TSH Receptor Expression Research Articles

Presence of TSHR in NK Cells and Action of TSH on NK Cells

Introduction: Thyroid-stimulating hormone receptor (TSHR) is widely expressed in human tissues and cells. TSHR is not only involved in thyroid disease but also in the neuroendocrine-immune regulatory network. However, no study has exclusively focused on the expression and function of TSHR in natural killer (NK) cells. Methods: We studied TSHR expression using reverse transcription PCR to verify TSHR mRNA transcripts in human and mouse NK cells. Human and mouse thyroid and liver tissues as well as peripheral blood mononuclear cells (PBMCs) or spleen lymphoid cells (SLCs) were used as controls. The TSHR protein levels in NK-92 cells were determined by immunofluorescence staining. The function of TSHR in NK cells was investigated by measuring the TSH-stimulated cAMP levels. Results: TSHR mRNA was detected in human and mouse NK cells as well as in NK-92 cells and had the same sequence as that of thyroid-derived, PBMC-derived, and liver-derived mRNA. The TSHR protein was also expressed in the cell membrane of NK-92 cells. Furthermore, the cAMP levels in NK-92 cells were significantly higher after adding 10² mIU/mL of bovine TSH at p < 0.05, which stimulated cAMP production in NK-92 cells. Conclusions: Our findings confirm that TSHR is present and functional in NK cells and provide key clues for the potential regulatory effects of TSH on TSHR in NK cells in the immune system.

The Growth Stimulatory Effects of Thyrotropin and Thyroid Hormones on Thyroid Cancer Depend on Expression of Thyrotropin Receptor and Integrins

Abstract Background: The long-term management of metastatic thyroid cancer (TC) consists of thyrotropin (TSH) suppression with supraphysiologic doses of thyroid hormones (TH) via a negative feedback loop. The goal of TSH suppression is to prevent TSH stimulation of the TSH receptor (TSHR), as it has been shown to promote proliferation of cancer cells. However, TH (T3 and T4) have also been shown to stimulate cancer cell proliferation via αvβ3 integrin signaling. Since both TSH and TH have mitogenic potential, we aimed to investigate which one is a more potent growth stimulus-TSH or TH by analyzing its growth stimulatory effects in TC models. Methods: We analyzed the mRNA expression of TSHR and ITGAV (αv), ITGB3 (β3) integrins in 496 human TC tissue samples, including 65 paired samples of normal tissue (NT) and the corresponding tumor included in The Cancer Genome Atlas (TCGA). We used 13 TC cell lines and analyzed the mRNA expression of 24 genes (4 thyroid-specific genes, 2 TH receptor genes, and 18 integrin genes) with an emphasis on the expression of cell surface receptors αv, β3 integrins, and TSHR. The protein expression of αv, β3, and TSHR was analyzed by immunoblotting. To test the effects of TH and TSH on cell proliferation and expression of αv, β3, and TSHR, cells were treated with varying concentrations of TSH (0.01, 0.1, 1, 10 mIU/mL), T3 (0.1, 1, 10 100 nM) and T4 (1, 10, 100, 1000 nM) for 72 h. Results: Analysis of the RNA seq data from TCGA revealed a significantly higher expression of TSHR in NT compared with TC (log fold change 0.59, p&amp;lt;0.001), lower expression of αv integrin in NT compared with TC (log fold change -0.3, p=0.001), and comparable expression of β3 integrin (log fold change 0.20, p=0.3). Based on the mRNA expression data of 13 TC cell lines, we selected 6 cell lines (FTC133, TPC1, XTC1, OCUT2, C643, THJ16T) characterized by variable αv, β3, and TSHR expression. The TPC1 and OCUT2 cells with high to moderate αVβ3 expression responded to T4 (1000nM; p&amp;lt;0.001) and T3 treatment (100nM; p&amp;lt;0.001) respectively, with increased proliferation, while the cell lines characterized by low to no β3 and/or low αV expression (FTC133, XTC1, C643, and THJ16T) did not change their growth rate in response to TH. The C643 and XTC1 cells characterized by a preserved low-to-moderate TSHR expression responded to TSH treatment (10mIU/mL) with increased proliferation (p&amp;lt;0.05), while the growth curve of cell lines with very low to no TSHR expression (FTC133, TPC1, OCUT1, THJ16T) was not affected. Analysis of the effects of TH and TSH on the mRNA expression of αV, β3, and TSHR was observed to be cell-line specific. Conclusion: The growth stimulatory effects of TSH and TH on TC cells depend on its concentration and expression of TSHR and αVβ3, respectively. Since TC is characterized by relatively lower TSHR and higher αV integrin expression than NT, treatment with supraphysiologic doses of TH in patients with metastatic TC needs to be individualized.

Thyroid‐stimulating hormone level is negatively associated with fertilization rate in patients with polycystic ovary syndrome undergoing in vitro fertilization

In this study, we investigated the effect of thyroid-stimulating hormone (TSH) level on the outcomes of in vitro fertilization (IVF) in patients with polycystic ovary syndrome (PCOS). Data pertaining to 60 patients who underwent IVF between May 2017 and May 2018 were included in the study. Thirty-two patients were diagnosed as PCOS (PCOS group) and 28 patients had tubal infertility (control group). Serum and follicular fluid TSH levels and follicular cAMP level were detected by ELISA. TSH receptor (TSHR) expression level in granulosa cells was quantified by RT-PCR and Western-blot. In the PCOS group, oocyte maturation rate and fertilization rate were significantly lower than those in the control group. Serum and follicular fluid TSH levels and ovarian cyclic AMP (cAMP) level were higher in PCOS group with an upregulation of ovarian TSHR. On multivariate linear regression analysis, fertilization rate showed a negative correlation with TSH levels in serum (B =-0.106, P=0.005) and follicular fluid (B =-0.107, P=0.001). In PCOS patients, TSH levels, both in serum and follicular fluid, were negatively correlated with IVF oocyte maturation rate and fertilization rate. The effect of TSH on controlled ovarian hyper-stimulated oocyte growth was likely mediated by the TSHR/cAMP signaling pathway.

Expression and Role of Thyrotropin Receptors in Proopiomelanocortin-Producing Pituitary Cells.

Background: Thyrotropin (TSH) is well known as the hormone of the anterior pituitary thyrotrophs responsible for acting in the thyroid gland, where it stimulates synthesis and release of thyroid hormones through Gs and Gq/11 protein coupled TSH receptors (TSHRs). Methods: In this study, we examined whether the functional TSHRs are also expressed in cultured rat pituitary cells, using double immunocytochemistry, quantitative reverse transcription-polymerase chain reaction analysis, cAMP and hormone measurements, and single-cell calcium imaging. Results: Double immunocytochemistry revealed the expression of TSHRs in cultured corticotrophs and melanotrophs, in addition to previously identified receptors in folliculostellate cells. The functional coupling of these receptors to the Gq/11 signaling pathway was not observed, as demonstrated by the lack of TSH activation of IP3-dependent calcium mobilization in these cells when bathed in calcium-deficient medium. However, TSH increased cAMP production in a time- and concentration-dependent manner and facilitated calcium influx in single corticotrophs and melanotrophs, indicating their coupling to the Gs signaling pathway. Consistent with these findings, TSH stimulated adrenocorticotropin and β-endorphin release in male and female pituitary cells in a time- and concentration-dependent manner without affecting the expression of proopiomelanocortin gene. Conclusions: These results indicate that TSH is a potential paracrine modulator of anterior pituitary corticotrophs and melanotrophs, controlling the exocytotic but not the transcriptional pathway in a cAMP/calcium influx-dependent manner.

BRAFV600E, hypothyroidism, and human relaxin in thyroid carcinogenesis.

BRAFV600E, a major driver of thyroid cancer, evaluated in the context of thyroid hormones and human relaxin. Immunohistochemical expressions of BRAFV600E, TSH, TSH receptor (TSHR), T4, T3 receptor (T3R), RLNH2, and its receptor, RXFP1, were evaluated in thyroid tumors from a retrospective U.S. population of 481 cancer cases diagnosed in 1983-2004. BRAFV600E was expressed in 52% of all thyroid tumors; expression of other markers ranged from 25% for T4 to 98% for RLNH2. Tumors predominantly exhibited hypothyroid-like conditions characterized by elevated TSH and TSHR and reduced T4. BRAFV600E prevalence was significantly higher in tumors expressing TSH, TSHR, T3R, and RXFP1 and lower in tumors expressing T4. The proportion of BRAFV600E mutation in classic papillary tumors significantly increased from 56 to 72% over the 21-year period of diagnoses, while expression of RXFP1, TSH, TSHR, and T3R decreased in non-tumor. Racial/ethnic differences were observed in thyroid hormone marker expression. Non-tumor expression of TSH, TSHR, and T3R were each associated with shorter overall survival, but did not remain significant after adjustment for demographic and clinical factors. Our study provides the first evidence of the potential interaction of BRAFV600E mutation, relaxin, and thyroid hormones in thyroid carcinogenesis. Moreover, our results suggest that hypothyroidism, influenced by RLNH2 activity, may underlie the development of the majority of thyroid cancers and mediate the role of BRAFV600E in thyroid carcinogenesis. BRAFV600E mutation is increasing in papillary thyroid cancers and may be contributing to the rising incidence of this malignancy.

Papillary Thyroid Cancer Tumor Spheres Cultured by Passaging Without Sorting Exhibit Cancer Stemness.

Cancer stem-like cell (CSC) markers and the role of CSCs derived from papillary thyroid carcinoma (PTC) in pathogenesis are unclear. This study aimed to investigate CSC properties using tumor spheres from passaged PTC cells but without sorting CSCs. To identify the properties of CSCs derived from PTC, the expression of SRY-box transcription factor 2(SOX2), octamer-binding transcription factor 4 (OCT4), Nanog homeobox (NANOG), thyroglobulin (TG), thyroid-stimulating hormone receptor (TSHR), E-cadherin, YES-associated protein 1 (YAP1), and signal transducer and activator of transcription 3 (STAT3) was investigated in tumor spheres serially passaged without sorting CSCs. The cultured tumor spheres had cancer stemness; high expression of OCT4, SOX2, NANOG, and YAP1; low expression of E-cadherin; and varied expression of TG, TSHR, and STAT3. PTC tumor spheres transfected with small interfering RNA targeting YAP1 had fewer CSC properties than the non-transfected tumor spheres did. Tumor spheres derived from PTC cells by passaging without sorting CSCs have more stem-like cell properties, and less differentiation potential. Thus, this simple and cost-effective method can be used for the enrichment of PTC stemness for employment in cell-based models, reducing the need for use of animal models.

OR16-05 Single-Cell Sequencing Identifies Novel Regulators of Thyrotrope Populations and POU1F1-Independent Thyroid-Stimulating Hormone Expression

We implemented single-cell RNA sequencing (scRNAseq) technology as a discovery tool to identify factors enriched in differentiated thyrotropes. Thyroid-stimulating hormone (TSH) is produced in the pars distalis of the anterior pituitary (AP) and primarily acts on the thyroid gland to regulate metabolism through T3/T4. However, TSH is also produced by cells in the pars tuberalis (PT), which is comprised of a thin layer of cells that extends rostrally from the pars distalis along the pituitary stalk to the median eminence in the hypothalamus. TSH produced by PT thyrotropes acts on hypothalamic tanycytes to regulate seasonal reproduction. PT thyrotropes likely descend from rostral tip thyrotropes that arise at e12.5 of mouse development, which transcribe the TSH beta subunit (Tshb) without detectable expression of the transcription factor POU1F1. POU1F1 is required for Tshb transcription in thyrotropes of the adenohypophysis, and it acts synergistically with GATA2 to drive cell fate. The molecular mechanisms driving Tshb expression independently of Pou1f1 in PT thyrotropes are unclear.Thyrotropes are the least abundant endocrine cell-type in the pituitary gland. We used genetic labeling and fluorescence-activated cell sorting (FACS) to enrich for thyrotropes for single-cell sequencing. We performed scRNAseq on 7-day-old GFP-positive pituitary cells from Tshb-Cre; R26-LSL-eYFP and intact whole pituitaries, recovering more than 15,000 cells altogether. We observe two distinct populations of cells expressing Tshb. The larger thyrotrope population has approximately twenty fold higher levels of Tshb and five fold higher Cga transcripts than the smaller population, and they are also distinguished by expression of Pou1f1, TSH-releasing hormone receptor (Trhr), and deiodinase 2 (Dio2), consistent with expectations for AP thyrotropes. The smaller thyrotrope population does not express Pou1f1, but those cells are characterized by expression of TSH receptor (Tshr) and melatonin receptor 1A (Mtnr1a), consistent with expectations for PT thyrotropes. They express mildly increased levels of Eya3 and Six1, although these genes are expressed in other cell-types including AP thyrotropes, stem cells, and gonadotropes. They have two-fold higher levels of Gata2 transcripts and uniquely express the transcription factor Sox14. SOX14 is a SoxB2 family transcription factor that counteracts the transcriptional activity of SoxB1 family members, such as Sox2. In conclusion, our scRNAseq has identified novel markers of PT thyrotropes and unveils novel insights into the similarities and differences in the development and function of pituitary thyrotrope subpopulations.

SAT-471 A Rare Case of Pretibial Myxedema Preceding Graves’ Hyperthyroidism

Introduction:Pretibial myxedema, also known as thyroid dermopathy, is typically seen as a manifestation of long-standing Graves’ disease. We report a rare case of pretibial myxedema as the initial presentation of Graves’ disease in a patient without ocular or overt hyperthyroid symptoms.Case:The patient is a 68 year old male with a past medical history of type 2 diabetes and hypertrophic cardiomyopathy, presenting with a one year history of a mildly pruritic, violaceous plaque of his left shin. He was referred to Dermatology Clinic, and biopsy came back as pretibial myxedema. Of note, his thyroid function studies were normal 6 months prior, and he had no symptoms of active thyroid disease at presentation. Additionally, he did not have any ocular abnormalities on exam. Upon repeat laboratory studies, he had low but detectable TSH, normal free T4, mildly elevated free T3, and elevated TSI/TRAb, overall consistent with early Graves’ hyperthyroidism. His pretibial myxedema was treated with high-dose topical steroids with improvement of his dermopathy. For his hyperthyroidism, he was started on low dose methimazole and never developed Graves’ ophthalmopathy.Discussion:Although the exact pathophysiology of pretibial myxedema is unknown, it is thought to be mediated by TSH receptor expression on fibroblasts, similar to Graves’ ophthalmopathy. It is usually seen in patients with active or longstanding Graves’ disease, often with high levels of thyroid receptor antibodies and concomitant Graves’ ophthalmopathy. However, there are a few cases of pretibial myxedema occurring in euthyroid patients with or without ocular symptoms, usually in the setting of elevated TSI, TRAb, or TPO antibodies. Our patient is a rare case of pretibial myxedema preceding active thyroid disease without ophthalmologic manifestations. Patients with hyper-pigmented, non-pitting plaques or nodules on pretibial regions should prompt providers to consider pretibial myxedema and test for thyroid studies, even in those without symptoms of hyperthyroidism or Graves’ ophthalmopathy.

TSH Receptor Homodimerization in Regulation of cAMP Production in Human Thyrocytes in vitro.

Thyrotropin hormone (TSH) was reported to exhibit biphasic regulation of cAMP production in human thyroid slices; specifically, upregulation at low TSH doses transitioning to inhibition at high doses. We observed this phenomenon in HEK293 cells overexpressing TSH receptors (TSHRs) but in only 25% of human thyrocytes (hThyros) in vitro. Because TSHR expression in hThyros in vitro was low, we tested the hypothesis that high, in situ levels of TSHRs were needed for biphasic cAMP regulation. We increased expression of TSHRs by infecting hThyros with adenoviruses expressing human TSHR (AdhTSHR), measured TSH-stimulated cAMP production and TSHR homodimerization. TSHR mRNA levels in hThyros in vitro were 100-fold lower than in human thyroid tissue. AdhTSHR infection increased TSHR mRNA expression to levels found in thyroid tissue and flow cytometry showed that cell-surface TSHRs increased more than 15-fold. Most uninfected hThyro preparations exhibited monotonic cAMP production. In contrast, most hThyro preparations infected with AdhTSHR expressing TSHR at in vivo levels exhibited biphasic TSH dose responses. Treatment of AdhTSHR-infected hThyros with pertussis toxin resulted in monotonic dose response curves demonstrating that lower levels of cAMP production at high TSH doses were mediated by Gi/Go proteins. Proximity ligation assays confirmed that AdhTSHR infection markedly increased the number of TSHR homodimers. We conclude that in situ levels of TSHRs as homodimers are needed for hThyros to exhibit biphasic TSH regulation of cAMP production.

Ameliorative effect of Aloe gel against L-T4-induced hyperthyroidism via suppression of thyrotropin receptors, inflammation and oxidative stress.

Untreated hyperthyroidism may develop serious complications. This attempt was made to investigate the potential of Aloe vera gel in regulating experimentally induced hyperthyroidism in rats. Female Wistar rats were made hyperthyroid with L-thyroxine (L-T4) at 0.5mg/kg/day, i.p. for 14days and the effects of Aloe vera methanolic fraction (AVMF) (50 or 500mg/kg/day, p.o.,) and a conventional antithyroid drug propylthiouracil (PTU) (10mg/kg, i.p.) for 30days were studied in those hyperthyroid rats. At the end, alterations in serum thyroid hormones and thyroid stimulating hormone (TSH); hepatic 5'mono-deiodinase-1(5'D1) activity, oxidative stress markers and antioxidants; serum inflammatory cytokines and the expression of thyrotropin receptor in thyroid gland were evaluated in all experimental animals. Hyperthyroid condition was confirmed by an increase in thyroid hormone levels and hepatic 5'D-1 activity with a decrease in TSH. However, either AVMF or PTU treatment in hyperthyroid rats decreased the levels of thyroid hormones and 5'D1 activity. AVMF administration in T4-induced rats also decreased the oxidative stress markers such as thiobarbituric acid reactive substances and lipid hydroperoxides and increased the antioxidant levels in liver tissues. Levels of liver marker enzymes, cytokines and different lipids were decreased in T4-induced AVMF treated rats. Further, a down regulation in the TSHR expression in thyroid was observed in AVMF or PTU treated groups. All these thyroid inhibiting effects were supported by an improvement in thyroid histology in hyperthyroid rats. It appears, about 15 compounds, as evidenced byLC-MS/MS study, mostly phenolics are involved in this anti-thyroid effects of the test compound.

No detection of TSH or TSHR in oral lichen planus lesions in patients with or without hypothyroidism

Objective: An association between hypothyroidism (HT) and oral lichen planus (OLP) has been reported. However, the mechanisms that could explain this association remain unresolved. This study aimed to evaluate the expression of thyroid-stimulating hormone (TSH) and thyroid-stimulating hormone receptor (TSHR) in healthy oral mucosa and in OLP lesions of individuals with and without HT.Material and methods: Immunohistochemical expression of TSH and TSHR was studied in oral mucosal biopsies obtained from 14 OLP patients with HT, 14 OLP patients without HT and 10 healthy controls without oral mucosal lesions. Gene expression of TSHR was investigated by using three different PCR techniques in oral mucosal samples from 7 OLP patients with HT, 3 OLP patients without HT, 9 healthy controls and in cultured human oral epithelial cells. Gene expression of TSH was examined by employing 2 PCR techniques in oral mucosal samples from 2 OLP patients with HT, 2 OLP patients without HT and 4 healthy controls.Results: TSH and TSHR stainings were negative in the studied oral mucosal specimens. Gene quantification assays demonstrated negative gene expression of TSH and TSHR in clinical and in vitro samples.Conclusions: These results suggest that TSH and TSHR may not be commonly involved in the pathogenetic mechanism that could explain the association between OLP and hypothyroidism.

Thyrotropin, but Not Thyroid-Stimulating Antibodies, Induces Biphasic Regulation of Gene Expression in Human Thyrocytes.

Background: Thyrotropin (TSH) and thyroid-stimulating antibodies (TSAbs) activate TSH receptor (TSHR) signaling by binding to its extracellular domain. TSHR signaling has been studied extensively in animal thyrocytes and in engineered cell lines, and differences in signaling have been observed in different cell systems. We, therefore, decided to characterize and compare TSHR signaling mediated by TSH and monoclonal TSAbs in human thyrocytes in primary culture. Methods: We used quantitative reverse transcription-polymerase chain reaction to measure mRNA levels of thyroid-specific genes thyroglobulin (TG), thyroperoxidase (TPO), iodothyronine deiodinase type 2 (DIO2), sodium-iodide symporter (NIS), and TSHR after stimulation by TSH or two monoclonal TSAbs, KSAb1 and M22. We also compared secreted TG protein after TSHR activation by TSH and TSAbs using an enzyme-linked immunosorbent assay. TSHR cell surface expression was determined using fluorescence activated cell sorting (FACS). Results: We found that TSH at low doses increases and at high doses (>1 mU/mL) decreases levels of gene expression for TSHR, TG, TPO, NIS, and DIO2. The biphasic effect of TSH on signaling was not caused by downregulation of cell surface TSHRs. This bell-shaped biphasic dose-response curve has been termed an inverted U-shaped dose-response curve (IUDRC). An IUDRC was also found for TSH-induced regulation of TG secretion. In contrast, KSAb1- and M22-induced regulation of TSHR, TG, TPO, NIS, and DIO2 gene expression, and secreted TG followed a monotonic dose-response curve that plateaus at high doses of activating antibody. Conclusions: Our data demonstrate that the physiological activation of TSHRs by TSH in primary cultures of human thyrocytes is characterized by a regulatory mechanism that may inhibit thyrocyte overstimulation. In contrast, TSAbs do not exhibit biphasic regulation. Although KSAb1 and M22 may not be representative of all TSAbs found in patients with Graves' disease, we suggest that persistent robust stimulation of TSHRs by TSAbs, unrelieved by a decrease at high TSAb levels, fosters chronic stimulation of thyrocytes in Graves' hyperthyroidism.

Insulin-like growth factor-1 enhances the expression of functional TSH receptor in orbital fibroblasts from thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO), an autoimmune disease, occurs in approximately 50 % of patients with Graves' hyperthyroidism. Thyroid-stimulating hormone receptor (TSHR) that is expressed in orbital fibroblasts is the autoimmune target in the development of TAO. In addition to thyroid-stimulating immunoglobulin (TSI), insulin-like growth factor (IGF)-1 is also involved in the development of TAO. IGF-1 has been reported to potentiate the effects of thyroid-stimulating hormone (TSH) and TSI on TSHR signaling. In the current study, we investigated the effects of IGF-1 on the cell surface expression of the functional TSHR and its possible mechanism of action in human orbital fibroblasts. Our results show that orbital fibroblasts from the TAO patients expressed higher levels of IGF-1 receptor (IGF-R), compared to control subjects. Treatment with IGF-1 enhanced the expression of surface TSHR in orbital fibroblasts from TAO patients, but not from control subjects. In addition, treatment with IGF-1 increased the level of TSHR at both the protein and mRNA levels. Furthermore, pre-treatment with IGF-1 potentiated TSH-induced cAMP production, compared to cells that were treated with only TSH. Our results also show that pre-treatment with cycloheximide, an inhibitor of mRNA translation, partially, but not completely, inhibited the expression of TSHR on the cell surfaces of orbital fibroblasts from TAO patients. These collective results show that IGF-1enhances the cell surface expression of functional TSHR, not only by increasing TSHR expression, but also by inducing TSHR translocation to the plasma membrane in orbital fibroblasts from TAO.

TSHR Signaling Stimulates Proliferation Through PI3K/Akt and Induction of miR-146a and miR-155 in Thyroid Eye Disease Orbital Fibroblasts.

PurposeTo investigate the molecular pathways that drive thyroid stimulating hormone receptor (TSHR)–induced cellular proliferation in orbital fibroblasts (OFs) from thyroid eye disease (TED) patients.MethodsOrbital fibroblasts from TED and non-TED patients were treated with TSH and changes in gene expression and proliferation were measured. To determine the role of TSHR, TSHR-specific siRNA was used to deplete TSHR levels. Proliferation was measured by bromodeoxyuridine (BrdU) incorporation. PI3K/Akt activation was analyzed by Western blot. The PI3K inhibitor LY294002 was used to investigate PI3K/Akt signaling in OF proliferation. Expression of TSHR, inflammatory cytokines, proliferation related genes and miR-146a and miR-155 were measured by qPCR.ResultsOrbital fibroblasts from TED patients proliferate significantly more than non-TED OFs in response to TSH. TSH-induced proliferation was dependent upon TSHR expression and required the PI3K/Akt signaling cascade. TSHR activation stimulated miR-146a and miR-155 expression. TED OFs produced significantly more miR-146a and miR-155 than non-TED OFs. MiR-146a and miR-155 targets, ZNRF3 and PTEN, which both limit cell proliferation, were decreased in TSH treated OFs.ConclusionsThese data reveal that TSHR signaling in TED OFs stimulates proliferation directly through PI3K/Akt signaling and indirectly through induction of miR-146a and miR-155. MiR-146a and miR-155 enhance TED OF proliferation by reducing expression of target genes that normally block cell proliferation. TSHR-dependent expression of miR-146a and miR-155 may explain part of the fibroproliferative pathology observed in TED.

Expression of proteins related to thyroid hormone function in the uterus is down-regulated at the day of implantation in hypothyroid pregnant rats.

Hypothyroidism has been linked to infertility, but the mechanisms underlying infertility-related hypothyroidism have yet to be fully elucidated. Therefore, in this study, effects of hypothyroidism on expression of the proteins related to thyroid hormone function in the uterus, which were thought to play a role implantation, including thyroid hormone receptor (TR), thyroid stimulating hormone receptor (TSHR), retinoic acid receptor (RAR) and extracellular kinase (ERK) were identified. Pregnant female rats were rendered hypothyroid by giving methimazole (MMI), orally. Following hypothyroid induction, rats were grouped into control (non-treated) and received subcutaneous thyroxine at 20, 40, and 80 μg/kg/day for five consecutive days. At Day 6, which is the day of implantation (GD 6), rats were sacrificed and the number of embryo implantation site in the uterus was calculated. Then, uterine horns were harvested and expression of the above proteins and their mRNAs were identified by Western blotting and real-time PCR, respectively. In non-treated hypothyroid pregnant rats, the number of embryo implantation sites decreased as compared to euthyroid and hypothyroid rats receiving thyroxine treatment. Similarly, expression of TRα-1, TRβ-1, TSHR, ERK1/2 and RAR proteins and mRNA in the uterus of non-treated hypothyroid rats also decreased (P < 0.05 when compared to euthyroid and thyroxine-treated hypothyroid rats). In conclusion, downregulated expression of the thyroid hormone related proteins in the uterus at the day of implantation might result in infertility as reported in hypothyroid condition.

Association of BRAF gene and TSHR with cervical lymph node metastasis of papillary thyroid microcarcinoma.

Differences in BRAF gene mutation frequency and thyroid-stimulating hormone receptor (TSHR) protein expression in thyroid tissues were detected to investigate their association with local tissue invasion and cervical lymph node metastasis potential of papillary thyroid microcarcinoma (PTMC). The BRAF gene mutation frequency and TSHR expression in PTMC patients were detected via qPCR and immunohistochemical method, and the association between them was discussed combined with the clinical and pathological parameters. Kruskal-Wallis test was used for the univariate correlation analyses and comparison of mutation rate and expression rate, and Chi-square test was used for the association of central lymph node metastasis with BRAF gene and TSHR. The BRAFV600E mutation only existed in patients with thyroid cancer. Τhe larger the number of metastatic central lymph nodes was, the higher the proportion of BRAFV600E mutation would be. Τhe BRAFV600E mutation was related to the primary lesion size, capsular infiltration and lymph node metastasis of PTMC (P<0.05). The expression of TSHR in PTMC tissues was < those in thyroid benign lesions and para-carcinoma normal tissues, which was positively associated with the central lymph node metastasis (P<0.05). Τhe low expression of TSHR was related to the primary lesion size, capsular infiltration and metastatic lymph nodes of PTMC (P<0.05). The BRAFV600E and TSHR may be involved in the occurrence and lymphatic metastasis of PTMC. The BRAFV600E mutation has no association with the TSHR protein expression (P=0.256), but the coincidence coefficient indicates that their diagnostic significance in PTMC is not similar, so BRAFV600E mutation and TSHR protein expression can be used jointly in the prediction of invasion and lymph node metastasis of PTMC, which may be more meaningful for clinical guidance.

Thyroid-Stimulating Hormone Receptor Expression on Primary Cultured Human Extraocular Muscle Myoblasts

ABSTRACT Purpose To isolate and culture human extraocular muscle (EOM) myoblasts and facilitate their differentiation to myotubes in vitro, and to determine whether these myoblasts express thyroid-stimulating hormone receptor (TSHR). Materials and methods Human EOM myoblasts were isolated from EOM samples, and identified by immunostaining for PAX7 and MYOD1 (markers of human skeletal myoblasts), and western blot for desmin (muscle marker). In addition, we investigated the expressions of SHOX2 (a genetic marker of EOM myoblasts) and HOXC10 (an exclusive marker of hind-limb muscle-derived myoblasts) by RT-PCR. Fusion index and myotube area were measured to quantify myotube differentiation. TSHR immunostaining and western blot were used to determine the presence of TSHR on human EOM myoblasts and investigate its expression during myogenesis. Results Human EOM myoblasts were immunopositive for PAX7 and MYOD1 staining, and had desmin expression during myogenesis. The EOM-specific gene SHOX2 was detected by RT-PCR, but HOXC10 was not detected. The significant change in both fusion index and myotubes were shown at 8 days after induction of differentiation myotubes. Immunostaining revealed TSHR was expressed on human EOM myoblasts and western blot demonstrated the presence of TSHR protein and highest TSHR protein expression was shown at 10 days after myogenic differentiation. Conclusions Human EOM myoblasts were cultured and underwent myogenic differentiation in vitro. TSHR protein was detected on human EOM myoblasts and increasing TSHR expression during myogenic differentiation.

Expression of sodium/iodide transporters and thyroid stimulating hormone receptors in thyroid cancer patients and its correlation with iodine nutrition status and pathology.

To investigate the relationship between the expression of sodium/iodide transporter (NIS) and thyroid stimulating hormone receptor (TSHR) and iodine nutritional status in patients with thyroid carcinoma. 146 cases of thyroid cancer in Shanghai Gongli Hospital, the Second Military Medical University between February and December 2014 were selected as thyroid cancer group, 120 cases of normal thyroid morphology examined by thyroid ultrasound at the same period were selected as normal group. General information and thyroid function of two groups were recorded and analyzed. H&E staining was used to perform histopathological study on both normal group and thyroid cancer group, and immunohistochemistry was used to detect NIS and TSHR protein expression and position. Reverse transcriptase polymerase chain reaction (RT-PCR) was used for quantitative detection of NIS and TSHR mRNA in the two groups, and the relationship between iodine nutrition and NIS and TSHR expression in thyroid cancer patients was studied. The expression of NIS and TSHR in each group was detected by Western blotting, and the difference in NIS and TSHR expression was analyzed by SPSS 17.0 statistical software. The difference of serum total triiodothyronine (TT3), total thyroxine (TT4), and thyroid stimulating hormone (TSH) levels between the normal group and the thyroid cancer group was statistically significant (p < 0.05). H&E staining showed that the histopathology of the thyroid cancer group was significantly different from that of the normal group. Immunohistochemistry showed the positive expression of NIS and TSHR in thyroid cancer group. The expression of NIS and TSHR mRNA and protein in thyroid cancer patients was significantly lower than that in normal group detected by RT-PCR and Western blotting. Analysis of variance showed that the difference of NIS and TSHR expression between the two groups was statistically significant (p < 0.05). These findings indicated that the expression of NIS and TSHR in thyroid cancer is closely related to iodine nutritional status, which has important research value.

A mechanistic linkage between oral lichen planus and autoimmune thyroid disease.

To determine the levels of antithyroid antibodies and thyroid hormones in the sera of patients with oral lichen planus (OLP), and to quantify the expression of thyroid proteins in OLP lesions. Venous blood samples were drawn from 110 patients with OLP who had no history of thyroid disease or levothyroxine supplementation (OLP+/LT4 -). A random population sample of 657 healthy subjects was used as the control group. Two additional groups were used as comparators. Immunohistochemical and qPCR analyses were performed on tissue specimens collected from the patients with OLP and thyroid disease and healthy subjects. No association was found between the presence of antithyroid antibodies and OLP. More patients in the OLP+/LT4 - group showed high levels of thyroid-stimulating hormone and low levels of free thyroxine than were seen in the control group. Thyroid-stimulating hormone receptor was more highly expressed in the OLP lesions of patients with thyroid disease than in the healthy oral mucosa. A significant number of patients with OLP who are not previously diagnosed with thyroid disease have thyroid parameters that are compatible with hypothyroidism. The expression of thyroid-stimulating hormone receptor in OLP lesions suggests that mechanisms related to autoimmune thyroid disease are involved in the aetiology of OLP.

Expression of BANCR promotes papillary thyroid cancer by targeting thyroid stimulating hormone receptor.

Papillary thyroid carcinoma (PTC) is the most common form of non-medullary thyroid cancer, accounting for ~80% of all cases of thyroid cancer. The aim of the present study was to explore the role of BRAF-activated long noncoding RNA (BANCR) in the development of PTC. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), the mRNA expression levels of BANCR, thyroid-stimulating hormone receptor (TSHR) and cyclin D1 between PTC and benign control thyroid nodule tissue samples from 60 patients were determined. Using RT-qPCR and western blot analysis, the expression levels of TSHR and cyclin D1 mRNA and protein were determined in cells transfected with BANCR-small interfering (si)RNA. An MTT assay and flow cytometry were used to analyze the effect of BANCR knockdown on the proliferation and cell cycle distribution of IHH-4 PTC cells. The expression of BANCR, TSHR and cyclin D1 was increased in the PTC group compared with the control group based on the RT-qPCR data. The transfection of IHH-4 cells with BANCR-siRNA induced the inhibition of TSHR and cyclin D1 expression compared with a transfection control. In addition, the proliferation of the IHH-4 cells transfected with BANCR-siRNA was suppressed, relative to the transfection control, and cells arrested in the G0/G1 phase, potentially due to the inhibition of the expression of cyclin D1. The data suggested that the expression of BANCR may promote the development of malignant thyroid nodules via the modulation of TSHR expression and its downstream effector, cyclin D1.