antigen (PCNA), G1/S-specific cyclin-D1 (Cyclin D1), B-cell lymphoma-2 (Bcl-2), Caspase-3, microtubule-associated proeins light chain 3 II/I (LC3-II/I), and recombinant human autophagy related 5 (ATG5). Thyroxine (T4) level was detected by enzyme-linked immunosorbent assay. The thyroid volume of rats in the model group was significantly increased compared to the normal control group (P<0.01), and pathological changes such as uneven size of follicular epithelial cells, disorderly arrangement, and irregular morphology occurred. The secretion of small vesicles by Golgi apparatus was reduced, and the expressions of receptor protein TSH-R and T4 were significantly increased (P<0.01), while the expressions of MST1, p-LATS1, p-YAP, Caspase-3, LC3-II/I, and ATG5 were significantly decreased (P<0.01). The expressions of Bcl-2, PCNA, and cyclin D1 were significantly increased (P<0.01). Compared with the model group, RS extracts reduced the volume of thyroid gland, improved pathological condition of the thyroid gland, promoted secretion of the secretory vesicles with double-layer membrane structure in thyroid Golgi, significantly inhibited the expression of TSH-R and T4 levels (P<0.01), upregulated MST1, p-LATS1, p-YAP, Caspase-3, LC3-II/I, and ATG5 expressions (P<0.01), and downregulated Bcl-2, PCNA, and Cyclin D1 expressions (P<0.01). XMU-MP-1 inhibited the intervention effects of RS extracts (P<0.01). RS extracts could inhibit proliferation and promote apoptosis and autophagy in thyroid tissues through MST1/Hippo pathway for treating hyperthyroidism.

Paternal developmental thyrotoxicosis disrupts neonatal leptin leading to increased adiposity and altered physiology of the melanocortin system.

The genetic code does not fully explain individual variability and inheritance of susceptibility to endocrine conditions, suggesting the contribution of epigenetic factors acting across generations. We used a mouse model of developmental thyrotoxicosis (Dio3-/- mouse) to analyze endocrine outcomes in the adult offspring of Dio3-/- males using standard methods for body composition, and baseline and fasting hormonal and gene expression determinations in serum and tissues of relevance to the control of energy balance. Compared to controls, adult females with an exposed father (EF females) exhibited higher body weight and fat mass, but not lean mass, a phenotype that was much milder in EF males. After fasting, both EF females and males exhibited a more pronounced decrease in body weight than controls. EF females also showed markedly elevated serum leptin, increased white adipose tissue mRNA expression of leptin and mesoderm-specific transcript but decreased expression of type 2 deiodinase. EF females exhibited decreased serum ghrelin, which showed more pronounced post-fasting changes in EF females than in control females. EF female hypothalami also revealed significant decreases in the expression of pro-opiomelanocortin, agouti-related peptide, neuropeptide Y and melanocortin receptor 4. These markers also showed larger changes in response to fasting in EF females than in control females. Adult EF females showed no abnormalities in serum thyroid hormones, but pituitary expression of thyrotropin-releasing hormone receptor 1 and thyroid gland expression of thyroid-stimulating hormone receptor, thyroid peroxidase and iodotyrosine deiodinase were increased at baseline and showed differential regulation after fasting, with no increase in Trhr1 expression and more pronounced reductions in Tshr, Tpo and Iyd. In EF males, these abnormalities were generally milder. In addition, postnatal day 14 (P14) serum leptin was markedly reduced in EF pups. A paternal excess of thyroid hormone during development modifies the endocrine programming and energy balance in the offspring in a sexually dimorphic manner, with baseline and dynamic range alterations in the leptin-melanocortin system and thyroid gland, and consequences for adiposity phenotypes. We conclude that thyroid hormone overexposure may have important implications for the non-genetic, inherited etiology of endocrine and metabolic pathologies.

Targeting the inward rectifier potassium channel 5.1 in thyroid cancer: artificial intelligence-facilitated molecular docking for drug discovery

BackgroundRecurrent and metastatic thyroid cancer is more invasive and can transform to dedifferentiated thyroid cancer, thus leading to a severe decline in the 10-year survival. The thyroid-stimulating hormone receptor (TSHR) plays an important role in differentiation process. We aim to find a therapeutic target in redifferentiation strategies for thyroid cancer.MethodsOur study integrated the differentially expressed genes acquired from the Gene Expression Omnibus database by comparing TSHR expression levels in the Cancer Genome Atlas database. We conducted functional enrichment analysis and verified the expression of these genes by RT-PCR in 68 pairs of thyroid tumor and paratumor tissues. Artificial intelligence-enabled virtual screening was combined with the VirtualFlow platform for deep docking.ResultsWe identified five genes (KCNJ16, SLC26A4, TG, TPO, and SYT1) as potential cancer treatment targets. TSHR and KCNJ16 were downregulated in the thyroid tumor tissues, compared with paired normal tissues. In addition, KCNJ16 was lower in the vascular/capsular invasion group. Enrichment analyses revealed that KCNJ16 may play a significant role in cell growth and differentiation. The inward rectifier potassium channel 5.1 (Kir5.1, encoded by KCNJ16) emerged as an interesting target in thyroid cancer. Artificial intelligence-facilitated molecular docking identified Z2087256678_2, Z2211139111_1, Z2211139111_2, and PV-000592319198_1 (-7.3 kcal/mol) as the most potent commercially available molecular targeting Kir5.1.ConclusionThis study may provide greater insights into the differentiation features associated with TSHR expression in thyroid cancer, and Kir5.1 may be a potential therapeutic target in the redifferentiation strategies for recurrent and metastatic thyroid cancer.

Expression and Clinical Significance of Thyroid-stimulating hormone receptor in the Subtypes of Papillary thyroid carcinomas.

To investigate the expression of TSH receptors (TSHR) in various subtypes of Papillary thyroid carcinomas (PTC) by immunohistochemistry. Retrospective analyses were carried out to the clinical data of 108 PTC patients randomly admitted into the Department of Thyroidthyroid surgery thyroid surgery and Breast Surgery, The Second Hospital of Hebei Medical University from March 2020 to December 2020. The archived paraffin blocks of the 108 cases as well as 18 contiguous normal thyroid tissues (control group) were taken from the Department of Pathology of The Second Hospital of Hebei Medical University. The pathological types of all PTC tissues were detected and the expression of TSHR was determined. TSHR expression was 86.11% positive in PTC tissues; with 85.00% positive in classical group; with 75.86% positive in micro group; with 84.61% positive in follicular group; with 83.33% positive in oncocytic group; with 50.00% positive in invasive group. TSHR expression was 100% in normal thyroid tissues. So TSHR expression in normal thyroid tissues is significantly higher than that in PTC; TSHR expression in microcarcinoma is stronger than in the other subtypes; there is no significant difference among the other subtypes. TSH suppression works better on microcarcinoma than on the other subtypes. And the effects on non-invasive subtypes are better than on invasive subtypes.

Elevated thyroid-stimulating hormone (TSH) is associated with atrial fibrillation and directly modulates electrophysiological properties of atrial cardiomyocytes

Abstract Background Thyroid dysfunction has a strong association with cardiac arrhythmias. However, subclinical hypothyroidism (SH) is often neglected in studies. SH is characterized by increased values of TSH and normal values of T3/T4. Recent studies indicate that high TSH levels are related to an increased incidence of cardiac electrical abnormalities, especially atrial fibrillation (AF). Purpose The present work aims to investigate direct effects of TSH on ion channel remodelling in cardiomyocytes and a possible causative role in the increased occurrence of arrhythmia in SH. Methods A retrospective monocentric study analysed medical records, ECGs and echocardiographies of 3134 patients between 2007 to 2020. Inclusion criteria comprised patient age ≥18 years, normal fT4, TSH values 4–75 mU/l and available ECGs. The patient cohort was divided into two groups based on current treatment recommendations for SH (TSH level 4–9.9 mU/l; 10–75 mU/l). Direct effects on ion channel remodelling were analysed in HL-1 and neonatal cardiomyocytes with confirmed expression of TSH receptors. TSH concentrations of 15, 30, and 60 mU/l simulated different degrees of hypothyroidism. RT-qPCR analyses, western blots and immunofluorescence stainings were performed after incubation with respective TSH concentrations for 24h. In vitro video analyses of automatism in NRCM before and after TSH incubation were performed. Statistical comparisons were calculated using student's t-tests and the chi-square test. In addition, a multivariate analysis of factors affecting the occurrence of AF in patients with SH was carried out. Results Elevated TSH levels were associated with higher prevalence of AF. In the group of patients with a TSH value of 10–75 mU/l, AF was observed in 42.8% of cases compared to 31.7% in patients with lower TSH levels (p&amp;lt;0.0001). In vitro, TSH application to cardiomyocytes led to significant changes in mRNA and protein expression of key ion channels and regulatory proteins. Incubation of HL-1 cells with 15 mU/l TSH showed a decrease in Kcne1 protein, while 60 mU/l TSH caused an upregulation of K2P3.1 (TASK-1), Kv7.1, and KCa2.1 expression. To further elucidate the TSH signaling cascade in cardiomyocytes, we investigated TSH effects on PKAα, CaMKIIδ and CaMKIIγ. PKAα was upregulated after 24h incubation with 60 mU/l TSH and exhibited an increase of the phosphorylated state. Finally, neonatal cardiomyocytes showed an increased beating rate after incubation with TSH. Conclusion The prevalence of atrial fibrillation is significantly increased in patients with SH. Our findings further reveal a direct effect of TSH on ion channel expression in cardiomyocytes as potential proarrhythmic mechanism. TSH induced changes in the expression of ion channels that determine action potential duration. Subclinical hypothyroidism contributes to the development of atrial fibrillation. Underlying mechanisms and therapeutic implications require further research. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Heidelberg University Medical School - Olympia Morata

Expression and Purification of the Human Thyroid-Stimulating Hormone Receptor.

The thyroid-stimulating hormone receptor (TSHR) is a Class A G protein-coupled receptor (GPCR) that mediates signalling through the hypothalamic-pituitary-thyroid axis. Inappropriate activation of TSHR by autoantibodies or mutations, results in human disease such as Grave's disease and Hashimito's thyroiditis. Therefore, there is a need to develop novel therapeutics targeting the TSHR. Understanding the structure and mechanism of activation of this receptor would help elucidate the pathogenesis of disease and aid drug development. Here, we describe a method for the expression of the human TSHR in a mammalian cell line generated through a lentiviral expression system. The receptor is then purified by affinity chromatography in the ligand-free state and is suitable for structure determination by single-particle electron cryo-microscopy (cryo-EM).

Expression of functional thyroid-stimulating hormone receptor in microglia

ObjectiveThe purpose of the present study was to clarify the expression of thyroid-stimulating hormone receptor (TSHR) in microglial cells, and to explore its function. Materials and methodsExpression of TSHR in microglia was determined by Western blot, immunocytofluorescence and double immunohistofluorescence. Cyclic adenosine 3′,5′-monophosphate (cAMP) production was measured after thyrotropin receptor stimulating antibody (TSAb) treatment. ResultsResults showed that TSHR protein was expressed and mainly located in the mouse microglia membrane. Moreover, TSAb stimulated cAMP production in mouse microglia (P<0.05). ConclusionsThis study demonstrated the presence of TSHR in microglial cells. Brain TSHR was able to respond specifically to TSAb stimulation, suggesting that TSHR expression is functional. As microglia are innate immune cells that maintain environmental stability in the central nervous system and play a key role in many neuroimmune diseases, expression of functional TSHR in microglia has important pathophysiological implications.

CAR-T Cells Targeting TSHR Demonstrate Safety and Potent Preclinical Activity Against Differentiated Thyroid Cancer.

Chimeric antigen receptor T cells (CAR-Ts) have demonstrated remarkable efficacy in hematological cancers but have not yet translated in treating solid tumors. The significant hurdles limiting CAR-T therapy were from a paucity of differentially expressed cell surface molecules on solid tumors that can be safely targeted. Here, we present TSH receptor (TSHR) as a putative target for CAR-T therapy of differentiated thyroid cancer (DTC). We undertook a large-scale screen on thyroid cancer tissues and multiple internal organs through bioinformatical analysis and immunohistochemistry to date TSHR expression. Using 3 previously described monoclonal antibodies, we generated 3 third-generation CAR-Ts. We tested anti-TSHR CAR-T in vitro activity by T-cell function and killing assay. Then we tested preclinical therapeutical efficacy in a xenograft mouse model of DTC and analyzed mice's physical conditions and histological abnormalities to evaluate anti-TSHR CAR-T's safety. TSHR is highly and homogeneously expressed on 90.8% (138/152) of papillary thyroid cancer, 89.2% (33/37) of follicular thyroid cancer, 78.2% (18/23) of cervical lymph node metastases, and 86.7% of radioactive iodine resistance diseases. We developed 3 novel anti-TSHR CAR-Ts from monoclonal antibodies M22, K1-18, and K1-70; all 3 CAR-Ts mediate significant antitumor activity in vitro. Among these, we demonstrate that K1-70 CAR-T can have therapeutical efficacy in vivo, and no apparent toxicity has been observed. TSHR is a latent target antigen of CAR-T therapy for DTC. Anti-TSHR CAR-T could represent a therapeutic option for patients with locoregional relapsed or distant metastases of thyroid cancer and should be tested in carefully designed clinical trials.

Thyroid-related ophthalmopathy development in concurrence with growth hormone administration

BackgroundThyroid stimulating hormone (TSH) receptor and local infiltrate lymphocytes have been considered as major pathological factors for developing thyroid-related ophthalmopathy. Overexpression of insulin-like growth factor-I (IGF-I) receptor has emerged as a promising therapeutic target for refractory patients. However, the relationship between activation of growth hormone (GH)/IGF-I receptor signaling and development or exacerbation of thyroid ophthalmopathy has not been elucidated. Herein we describe a case that provides further clarification into the association between thyroid-related ophthalmopathy and GH/IGF-I receptor signaling.Case presentationA 62-year-old Japanese female diagnosed with thyroid-related ophthalmopathy was admitted to Kurume University Hospital. She had received daily administration of GH subcutaneously for severe GH deficiency; however, serum IGF-I levels were greater than + 2 standard deviation based on her age and sex. She exhibited mild thyrotoxicosis and elevation in levels of TSH-stimulating antibody. Discontinuation of GH administration attenuated the clinical activity scores of her thyroid-related ophthalmopathy. Additionally, concomitant use of glucocorticoid and radiation therapies resulted in further improvement of thyroid-related ophthalmopathy. The glucocorticoid administration was reduced sequentially, followed by successful termination. Thereafter, the patient did not undergo recurrence of thyroid-related ophthalmopathy and maintained serum IGF-I levels within normal physiological levels.ConclusionsWe describe here a case in which development of thyroid-related ophthalmopathy occurred upon initiation of GH administration. GH/IGF-I signaling was highlighted as a risk factor of developing thyroid-related ophthalmopathy. Additionally, aberrant TSH receptor expression was suggested to be a primary pathophysiological mechanism within the development of thyroid-related ophthalmopathy. Physicians should be aware of the risks incurred via GH administration, especially for patients of advanced age, for induction of thyroid-related ophthalmopathy.