RESUMEN
This study aimed to investigate the expression of microRNAs (miRNAs) -146b-3p, -221-5p, -222-3p, and -21a-3p and the methylation pattern of the thyroid-stimulating hormone receptor (TSHR) gene in blood plasma samples from papillary thyroid cancer (PTC) patients before and after thyroidectomy compared to healthy controls (HCs). This study included 103 participants, 46 PTC patients and 57 HCs, matched for gender and age. Significantly higher preoperative expression levels of miRNAs and TSHR methylation were determined in the PTC patients compared to HCs. Post-surgery, there was a notable decrease in these biomarkers. Elevated TSHR methylation was linked to larger tumor sizes and lymphovascular invasion, while increased miRNA-222-3p levels correlated with multifocality. Receiver operating characteristic (ROC) analysis showed AUCs below 0.8 for all candidate biomarkers. However, significant changes in the expression of all analyzed miRNAs and TSHR methylation levels indicate their potential to differentiate PTC patients from healthy individuals. These findings suggest that miRNAs and TSHR methylation levels may serve as candidate biomarkers for early diagnosis and monitoring of PTC, with the potential to distinguish PTC patients from healthy individuals. Further research is needed to validate these biomarkers for clinical application.
Asunto(s)
Biomarcadores de Tumor , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , MicroARNs , Receptores de Tirotropina , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides , Humanos , MicroARNs/sangre , MicroARNs/genética , Femenino , Masculino , Cáncer Papilar Tiroideo/genética , Cáncer Papilar Tiroideo/sangre , Cáncer Papilar Tiroideo/diagnóstico , Cáncer Papilar Tiroideo/patología , Persona de Mediana Edad , Neoplasias de la Tiroides/sangre , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/diagnóstico , Neoplasias de la Tiroides/patología , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Adulto , Receptores de Tirotropina/genética , Estudios de Casos y Controles , Curva ROCRESUMEN
Congenital hypothyroidism (CHT) is a diverse condition with various genetic etiologies. This study aimed to investigate the utility of next-generation sequencing (NGS) analysis in guiding treatment decisions and predicting prognosis for CHT patients with gland in situ (GIS). A retrospective analysis was conducted on 33 CHT patients with GIS who underwent NGS analysis at a single institution between 2018 and 2023. Patients were classified as having permanent (PCH), transient congenital hypothyroidism, or ambiguous congenital hypothyroidism (ACH) CHT based on their response to levothyroxine discontinuation at 3 years of age. Among the 33 patients, genetic variants were identified in 26, with the most prevalent variants found in DUOX2 (26.92%), TSHR (30.77%), TG (19.35%), and DUOXA2 (19.23%). Patients with high initial thyroid-stimulating hormone levels (>50 mIU/L) and low free thyroxine levels (<0.89 ng/dL) at diagnosis tended to have compound heterozygous or homozygous variants in DUOX2, DUOXA2, and TG, and were more likely to develop PCH. In contrast, patients with heterozygous variants in these genes often exhibited ACH. TSHR variants were associated with diverse clinical manifestations, ranging from PCH to ACH, and were more common in patients with initial thyroid-stimulating hormone levels <50 mIU/L. The study highlights the potential utility of NGS analysis in predicting the clinical course and guiding treatment decisions for CHT patients with GIS. Genetic analysis may aid in determining the appropriate duration of levothyroxine therapy and monitoring strategies, particularly in cases where traditional clinical indicators are inconclusive.
Asunto(s)
Hipotiroidismo Congénito , Oxidasas Duales , Secuenciación de Nucleótidos de Alto Rendimiento , Receptores de Tirotropina , Tiroxina , Humanos , Hipotiroidismo Congénito/genética , Hipotiroidismo Congénito/tratamiento farmacológico , Hipotiroidismo Congénito/diagnóstico , Hipotiroidismo Congénito/sangre , Femenino , Masculino , Estudios Retrospectivos , Oxidasas Duales/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Tiroxina/uso terapéutico , Receptores de Tirotropina/genética , Preescolar , Lactante , Recién Nacido , Tiroglobulina/genética , Tiroglobulina/sangre , Proteínas de la MembranaRESUMEN
Thyroid stimulating hormone (TSH), a glycoprotein synthesized and secreted from thyrotrophs of the pituitary gland, is composed of a glycoprotein hormone common alpha subunit (CGA) and a specific beta subunit (TSHB). The major biological function of TSH is to stimulate thyroidal follicles to synthesize and secrete thyroid hormones through activating its cognate receptor, the thyroid stimulating hormone receptor (TSHR). In the present study, polyclonal antisera against ricefield eel Tshb and Tshr were generated respectively, and the expression of Tshb and Tshr was examined at mRNA and protein levels. RT-PCR analysis showed that tshb mRNA was expressed mainly in the pituitary as well as in some extrapituitary tissues including the ovary and testis. Tshr mRNA was also expressed in a tissue-specific manner, with transcripts detected in tissues including the kidney, ovary, and testis. The immunoreactive Tshb signals in the pituitary were shown to be localized to the inner areas of adenohypophysis which are close to the neurohypophysis of adult ricefield eels. Tshb-immunoreatvie cells in the pituitary of ricefield eel larvae were firstly observed at hatching. The expression of immunoreactive Tshb and Cga was also detected in ricefield eel ovary and testis together with Tshr. In the ovary, immunoreactive Tshb, Cga, and Tshr were observed in oocytes and granulosa cells. In the testis, immunoreactive Tshb was mainly observed in Sertoli cells while immunoreactive Cga and Tshr were detected in germ cells as well as somatic cells. Results of the present study suggest that Tsh may be synthesized both in the ovary and testis locally, which may play paracrine and/or autocrine roles in gonadal development in ricefield eels.
Asunto(s)
Anguilas , Receptores de Tirotropina , Animales , Receptores de Tirotropina/metabolismo , Receptores de Tirotropina/genética , Femenino , Masculino , Anguilas/metabolismo , Anguilas/genética , Testículo/metabolismo , Gónadas/metabolismo , Comunicación Paracrina/fisiología , Ovario/metabolismo , Hipófisis/metabolismo , Tirotropina de Subunidad beta/metabolismo , Tirotropina de Subunidad beta/genética , Comunicación Autocrina/fisiologíaRESUMEN
Thyroid cancer is the most common endocrine cancer, with differentiated thyroid cancers (DTCs) accounting for 95% of diagnoses. While most DTC patients are diagnosed and treated with radioiodine (RAI), up to 20% of DTC patients become RAI refractory (RAI-R). RAI-R patients have significantly reduced survival rates compared to patients who remain RAI-avid. This study explores [89Zr]Zr-TR1402 as a thyroid-stimulating hormone receptor (TSHR)-targeted PET radiopharmaceutical for DTC. [89Zr]Zr-TR1402 was synthesized with a molar activity of 25.9 MBq/nmol by conjugating recombinant human TSH (rhTSH) analogue TR1402 to chelator p-SCN-Bn-deferoxamine (DFO) in a molar ratio of 3:1 (DFO/TR1402) and radiolabeling with 89Zr (t1/2 = 78.4 h, ß+ = 22.7%). As TSHR is absent in commonly available DTC-derived cell lines, TSHR was reintroduced via stable transduction by delivering a lentivirus containing the full-length coding region of the human TSHR gene. Receptor-mediated uptake of [89Zr]Zr-TR1402 was evaluated in vitro in stably transduced TSHR+ and wild-type TSHR- DTC cell lines. In vivo PET imaging was performed on Days 1-3 postinjection in male and female athymic nude mice bearing TSHR+ and TSHR- xenografts, along with ex vivo biodistribution on Day 3 postinjection. In vitro uptake of 1 nM [89Zr]Zr-TR1402 was significantly higher in TSHR+ THJ529T (P < 0.0001) and FTC133 (P < 0.01) cells than in TSHR- THJ529T and FTC133 cells. This uptake was shown to be specific in both TSHR+ THJ529T (P < 0.0001) and TSHR+ FTC133 (P < 0.0001) cells by blocking uptake with 250 nm DFO-TR1402. In vivo PET imaging showed accumulation of [89Zr]Zr-TR1402 in TSHR+ tumors, which was the highest on Day 1. In the male FTC133 xenograft model, ex vivo biodistribution confirmed a significant difference (P < 0.001) in uptake between FTC133+ (1.3 ± 0.1%ID/g) and FTC133- (0.8 ± 0.1%ID/g) tumors. A significant difference (P < 0.05) in uptake was also seen in the male THJ529T xenograft model between THJ529T+ (1.8 ± 0.6%ID/g) and THJ529T- (0.8 ± 0.4%ID/g) tumors. The in vitro and in vivo accumulation of [89Zr]Zr-TR1402 in TSHR-expressing DTC cell lines support the continued preclinical optimization of this approach.
Asunto(s)
Ratones Desnudos , Tomografía de Emisión de Positrones , Receptores de Tirotropina , Neoplasias de la Tiroides , Circonio , Animales , Neoplasias de la Tiroides/diagnóstico por imagen , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Humanos , Ratones , Circonio/química , Tomografía de Emisión de Positrones/métodos , Línea Celular Tumoral , Femenino , Receptores de Tirotropina/metabolismo , Receptores de Tirotropina/genética , Radiofármacos/farmacocinética , Radiofármacos/química , Distribución Tisular , Masculino , Radioisótopos/químicaRESUMEN
Thyroid dyshormonogenesis (TDH) is responsible for 15%-25% of congenital hypothyroidism (CH) cases. Pathogenetic variants of this common inherited endocrine disorders vary geographically. Unraveling the genetic underpinnings of TDH is essential for genetic counseling and precise therapeutic strategies. This study aims to identify genetic variants associated with TDH in Southern Taiwan using whole exome sequencing (WES). We included CH patients diagnosed through newborn screening at a tertiary medical center from 2011 to 2022. Permanent TDH was determined based on imaging evidence of bilateral thyroid structure and the requirement for continuous medication beyond 3 years of age. Genomic DNA extracted from blood was used for exome library construction, and pathogenic variants were detected using an in-house algorithm. Of the 876 CH patients reviewed, 121 were classified as permanent, with 47 (40%) confirmed as TDH. WES was conducted for 45 patients, and causative variants were identified in 32 patients (71.1%), including DUOX2 (15 cases), TG (8 cases), TSHR (7 cases), TPO (5 cases), and DUOXA2 (1 case). Recurrent variants included DUOX2 c.3329G>A, TSHR c.1349G>A, TG c.1348delT, and TPO c.2268dupT. We identified four novel variants based on genotype, including TSHR c.1135C>T, TSHR c.1349G>C, TG c.2461delA, and TG c.2459T>A. This study underscores the efficacy of WES in providing definitive molecular diagnoses for TDH. Molecular diagnoses are instrumental in genetic counseling, formulating treatment, and developing management strategies. Future research integrating larger population cohorts is vital to further elucidate the genetic landscape of TDH.
Asunto(s)
Hipotiroidismo Congénito , Secuenciación del Exoma , Yoduro Peroxidasa , Receptores de Tirotropina , Humanos , Taiwán , Femenino , Masculino , Hipotiroidismo Congénito/genética , Hipotiroidismo Congénito/diagnóstico , Recién Nacido , Yoduro Peroxidasa/genética , Receptores de Tirotropina/genética , Oxidasas Duales/genética , Tiroglobulina/genética , Proteínas de Unión a Hierro/genética , Preescolar , Variación Genética , Mutación , Disgenesias Tiroideas/genética , Disgenesias Tiroideas/diagnóstico , Lactante , AutoantígenosRESUMEN
Thyroid-stimulating hormone receptor autoantibodies (TRAbs) play a crucial role as pathogenic antibodies in both the diagnosis and management of Graves' disease (GD). GD, an autoimmune disease resulting from a combination of genetic and environmental factors, is the most common cause of hyperthyroidism. With advancements in technology for TRAb detection and the availability of automated commercial kits, TRAb has become an essential clinical laboratory marker for the diagnosis of GD, as well as extra-thyroidal manifestations like Graves' ophthalmopathy (GO). This article provides a comprehensive review of TRAb, encompassing its clinical assays along with its significance in the clinical setting.
Asunto(s)
Autoanticuerpos , Enfermedad de Graves , Receptores de Tirotropina , Humanos , Autoanticuerpos/inmunología , Receptores de Tirotropina/inmunología , Enfermedad de Graves/inmunología , Enfermedad de Graves/diagnósticoRESUMEN
The present report describes for the first time a case of diffuse hyperthyroidism in a 30-year-old female patient who had normal levels of thyroid-stimulating hormone receptor antibodies (TSHR-Ab), slightly elevated plasma levels of thyroid hormones, and slightly increased thyroid blood flow. Seven years before, after severe stress, she had Graves' disease with elevated plasma levels of TSHR-Ab. The patient's recent medical history included mental stress and autonomic dysfunction. This report describes a mild form of hyperthyroidism in terms of elevated plasma levels of thyroid hormones and Doppler ultrasonography data; this condition was first defined as 'minor hyperthyroidism'. The examination data suggest a probable secondary role of the immune system and primary role of the autonomic nervous system in the pathogenesis of Graves' disease.
Asunto(s)
Hipertiroidismo , Receptores de Tirotropina , Humanos , Femenino , Adulto , Hipertiroidismo/sangre , Hipertiroidismo/inmunología , Receptores de Tirotropina/inmunología , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Enfermedad de Graves/inmunología , Enfermedad de Graves/sangre , Inmunoglobulinas Estimulantes de la Tiroides/sangre , Hormonas Tiroideas/sangreRESUMEN
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.
Asunto(s)
Oftalmopatía de Graves , Inhibidores de Puntos de Control Inmunológico , Humanos , Oftalmopatía de Graves/inmunología , Oftalmopatía de Graves/etiología , Oftalmopatía de Graves/patología , Animales , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Proteínas de Punto de Control Inmunitario/metabolismo , Proteínas de Punto de Control Inmunitario/genética , Autoanticuerpos/inmunología , Receptor IGF Tipo 1/inmunología , Receptor IGF Tipo 1/metabolismo , Receptores de Tirotropina/inmunología , Receptores de Tirotropina/metabolismoRESUMEN
Background: Familial non-autoimmune hyperthyroidism is a rare disorder characterized by the absence of thyroid autoimmunity, particularly TSH receptor antibody [TRAb]. Objective: The aim of this study was to describe a novel TSHR mutation identified in a family of two siblings and their father. Methods: Two siblings presented for endocrine assessment at ages 7 and 14 years with mild T3 toxicosis, and the father presented at 30 years of age with non-autoimmune thyrotoxicosis. Both siblings were treated with oral antithyroid therapy to achieve reasonable symptom control and thyroid function normalization. The father was treated with oral antithyroid therapy, radioactive iodine, thyroidectomy, and thyroid replacement therapy. Peripheral blood DNA was extracted from both affected siblings and father. Mutation analysis of TSHR was carried out by PCR and Sanger sequencing of both strands of the extracted DNA. Results: Both siblings and their father were heterozygous for the missense TSHR variant c.1855G>C, p.[Asp619His], in exon 10. Conclusions: This novel TSHR variant is associated with T3 toxicosis during childhood. Therefore, early identification and treatment may improve patient outcomes.
Asunto(s)
Hipertiroidismo , Neoplasias de la Tiroides , Humanos , ADN , Hipertiroidismo/genética , Radioisótopos de Yodo , Mutación , Receptores de Tirotropina/genéticaRESUMEN
Background: There is no doubt that both Hashimoto thyroiditis and Graves' disease are autoimmune thyroid diseases (AITDs), but the relationship between anti-nuclear antibody (ANA) and AITDs is poorly studied. The association between thyroid autoantibody levels and ANA positivity was evaluated to assess the role of ANA in AITDs. Methods: We conducted an analysis using data from 1,149,893 patients registered at our hospital and 53,021 patients registered in the National Health and Nutrition Examination Survey databases. We focused on patients with data for thyroid peroxidase antibody (TPOAb)/ANA, TPOAb/immunoglobulin G (IgG), thyroid-stimulating hormone (TSH) receptor antibody (TRAb)/ANA, TRAb/IgG, TSH/ANA, or TSH/IgG. Results: ANA positivity rates were 12.88% and 21.22% in TPOAb/ANA and TSH/ANA patients, respectively. In TPOAb/IgG and TSH/IgG data, high IgG levels (≥15 g/L) were detected in 2.23% and 4.06% of patients, respectively. There were significant differences in ANA positivity rates and high IgG proportions among patients with different TPOAb and TSH levels. TPOAb level was correlated with ANA positivity rate and high IgG proportion, and TSH level was correlated with ANA positivity rate. Regression analysis showed positive correlations between TPOAb levels and ANA positivity risk or high IgG risk, TSH levels and high IgG risk, and elevated TSH and ANA positivity risk. Of patients with TRAb/ANA data, 35.99% were ANA-positive, and 13.93% had TRAb levels ≥1.75IU/L; 18.96% of patients with TRAb/IgG data had high IgG levels, and 16.51% had TRAb levels ≥1.75IU/L. ANA positivity rate and high IgG proportion were not significantly different among different TRAb levels. TRAb levels, ANA positivity risk and high IgG risk were not correlated. Conclusion: ANA positivity and high IgG are related to Hashimoto thyroiditis but not Graves' disease, which implies distinct pathophysiological mechanisms underlying the AITDs.
Asunto(s)
Enfermedad de Graves , Enfermedad de Hashimoto , Humanos , Encuestas Nutricionales , Autoanticuerpos , Enfermedad de Graves/diagnóstico , Receptores de Tirotropina , Inmunoglobulina G , TirotropinaRESUMEN
Background: Thyroid stimulating immunoglobulins (TSI) play a central role in the pathogenesis of Graves' orbitopathy (GO), while soluble interleukin-2 receptor (sIL-2R) is a marker for T-cell activity. We investigated TSI and sIL-2R levels in relation to thyroid function, disease activity and severity and response to treatment with intravenous methylprednisolone (IVMP) in patients with GO. Methods: TSI (bridge-based TSI binding assay), sIL-2R, TSH and fT4 levels were measured in biobank serum samples from 111 GO patients (37 male, 74 female; mean age 49.2 years old) and 25 healthy controls (5 male, 20 female; mean age 39.8 years old). Clinical characteristics and response to treatment were retrospectively retrieved from patient files. Results: Higher sIL-2R levels were observed in GO patients compared to controls (p < 0.001). sIL-2R correlated with fT4 (r = 0.26), TSH (r = -0.40) and TSI (r = 0.21). TSI and sIL-2R concentrations were higher in patients with active compared to inactive GO (p < 0.001 and p < 0.05, respectively). Both TSI and sIL-2R correlated with total clinical activity score (CAS; r = 0.33 and r = 0.28, respectively) and with several individual CAS items. Cut-off levels for predicting active GO were 2.62 IU/L for TSI (AUC = 0.71, sensitivity 69%, specificity 69%) and 428 IU/mL for sIL-2R (AUC = 0.64, sensitivity 62%, specificity 62%). In multivariate testing higher TSI (p < 0.01), higher age (p < 0.001) and longer disease duration (p < 0.01) were associated with disease activity. TSI levels were higher in patients with a poor IVMP response (p = 0.048), while sIL-2R levels did not differ between responders and non-responders. TSI cut-off for predicting IVMP response was 19.4 IU/L (AUC = 0.69, sensitivity 50%, specificity 91%). In multivariate analysis TSI was the only independent predictor of response to IVMP (p < 0.05). Conclusions: High TSI levels are associated with active disease (cut-off 2.62 IU/L) and predict poor response to IVMP treatment (cut-off 19.4 IU/L) in GO. While sIL-2R correlates with disease activity, it is also related to thyroid function, making it less useful as an additional biomarker in GO.
Asunto(s)
Oftalmopatía de Graves , Humanos , Masculino , Femenino , Persona de Mediana Edad , Adulto , Inmunoglobulinas Estimulantes de la Tiroides , Oftalmopatía de Graves/tratamiento farmacológico , Estudios Retrospectivos , Receptores de Tirotropina , TirotropinaRESUMEN
Background: Manifestations of thyroid-associated ophthalmopathy (TAO) vary greatly. Few tools and indicators are available to assess TAO, restricting personalized diagnosis and treatment. Aim: To identify an aptamer targeting thyroid-stimulating hormone receptor (TSHR) and utilize this aptamer to evaluate clinical activity in patients with TAO. Methods: An aptamer targeting TSHR was developed by exponential enrichment and systematic evaluation of TSHR ligands. After truncation and optimization, the affinity, equilibrium dissociation constant, and serum stability of this aptamer were evaluated. The affinity of the TSHR-targeting aptamer to isolated fibrocytes was assessed, as was aptamer internalization by fibrocytes. The mechanism of binding was determined by molecular docking. The correlation between disease manifestations and the percentage of TSHR-positive cells was assessed by correlation analysis. Results: The aptamer TSHR-21-42 was developed to bind to TSHR, with the equilibrium dissociation constant being 71.46 Kd. Isolated fibrocytes were shown to bind TSHR-21-42 through TSHR, with its affinity maintained at various temperatures and ion concentrations. TSHR-21-42 could compete with anti-TSHR antibody, both for binding site to TSHR and uptake by cells after binding. In addition, TSHR-21-42 could bind to leukocytes in peripheral blood, with this binding differing in patients with TAO and healthy control subjects. The percentage of TSHR-positive monocytes, as determined by binding of TSHR-21-42, correlated positively with clinical activity score in patients with TAO, indicating that TSHR-21-42 binding could assess the severity of TAO. Conclusion: This aptamer targeting TSHR may be used to objectively assess disease activity in patients with TAO, by evaluating the percentages of TSHR positive cells in peripheral blood.
Asunto(s)
Aptámeros de Nucleótidos , Monocitos , Receptores de Tirotropina , Humanos , Aptámeros de Nucleótidos/química , Monocitos/metabolismo , Receptores de Tirotropina/metabolismo , Femenino , Simulación del Acoplamiento Molecular , Masculino , Adulto , Persona de Mediana Edad , Técnica SELEX de Producción de Aptámeros/métodosRESUMEN
Differentiated thyroid cancer (DTC) is the predominant type of thyroid cancer, with some patients experiencing relapse, distant metastases, or refractoriness, revealing limited treatment options. Chimeric antigen receptor (CAR)-modified Natural Killer (NK) cells are revolutionary therapeutic agents effective against various resistant cancers. Thyroid-stimulating hormone receptor (TSHR) expression in DTC provides a unique tumor-specific target for CAR therapy. Here, we developed an innovative strategy for treating DTC using modified NK-92 cells armed with a TSHR-targeted CAR. The modified cells showed enhanced cytotoxicity against TSHR-positive DTC cell lines and exhibited elevated degranulation and cytokine release. After undergoing irradiation, the cells effectively halted their proliferative capacity while maintaining potent targeted killing ability. Transfer of these irradiation-treated cells into NSG mice with DTC tumors resulted in profound tumor suppression. NK-92 cells modified with TSHR-CAR offer a promising, off-the-shelf option for advancing DTC immunotherapy.
Asunto(s)
Células Asesinas Naturales , Receptores Quiméricos de Antígenos , Receptores de Tirotropina , Neoplasias de la Tiroides , Receptores de Tirotropina/inmunología , Receptores de Tirotropina/metabolismo , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/terapia , Neoplasias de la Tiroides/inmunología , Humanos , Animales , Células Asesinas Naturales/inmunología , Línea Celular Tumoral , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Ratones , Diferenciación Celular , Ensayos Antitumor por Modelo de Xenoinjerto , Ratones Endogámicos NOD , Proliferación Celular , Citotoxicidad Inmunológica , Inmunoterapia Adoptiva/métodosRESUMEN
Graves' disease is a type of autoimmune hyperthyroidism caused by thyroid-stimulating antibodies (TSAb).1 The combination of a porcine thyroid cell bioassay and cyclic adenosine monophosphate (cAMP) immunoassay (TSAb-enzyme immunoassay; EIA) is a clinically approved TSAb measurement method. Due to the requirement of multiple procedures and a long assay time of 6 h in the TSAb-EIA, a simplified and rapid assay is desired. Herein, we developed a rapid homogeneous TSAb bioassay (rapid-TSAb assay) using the human embryonic kidney cell line (HEK293), engineered to express the human thyroid-stimulating hormone receptor (TSHR), along with a cAMP-dependent luminescence biosensor. The measurement consists of three steps: thawing frozen cells, blood sample addition, and luminescence detection. The procedures can be conducted within 1 h. The World Health Organization International Standard TSAb (NIBSC 08/204) stimulated the cells co-expressing TSHR and cAMP biosensor. The intra- and inter-assay coefficients of variance were < 10%. Stimulation activity using wild-type TSHR and chimeric TSHR (Mc4) almost completely correlated with the tested Graves' disease and normal samples. In the rapid-TSAb assay, the evaluation of 39 samples, including TSHR antibody-positive sera, yielded a sensitivity of 100.0% and a specificity of 90.9%, compared to the TSAb-EIA control. The rapid-TSAb assay enables simple and rapid measurement of TSAb and is promising for improving the diagnosis of autoimmune thyroid diseases.
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Enfermedad de Graves , Receptores de Tirotropina , Animales , Porcinos , Humanos , Células HEK293 , Inmunoglobulinas Estimulantes de la Tiroides , Receptores Acoplados a Proteínas G , Tirotropina , Bioensayo/métodos , AutoanticuerposRESUMEN
Subclinical hyperthyroidism is defined biochemically as a low or undetectable thyroid-stimulating hormone (TSH) with normal thyroid hormone levels. Low TSHR signaling is considered to associate with cognitive impairment. However, the underlying molecular mechanism by which TSHR signaling modulates memory is poorly understood. In this study, we found that Tshr-deficient in the hippocampal neurons impairs the learning and memory abilities of mice, accompanying by a decline in the number of newborn neurons. Notably, Tshr ablation in the hippocampus decreases the expression of Wnt5a, thereby inactivating the ß-catenin signaling pathway to reduce the neurogenesis. Conversely, activating of the Wnt/ß-catenin pathway by the agonist SKL2001 results in an increase in hippocampal neurogenesis, resulting in the amelioration in the deficits of memory caused by Tshr deletion. Understanding how TSHR signaling in the hippocampus regulates memory provides insights into subclinical hyperthyroidism affecting cognitive function and will suggest ways to rationally design interventions for neurocognitive disorders.
Asunto(s)
Hipertiroidismo , beta Catenina , Ratones , Animales , beta Catenina/metabolismo , Receptores de Tirotropina/genética , Receptores de Tirotropina/metabolismo , Vía de Señalización Wnt/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Hipocampo/metabolismo , Neurogénesis/fisiología , Hipertiroidismo/metabolismoRESUMEN
BACKGROUND: Central hypothyroidism (CH) is characterized by low T4 levels and reduced levels or bioactivity of circulating TSH. However, there is a lack of studies on CH-related intestinal maldevelopment. In particular, the roles of TH and TSH/TSHR signaling in CH-related intestinal maldevelopment are poorly understood. Herein, we utilized Tshr-/- mice as a congenital hypothyroidism model with TH deprival and absence of TSHR signaling. METHODS: The morphological characteristics of intestines were determined by HE staining, periodic acid-shiff staining, and immunohistochemical staining. T4 was administrated into the offspring of homozygous mice from the fourth postnatal day through weaning or administrated after weaning. RT-PCR was used to evaluate the expression of markers of goblet cells and intestinal digestive enzymes. Single-cell RNA-sequencing analysis was used to explore the cell types and gene profiles of metabolic alternations in early-T4-injected Tshr-/- mice. KEY FINDINGS: Tshr deletion caused significant growth retardation and intestinal maldevelopment, manifested as smaller and more slender small intestines due to reduced numbers of stem cells and differentiated epithelial cells. Thyroxin supplementation from the fourth postnatal day, but not from weaning, significantly rescued the abnormal intestinal structure and restored the decreased number of proliferating intestinal cells in crypts of Tshr-/- mice. Tshr-/- mice with early-life T4 injections had more early goblet cells and impaired metabolism compared to Tshr+/+ mice. SIGNIFICANCE: TH deprival leads to major defects of CH-associated intestinal dysplasia while TSH/TSHR signaling deficiency promotes the differentiation of goblet cells and impairs nutrition metabolism.
Asunto(s)
Hipotiroidismo , Hormonas Tiroideas , Tirotropina , Animales , Ratones , Hipotiroidismo/complicaciones , Hipotiroidismo/metabolismo , Receptores Acoplados a Proteínas G , Receptores de Tirotropina/genética , Receptores de Tirotropina/metabolismo , Transducción de Señal , Hormonas Tiroideas/metabolismo , Intestinos/patologíaRESUMEN
Background: Genetic defects in the human thyroid-stimulating hormone (TSH) receptor (TSHR) gene can cause congenital hypothyroidism (CH). However, the biological functions and comprehensive genotype-phenotype relationships for most TSHR variants associated with CH remain unexplored. We aimed to identify TSHR variants in Chinese patients with CH, analyze the functions of the variants, and explore the relationships between TSHR genotypes and clinical phenotypes. Methods: In total, 367 patients with CH were recruited for TSHR variant screening using whole-exome sequencing. The effects of the variants were evaluated by in-silico programs such as SIFT and polyphen2. Furthermore, these variants were transfected into 293T cells to detect their Gs/cyclic AMP and Gq/11 signaling activity. Results: Among the 367 patients with CH, 17 TSHR variants, including three novel variants, were identified in 45 patients, and 18 patients carried biallelic TSHR variants. In vitro experiments showed that 10 variants were associated with Gs/cyclic AMP and Gq/11 signaling pathway impairment to varying degrees. Patients with TSHR biallelic variants had lower serum TSH levels and higher free triiodothyronine and thyroxine levels at diagnosis than those with DUOX2 biallelic variants. Conclusions: We found a high frequency of TSHR variants in Chinese patients with CH (12.3%), and 4.9% of cases were caused by TSHR biallelic variants. Ten variants were identified as loss-of-function variants. The data suggest that the clinical phenotype of CH patients caused by TSHR biallelic variants is relatively mild. Our study expands the TSHR variant spectrum and provides further evidence for the elucidation of the genetic etiology of CH.
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Hipotiroidismo Congénito , Humanos , China , Hipotiroidismo Congénito/diagnóstico , Hipotiroidismo Congénito/genética , AMP Cíclico , Oxidasas Duales/genética , Mutación , Fenotipo , Receptores de Tirotropina/genética , TirotropinaRESUMEN
The receptor for thyroid stimulating hormone (TSHR), a GPCR, is the primary antigen in autoimmune hyperthyroidism (Graves' disease) caused by stimulating TSHR antibodies. While we have previously published a full length model of the TSHR, including its leucine rich domain (LRD), linker region (LR) and transmembrane domain (TMD), to date, only a partial LRD (aa 21-261) stabilized with TSHR autoantibodies has been crystallized. Recently, however, cryo-EM structures of the full-length TSHR have been published but they include only an incomplete LR. We have now utilized the cryo-EM models, added disulfide bonds to the LR and performed longer (3000 ns) molecular dynamic (MD) simulations to update our previous model of the entire full-length TSHR, with and without the presence of TSH ligand. As in our earlier work, the new model was embedded in a lipid membrane and was solvated with water and counterions. We found that the 3000 ns Molecular Dynamic simulations showed that the structure of the LRD and TMD were remarkably constant while the LR, known more commonly as the "hinge region", again showed significant flexibility, forming several transient secondary structural elements. Analysis of the new simulations permitted a detailed examination of the effect of TSH binding on the structure of the TSHR. We found a structure-stabilizing effect of TSH, including increased stability of the LR, which was clearly demonstrated by analyzing several intrinsic receptor properties including hydrogen bonding, fluctuation of the LRD orientation, and radius of gyration. In conclusion, we were able to quantify the flexibility of the TSHR and show its increased stability after TSH binding. These data indicated the important role of ligands in directing the signaling structure of a receptor.
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Receptores de Tirotropina , Tirotropina , Receptores de Tirotropina/química , Receptores de Tirotropina/metabolismo , Ligandos , Tirotropina/química , Tirotropina/metabolismo , LeucinaRESUMEN
CONTEXT: Thyroid-stimulating hormone (or thyrotropin) receptor (TSHR) could be a selective target for small molecule ligands to treat thyroid cancer (TC). OBJECTIVE: We report a novel, orally efficacious ligand for TSHR that exhibits proliferation inhibitory activity against human TC in vitro and in vivo, and inhibition of metastasis in vivo. METHODS: A35 (NCATS-SM4420; NCGC00241808) was selected from a sublibrary of >200 TSHR ligands. Cell proliferation assays including BrdU incorporation and WST-1, along with molecular docking studies were done. In vivo activity of A35 was assessed in TC cell-derived xenograft (CDX) models with immunocompromised (NSG) mice. Formalin-fixed, paraffin-embedded sections of tumor and lung tissues were observed for the extent of cell death and metastasis. RESULTS: A35 was shown to stimulate cAMP production in some cell types by activating TSHR but not in TC cells, MDA-T32, and MDA-T85. A35 inhibited proliferation of MDA-T32 and MDA-T85 in vitro and in vivo, and pulmonary metastasis of MDA-T85F1 in mice. In vitro, A35 inhibition of proliferation was reduced by a selective TSHR antagonist. Inhibition of CDX tumor growth without decreases in mouse weights and liver function showed A35 to be efficacious without apparent toxicity. Lastly, A35 reduced levels of Ki67 in the tumors and metastatic markers in lung tissues. CONCLUSION: We conclude that A35 is a TSHR-selective inhibitor of TC cell proliferation and metastasis, and suggest that A35 may be a promising lead drug candidate for the treatment of differentiated TC in humans.
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Proliferación Celular , Receptores de Tirotropina , Neoplasias de la Tiroides , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Humanos , Ratones , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Proliferación Celular/efectos de los fármacos , Receptores de Tirotropina/antagonistas & inhibidores , Receptores de Tirotropina/metabolismo , Ligandos , Línea Celular Tumoral , Administración Oral , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Antineoplásicos/administración & dosificación , Metástasis de la Neoplasia , Simulación del Acoplamiento Molecular , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , FemeninoRESUMEN
NOTCH2 is expressed in pituitary stem cells and is necessary for stem cell maintenance, proliferation, and differentiation. However, the pathways NOTCH2 engages to affect pituitary development remain unclear. In this study, we hypothesized that glycoprotein hormone subunit A2 (GPHA2), a corneal stem cell factor and ligand for the thyroid stimulating hormone receptor (TSHR), is downstream of NOTCH2 signaling. We found Gpha2 is expressed in quiescent pituitary stem cells by RNAscope in situ hybridization and scRNA seq. In Notch2 conditional knockout pituitaries, Gpha2 mRNA is reduced compared with control littermates. We then investigated the possible functions of GPHA2. Pituitaries treated with a GPHA2 peptide do not have a change in proliferation. However, in dissociated adult pituitary cells, GPHA2 increased pCREB expression and this induction was reversed by co-treatment with a TSHR inhibitor. These data suggest GPHA2 is a NOTCH2 related stem cell factor that activates TSHR signaling, potentially impacting pituitary development.