Thyroid Plasma Membranes Research Articles

TSH Receptor Oligomers Associated With the TSH Receptor Antibody Reactome.

The TSH receptor (TSHR) and its many forms are the primary antigens of Graves' disease as evidenced by the presence of TSHR antibodies of differing biological activity. The TSH holoreceptor undergoes complex posttranslational changes including cleavage of its ectodomain and oligomer formation. We have previously shown that the TSHR exists in both monomeric and dimeric structures in the thyroid cell membrane and have demonstrated, by modeling, that the transmembrane domains (TMD) can form stable dimeric structures. Based on these earlier simulations of the TSHR-TMD structure and our most recent model of the full-length TSHR, we have now built models of full-length TSHR multimers with and without TSH ligand in addition to multimers of the extracellular leucine-rich domain, the site of TSH and autoantibody binding. Starting from these models we ran molecular dynamics simulations of the receptor oligomers solvated with water and counterions; the full-length oligomers also were embedded in a dipalmitoylphosphatidylcholine bilayer. The full-length TSHR dimer and trimer models stayed in the same relative orientation and distance during 2000 ns (or longer) molecular dynamics simulation in keeping with our earlier report of TMD dimerization. Simulations were also performed to model oligomers of the leucine-rich domain alone; we found a trimeric complex to be even more stable than the dimers. These data provide further evidence that different forms of the TSHR add to the complexity of the immune response to this antigen that, in patients with autoimmune thyroid disease, generate an autoantibody reactome with multiple types of autoantibody to the TSHR.

Adherens Junction Integrity Is a Critical Determinant of Sodium Iodide Symporter Residency at the Plasma Membrane of Thyroid Cells.

While most cases of differentiated thyroid carcinoma (DTC) are associated with a good prognosis, a significant number progress to advanced disease exhibiting aggressive clinical characteristics and often becoming refractory to radioactive iodine (RAI) treatment, the current gold-standard therapeutic option for metastatic disease. RAI-refractoriness is caused by defective functional expression of the sodium-iodide symporter (NIS), which is responsible for the active transport of iodide across the plasma membrane (PM) into thyroid follicles. NIS deficiency in these tumors often reflects a transcriptional impairment, but also its defective targeting and retention at the cells' PM. Using proteomics, we previously characterized an intracellular signaling pathway derived from SRC kinase that acts through the small GTPase RAC1 to recruit and bind the actin-anchoring adaptor EZRIN to NIS, regulating its retention at the PM of both non-transformed and cancer thyroid cells. Here, we describe how by reanalyzing the proteomics data, we identified cell-cell adhesion as the molecular event upstream the pathway involved in the anchoring and retention at the PM. We show that by interacting with NIS at the PM, adherens junction (AJ)-associated P120-catenin recruits and is phosphorylated by SRC, allowing it to recruit RAC1 to the complex. This enables SRC-phosphorylated VAV2 exchange factor to activate RAC1 GTPase, inducing NIS retention at the PM, thus increasing its abundance and function at the surface of thyroid cells. Our findings indicate that the loss of epithelial cell-cell adhesion may contribute to RAI refractoriness, indicating that in addition to stimulating NIS expression, successful resensitization therapies might require the employment of agents that improve cell-cell adhesion and NIS PM retention in refractory TC cells.

Vitamin D and interferon-γ co-operate to increase the ACE-2 receptor expression in primary cultures of human thyroid cells.

BackgroundA more severe course of COVID-19 was associated with low levels of Vitamin D (VitD). Moreover in vitro data showed that VitD up-regulates the mRNA of the Angiotensin Converting Enzyme 2 (ACE-2), the SARS-COV-2 receptor in different type of cells. ACE-2 is expressed in several type of tissues including thyroid cells, on which its mRNA was shown to be up-regulated by interferon-gamma (IFN-γ). The aim of the present study was to investigate if treatment with VitD alone or in combination with IFN-γ would increase ACE-2 both at mRNA and protein levels in primary cultures of human thyrocytes.Materials and methodsPrimary thyroid cell cultures were treated with VitD and IFN-γ alone or in combination for 24 h. ACE-2 mRNA levels were measured by Real-time Polymerase Chain Reaction (RT-PCR). The presence of ACE-2 on thyroid cell membrane was assessed by immunocytochemistry basally and after the previous mentioned treatments.ResultsACE-2 mRNA levels increased after treatment with VitD and IFN-γ alone. The combination treatment (VitD + IFN-γ) showed an additive increase of ACE-2-mRNA. Immunocytochemistry experiments showed ACE-2 protein on thyroid cells membrane. ACE-2 expression increased after treatment with VitD and IFN-γ alone and further increased by the combination treatment with VitD + IFN-γ.ConclusionsVitD would defend the body by SARS-COV2 both by regulating the host immune defense and by up-regulating of the expression of the ACE-2 receptor. The existence of a co-operation between VitD and IFN-γ demonstrated in other systems is supported also for ACE-2 up-regulation. These observations lead to an increased interest for the potential therapeutic benefits of VitD supplementation in COVID-19.

Altered production of membrane-associated proteins, providing iodine accumulation by thyroid gland, after exposure to endocrine disruptor DDT

Exposure to endocrine disruptors has a sufficient impact in rise of thyroid disorders. Iodine accumulation by the thyroid is provided by sodium iodide symporter, a glycoprotein, located in the basolateral membrane of thyroid cells. Therefore, sodium iodide symporter might be a putative target for endocrine disruptors. Objective of the research – investigation of sodium iodide symporter production after long-term exposure to endocrine disruptor dichlorodiphenyltrichloroethane (DDT) in different periods on postnatal development. Newly borne and 7-week old male Wistar rats were exposed to low doses of DDT during 6 and 10 weeks. Expression of sodium iodide symporter in thyroid cells was evaluated by immunohistochemistry. Quantification of sodium iodide symporter, thyroid hormones (T3 and T4) and thyroid stimulating hormone (TSH) in serum was performed by enzyme-linked immunosorbent assay. The investigation also included morphological examination of thyroid slides and morphometry. Adult rats after 6 weeks of exposure showed diminished level of total T4 and increase in T3 production, associated with significantly lowered TSH and sodium iodide symporter serum concentration. Rats, exposed to endocrine disruptor since birth, demonstrated slight decrease in T4 and sodium iodide symporter. Reduced level of free T3 and elevated serum TSH were found in adult rats after 10 weeks of exposure. Exceeded level of serum sodium iodide symporter was revealed. It was provided by increased number of thyroid cells, producing this protein, due to formation of numerous microfollicles in the thyroid gland. Young rats after 10 weeks of exposure demonstrated diminished total and free T3 levels and elevated TSH. No up-regulation of sodium iodide symporter expression was found. Morphological examination revealed enlarged follicles and no microfollicular rearrangement of thyroid parenchyma. Considered that low dose exposure to endocrine disruptor DDT irreversibly depresses expression of membrane glycoprotein sodium iodide symporter, which provides iodine uptake by the thyroid gland. After-birth exposure impairs compensatory activation of proliferation and increase in number of sodium iodide symporter-producing cells.

TNFα-mediated activation of NF-κB downregulates sodium-iodide symporter expression in thyroid cells.

The sodium-iodide symporter (NIS) mediates transport of iodide across the basolateral membrane of thyroid cells. NIS expression in thyroid cancer (TC) cells allows the use of radioactive iodine (RAI) as a diagnostic and therapeutic tool, being RAI therapy the systemic treatment of choice for metastatic disease. Still, a significant proportion of patients with advanced TC lose the ability to respond to RAI therapy and no effective alternative therapies are available. Defective NIS expression is the main reason for impaired iodide uptake in TC and NIS downregulation has been associated with several pathways linked to malignant transformation. NF-κB signaling is one of the pathways associated with TC. Interestingly, NIS expression can be negatively regulated by TNF-α, a bona fide activator of NF-κB with a central role in thyroid autoimmunity. This prompted us to clarify NF-kB’s role in this process. We confirmed that TNF-α leads to downregulation of TSH-induced NIS expression in non-neoplastic thyroid follicular cell-derived models. Notably, a similar effect was observed when NF-κB activation was triggered independently of ligand-receptor specificity, using phorbol-myristate-acetate (PMA). TNF-α and PMA downregulation of NIS expression was reverted when NF-κB-dependent transcription was blocked, demonstrating the requirement for NF-kB activity. Additionally, TNF-α and PMA were shown to have a negative impact on TSH-induced iodide uptake, consistent with the observed transcriptional downregulation of NIS. Our data support the involvement of NF-κB-directed transcription in the modulation of NIS expression, where up- or down-regulation of NIS depends on the combined output to NF-κB of several converging pathways. A better understanding of the mechanisms underlying NIS expression in the context of normal thyroid physiology may guide the development of pharmacological strategies to increase the efficiency of iodide uptake. Such strategies would be extremely useful in improving the response to RAI therapy in refractory-TC.

Different expression of sodium-iodide importer (NIS) between lactating breast and thyroid tissues may be due to structural difference of thyroid-stimulating hormone receptor (TSHR).

Thyroid-stimulating hormone (TSH) binds TSH receptor (TSHR) on thyroid cell membranes, which will lead activation of cyclic adenosine 3',5'-monophosphate/protein kinase A signaling pathway. Through this pathway, TSHR regulates the expression of sodium-iodide symporter (NIS) to complete iodine intake. In recent studies, it is found that TSHR is widely expressed in a variety of extra-thyroidal tissues. TSHR expressions as well as distribution in normal mammary gland tissues have not been reported. The physiological mechanism of the TSHR in the extra-thyroidal tissues has also been controversial. In this study, immunohistochemistry and immunofluorescence were used to characterize the expression distribution of TSHR protein in lactating breast. DNA sequence of TSHR cDNA from mice lactating breast was determined and then compared with TSHR cDNA from mice thyroidal tissue. A 173 amino acid (AA) fragment deletion was found in the extra-cellular domain of lactating breast TSHR. The expression levels of NIS mRNA were compared between two tissues, and the level of NIS mRNA in lactating breasts was lower than the one in thyroidal tissues. The lower expression of NIS in lactating breast may be due to the 173 AA deletion in the TSHR resulting the lower binding of TSH to the TSHR. For the first time, this finding may explain the reason of the lower NIS expression in lactating breast.

Helicobacter Pylori Infection: Association with Hashimoto’s Thyroiditis

Autoimmune thyroid diseases (ATDS) are multifactorial conditions that result from genetic predisposition in combination with environmental risk factors. Helicobacter Pylori (HP) infection as an environmental risk factor has been proposed to initiate the antigenic components of the thyroid cell membrane and may play a leading role in the onset of the auto immune diseases such as Hashimoto's thyroiditis (HT). 1.1. The Aim: To investigate the association between H.P infection and HT and the prevalence of cag. A positive strains H.P in the patients. 1.2. Methods:The study include 40 female patients with Hashimoto's thyroiditis as a case group and 30 healthy female as control group. HP antigen in stool samples using unamplified enzyme immunoassay and Cag. Ainserum samples using an enzyme linked immunoassay method (ELISA). Anti-thyroid peroxidase antibodies (anti TPO) and thyroid function tests thyroid stimulating hormone (TSH) and thyroxine (T4) were determined. 1.3. Results:There is statistically significant association between H P and HT (Odd ratio = 8.1, 95% confidence interval: 3-30.1, p<0.001). Also statistically significant association between presence of Cag-A positive strains of HP and HT (odd ratio=5.3, p=0.001). To establish the significant correlation between HP and HT further and more specialized studies are needed. 1.4. Conclusion:There was a significant association between Hashimoto's thyroiditis and H.P infection and significant prevalence of cag. A positive strain of H.P positive patients and HT

Anoctamin-1/TMEM16A is the major apical iodide channel of the thyrocyte.

Iodide is captured by thyrocytes through the Na(+)/I(-) symporter (NIS) before being released into the follicular lumen, where it is oxidized and incorporated into thyroglobulin for the production of thyroid hormones. Several reports point to pendrin as a candidate protein for iodide export from thyroid cells into the follicular lumen. Here, we show that a recently discovered Ca(2+)-activated anion channel, TMEM16A or anoctamin-1 (ANO1), also exports iodide from rat thyroid cell lines and from HEK 293T cells expressing human NIS and ANO1. The Ano1 mRNA is expressed in PCCl3 and FRTL-5 rat thyroid cell lines, and this expression is stimulated by thyrotropin (TSH) in rat in vivo, leading to the accumulation of the ANO1 protein at the apical membrane of thyroid follicles. Moreover, ANO1 properties, i.e., activation by intracellular calcium (i.e., by ionomycin or by ATP), low but positive affinity for pertechnetate, and nonrequirement for chloride, better fit with the iodide release characteristics of PCCl3 and FRTL-5 rat thyroid cell lines than the dissimilar properties of pendrin. Most importantly, iodide release by PCCl3 and FRTL-5 cells is efficiently blocked by T16Ainh-A01, an ANO1-specific inhibitor, and upon ANO1 knockdown by RNA interference. Finally, we show that the T16Ainh-A01 inhibitor efficiently blocks ATP-induced iodide efflux from in vitro-cultured human thyrocytes. In conclusion, our data strongly suggest that ANO1 is responsible for most of the iodide efflux across the apical membrane of thyroid cells.

Serum-negative autoimmune thyroiditis: what’s in a name?

Chronic autoimmune thyroiditis (CAT) is the main cause of hypothyroidism in the general population. Autoantibodies to thyroperoxidase (TPO) and to thyroglobulin (Tg) are the circulating hallmark of this autoimmune thyroid disease. Positive tests for TPO Ab and Tg Ab are present in nearly 90 (TPOAb) and 60 % (Tg Ab) of hypothyroid patients with CAT. This implies that despite the high sensitivity of modern assay methods for thyroid auto Ab, there still is a considerable number of patients presenting with hypothyroidism in whom the humoral markers of thyroid autoimmunity are found to be negative. The widespread use of ultrasound in the diagnostic work-up of thyroid diseases allowed to ascertain that most of these patients display a hypoechoic pattern of the thyroid at ultrasound [1]. Further characterization of this echographic finding indicated that this peculiar echopattern is mainly due to lymphocytes infiltrating the thyroid parenchyma, as confirmed by fine needle aspiration biopsy. On this basis, a new clinical entity was identified, the so-called serumnegative chronic autoimmune thyroiditis (SN-CAT). There are no published studies on large series of patients with SN-CAT, which however is not a rare clinical entity. Although epidemiologic data are lacking, the prevalence of the disease can be roughly estimated at around 5 % of patients presenting with hypothyroidism by taking into consideration the rate of hypothyroid patients showing positive tests for thyroid autoantibodies in published studies [2]. It is likely that in the last two decades, a raise in the prevalence of SN-CAT occurred due to the routine use of ultra-sensitive TSH measurements, which increased the detection rate of patients with subclinical hypothyroidism, and to the widespread use of ultrasound in clinical endocrine practice. A brief discussion on the mechanisms involved in autoimmune thyroid diseases may provide arguments for explaining the physiopathology of SN-CAT. Early studies showed that TPO Ab fix complement and bind TPO expressed on the thyroid cell membrane, thus producing in vitro complement-mediated cytotoxicity of cultured human thyroid cells [3]. However, the notion that in vivo TPO is expressed at the inner follicular pole of thyroid cells, where it is un-accessible to circulating TPO-Ab, clearly indicated that the cytotoxic effect of these autoantibodies is a secondary phenomenon, only occurring when the follicle integrity is already disrupted by auto-reactive T cells. Studies in mothers with circulating TPO Ab confirmed this assumption, because their newborns had a normal thyroid gland in spite of the trans-placental passage of TPO Ab [4]. Nowadays, an array of experimental data demonstrate the prominent pathogenic role of cellular immune responses in autoimmune thyroid diseases and the involvement of proinflammatory cytokines and chemokines in producing thyroid cell apoptosis [5]. Thus, it became evident that chronic autoimmune inflammation does not require the presence of auto-Ab to induce thyroid damage [5]. Whichever its physiopathology, when SN-CAT leads to hypothyroidism there is no difference in treating the condition as compared to treating hypothyroidism due to any other etiology. On the other hand, several methodological and clinical aspects should be taken into consideration to render a correct diagnosis of SN-CAT. This is mainly based on the occurrence of a raised serum TSH associated with a hypoechoic pattern of the thyroid parenchyma at M. Rotondi F. Coperchini F. Magri L. Chiovato (&) Unit of Internal Medicine and Endocrinology, Laboratory for Endocrine Disruptors, Fondazione Salvatore Maugeri I.R.C.C.S, University of Pavia, Via S. Maugeri 10, 27100 Pavia, Italy e-mail: luca.chiovato@fsm.it

Diverse β subunits of heterotrimeric G proteins are present in thyroid plasma membranes

The functioning of heterotrimeric G protein α subunits in the transduction of hormonal signals to appropriate intracellular responses is well recognized. Much less is known about the distribution of isoforms and functions of G protein β subunits. Here, using specific antibodies, we documented that in plasma membranes of the thyroid cell line Nthy-ori 3-1 all Gβ isoforms-Gβ1, Gβ2, Gβ3, Gβ4 and Gβ5 are present, while the Gβ3 occurs in minute amount. In plasma membrane fraction isolated from pooled postoperative thyroids of patients with nodular goiter and Graves’ disease, the Gβ1, Gβ2, Gβ4 and Gβ5 subunits were found, whereas Gβ3 could not be detected.Competition studies revealed that the Gβ2 is the principal Gβ subunit in membranes from cultured thyroid cells, originated from normal thyroid, as well as in membranes from patients’ thyroids. This suggests that Gβ2 subunit cooperates with Gαs subunit, the most active of the Gα variants, during stimulation of adenylate cyclase which constitutes the main route of physiological thyroid stimulation.

Syndromes of resistance to TSH

The resistance to TSH action is a genetic disease characterized by molecular defects hampering the adequate transmission of TSH stimulatory signal into thyroid cells. In principle the defect may affect every step along the cascade of events following the binding of TSH to its receptor (TSHR) on thyroid cell membranes. The phenotypic expressivity of TSH resistance is highly variable going from severe congenital hypothyroidism (CH) with thyroid hypoplasia to mild hyperthyrotropinemia (hyperTSH) associated with an apparent euthyroid state. More severe forms follow a recessive pattern of inheritance and occur in patients with biallelic mutations both causing a severe loss of TSHR function. Differential diagnosis in these cases includes the exclusions of other causes of isolated thyroid dysgenesis. Mildest forms may instead occur in patients with monoallelic TSHR defects following a dominant mode of inheritance. In these cases we described the dominant negative effect exerted by some mutants on the activity of the receptor encoded by the wild type allele. In these cases, differential diagnosis involves the exclusion of autoimmune thyroid disease or pseudohypoparathyroidism associated with defects at the GNAS locus. This review will focus on the variable clinical expression of this disease.

Identification of a Correlation between Helicobacter pylori Infection and Graves’ Disease

Viral and bacterial antigens have been suspected to be able to mimic the antigenic profile of the thyroid cell membrane and to play an important role in the onset of the autoimmune diseases, such as Graves' disease and Hashimoto thyroiditis. The Helicobacter pylori infection is worldwide diffused and is present in the developed countries up to 50% of the population. The presence of the cytotoxin-associated gene A antigens identifies the most virulent strains of the bacterium. Previous studies have demonstrated the possible correlation between the Helicobacter pylori and Hashimoto's thyroiditis but these results are controversial. We studied the prevalence rate of this bacterium in the Graves' disease and two selected subgroups such as the hyperthyroid patients, at the first time of diagnosis, and the euthyroid methimazole-treated patients. We analyzed Helicobacter pylori in fresh stool samples with an enzyme immunoassay method and the presence of cytotoxin-associated gene A antigens with a serological test. Our results show that a significative increased rate of prevalence is present in Graves' patients, when the disease is ongoing, with an overall prevalence of the strains expressing the cytotoxin-associated gene A antigens compared to the control group. The association between the Helicobacter pylori and Graves' disease suggests a possible role of this bacterium in the onset and/or the maintenance of the disease.

Phenotypic changes of the thyrocyte membrane in papillary thyroid carcinoma. A three-dimensional study

Aim of the study was to assess the presence of structural changes in the complex carbohydrate chains of thyroid epithelia undergoing neoplastic transformation. We investigated thyroid cells from neoplastic lesions using a panel of lectins with specific affinity for distinct carbohydrate residues. Sixty samples of thyroid tissue, including normal, hyperplastic and neoplastic lesions were obtained from surgical specimens and blindly evaluated with lectin stains. Confocal microscopy was used to obtain three-dimensional (3-D) images of the samples with a positive reaction. Wheat germ agglutinin (WGA) was consistently positive on the apical membrane of papillary thyroid carcinomas (PTC), was weakly expressed in follicular carcinomas (FC) and resulted negative in normal thyrocytes and in benign conditions. The 3-D microscopy model showed that the WGA staining pattern in light microscopy corresponds to a continuous layer on the luminal surface of both papillary and tubular structures of PTC cells. The other lectins under evaluation did not provide any significant result. In conclusion, in PTC the apical border of thyrocytes showed a strong, specific and consistent staining with WGA. These findings may be related to a modified interaction of thyroglobulin molecule with thyroid cell membrane and with the expression of molecules that are involved in the process of tumorigenesis and tumor progression.

Evaluation of activation of G proteins in response to thyroid stimulating hormone in thyroid gland cells from euthyroid and hyperthyroid cats

To evaluate alterations in ligand-stimulated activity of G proteins in thyroid gland cells of hyperthyroid cats. Membranes of thyroid gland cells isolated from 5 hyperthyroid cats and 3 age-matched euthyroid (control) cats immediately after the cats were euthanatized. Isolated thyroid cell membranes were treated with thyroid-stimulating hormone (TSH), and activation of G protein was quantified by measurement of the binding of guanosine triphosphate gamma labeled with sulfur 35 (GTPgamma(35)S). The separate effects of G-protein inhibitory (G(i)) and G-protein stimulatory (G(s)) proteins were determined by the use of pertussis toxin and cholera toxin, respectively. Thyroid cell membranes from hyperthyroid cats had higher basal GTPgamma(35)S binding than did thyroid cell membranes from euthyroid cats. Thyroid cell membranes from hyperthyroid and euthyroid cats had a concentration-dependent increase in TSH-stimulated GTPgamma(35)S binding over the TSH range of 0 to 100 mU/mL, with maximal activity at 1 to 100 mU/mL for both. The percentage increase in GTPgamma(35)S binding stimulated by TSH was similar in magnitude between the membranes from hyperthyroid and euthyroid cats. The TSH-stimulated activation of G(s) and G(i) was not different between euthyroid and hyperthyroid cats. Ligand-stimulated activation of G proteins was the same in thyroid cell membranes obtained from hyperthyroid and euthyroid cats. Therefore, alterations in inherent G(s) or G(i) activities did not appear to be part of the pathogenesis of hyperthyroidism in cats.

Association of Duoxes with Thyroid Peroxidase and Its Regulation in Thyrocytes

Thyroid hormone synthesis requires H(2)O(2) produced by dual oxidases (Duoxes) and thyroperoxidase (TPO). Defects in this system lead to congenital hypothyroidism. H(2)O(2) damage to the thyrocytes may be a cause of cancer. The objective of the study was to investigate whether Duox and TPO, the H(2)O(2) producer and consumer, might constitute a complex in the plasma membrane of human thyroid cells, thus maximizing efficiency and minimizing leakage and damage. The interaction between Duox and TPO was studied by coimmunoprecipitation and Western blotting of plasma membranes from incubated follicles prepared from freshly resected human thyroid tissue from patients undergoing thyroidectomy, and COS-7 cells transiently transfected with the entire Duoxes or truncated [amino (NH2) or carboxyl (COOH) terminal]. The following results were reached: 1) Duox and TPO from membranes are coprecipitated, 2) this association is up-regulated through the Gq-phospholipase C-Ca(2+)-protein kinase C pathway and down-regulated through the Gs-cAMP-protein kinase A pathway, 3) H(2)O(2) increases the association of Duox1 and Duox2 to TPO in cells and in membranes, and 4) truncated NH(2)- or COOH-terminal Duox1 and Duox2 proteins show different binding abilities with TPO. Coimmunoprecipitations show that Duox and TPO locate closely in the plasma membranes of human thyrocytes, and this association can be modulated by H(2)O(2), optimizing working efficiency and minimizing H(2)O(2) spillage. This association could represent one part of a postulated pluriprotein complex involved in iodination. This suggests that defects in this association could impair thyroid hormone synthesis and lead to thyroid insufficiency and cell damage.

Comparison of Autoantibodies Level in Thyroid Autoimmune Diseases Estimated by Different Methods

Autoantibodies to different thyroid cell components in serum of patients with thyroid disease have been estimated by several methods. The TSH receptor antibodies (TRAb) have been detected in 77% of untreated Graves-Basedow, in 70% of thionamide drugs treated Graves-Basedow patients up to four months of the treatment and in 48% of patients with Hashimoto's thyroiditis. Microsomal antibodies (MAb) and thyroid plasma membrane antibodies (ATMA) have been detected in 84% and 68% respectively, in thionamide drugs treated Graves-Basedow and in 91% and 72% in Hashimoto's thyroiditis. The thyroglobulin antibodies have been detected in 42% of untreated Graves-Basedow, in 47% of thionamide drugs treated Graves-Basedow and in 77% of Hashimoto's thyroiditis. In 19% to 21% of patients with toxic nodular goiter underlying autoimmunological process have been established by the detection of high levels of serum autoantibodies (ATMA and MAb). In the non-toxic nodular goiter, non-thyroid autoimmune diseases and healthy blood donors only thyroglobulin antibodies were detected in a significant number of patients. Other types of antibodies (ATMA or MAb) were detected only in sporadic cases of non-thyroid autoimmune diseases.

Molecular nuclear medicine using sodium/iodide symporter

Iodide uptake occurs across the membrane of thyroid cells via an active transporter, sodium/iodide symporter (NIS). Decreased NIS expression levels account for the reduced iodide uptake in thyroid carcinomas. We found that thyroid cancer patients with positive immunostaining for NIS responded to I-131 therapy better than did the patients with negative immunostaining. Thus, NIS gene can be used for radionuclide gene therapy. Targeted expression of functional NIS in cancer cells would enable these cells to concentrate iodide from plasma and would, therefore, offer the possibility of radioiodine therapy. We and others have shown that gene transfer of NIS into a variety of cell types confers increased radioiodine uptake up to several hundred-fold that of controls. Imaging reporter gene is useful in noninvasively determining the location, duration and magnitude of transgene expression in living animal. We could monitor not only exogenous gene expression, but also endogenous gene expression such as p53 and nuclear receptor activity such as retinoic acid receptor using cis-NIS reporter imaging gene system. This reporter gene imaging can also be used for monitoring cancer cells, stem cells and immune cells. In conclusion, the NIS has the potential to expand molecular nuclear medicine in the near future.

Functional assessment of the thyrotropin receptor-beta subunit.

Posttranslational processing of the TSH receptor (TSHR) involves proteolysis of a single chain holoreceptor into TSHR-alpha (or A) and TSHR-beta (or B) subunits, which remain associated via disulfide bonds and which may then form oligomers. As both uncleaved and cleavage-derived forms of this receptor have been reported to bind TSH and transduce signals, reasons for this cleavage into alpha- and beta-subunits have remained enigmatic. Recently we suggested that TSHR cleavage was related to receptor oligomerization and now we have asked if cleavage influenced the binding of G proteins to this receptor. Furthermore, as TSHR-alpha subunits are subject to shedding from the cell surface membrane, we have examined whether the remaining TSHR-beta subunits could mediate signaling themselves, either constitutively and /or ligand-induced. We found that only the cleaved form of the TSHR in transfected Chinese hamster ovary cells was able to bind Gsalpha protein, suggesting that cleavage of the native TSH receptor was associated with receptor activation. We also found that independently expressed TSHR-beta subunits on stable cell lines were unable to mediate either constitutive or TSH-induced signaling, as monitored by their inability to induce cAMP accumulation. These data suggested that receptor cleavage was intimately associated with receptor activation in the wild-type TSH receptor and that the residual TSHR-beta subunits left on the thyroid cell membrane, after TSHR cleavage and subsequent TSHR-alpha shedding, were essentially silent and did not participate in signal transduction.

Characterization of ThOX Proteins as Components of the Thyroid H2O2-Generating System

We have recently cloned two thyroid-specific cDNAs encoding new members of the NADPH oxidase family. ThOX1 and ThOX2 proteins are colocalized with thyroperoxidase at the apical membrane of human thyroid cells. In the present study we have determined their subcellular localization and maturation in relation to their enzymatic activity. A majority of ThOX proteins accumulated inside the cell and only a small fraction was expressed at the surface. Western blots demonstrated that ThOX's are glycoproteins of 180,000 and 190,000. When totally deglycosylated the molecular weight of both ThOX1 and ThOX2 drops to 160,000. Ca2+ stimulates the basal H2O2 generation in PC Cl3 cells at a level corresponding to 20% of the leukocyte H2O2 production stimulated by PMA. Nonthyroid cell lines transfected with ThOX1 and ThOX2 show only a single immunoreactive band in Western blot analysis, corresponding to the protein of 180,000. This “immature” protein remains exclusively intracellular and does not present any enzymatic activity. This is not modified by coexpression of thyroperoxidase and p22Phox. Transfection of ThOX cDNAs into PLB-XCGD cells does not reconstitute their NADPH oxidase activity. We conclude that (1) the thyroid contains some elements of the leukocyte H2O2-generating system but not all of them; (2) ThOX's are predominantly or exclusively located inside the cell in thyrocytes or in transfected cells, respectively, and as such they are inactive; (3) ThOX's cannot replace gp91Phox in the leukocyte; and (4) the thyroid H2O2-generating system is analogous to the leukocyte system with regard to ThOX's and gp91Phox but very different in other aspects. Additional thyroid-specific components are probably required to get complete protein processing and full enzymatic activity in the thyroid.

Muscarinic receptor subtypes and calcium signaling in Fischer rat thyroid cells

1 A specific and saturable binding site for [ 3H]N-methyl-scopolamine ([ 3H]NMS) was observed in plasma membrane of Fischer rat thyroid (FRT) cells with an equilibrium dissociation constant ( K d ) of 0.11 ± 0.02 nM and a concentration of receptor sites ( B max) of 14.1 ± 3.9 fmol/mg protein. Pharmacological characterization of this binding site using pirenzepine, himbacine, (11(2-diethyl-amino)methyl)-1-piperidinylacetyl-5-11-dihydro-6 H-pyrido(14) benzodiazepine (AF-DX 116), dicyclomine, 4-diphenylacetoxy- N-methylpiperidine methiodide (4-DAMP), and hexahydro-sila-difenidol (HHSD) showed clear differences, in terms of affinities, between these muscarinic receptor antagonists. The order of potency for inhibiting [ 3H]NMS binding was HHSD = dicyclomine > 4-DAMP > pirenzepine = himbacine > AF-DX 116. These findings suggest that the muscarinic receptors found in FRT cells belong to the M 3 subtype. Stimulation of FRT cells with carbachol produced a biphasic and dose-dependent increase in the intracellular calcium concentration ([Ca 2+] i), which was blocked in pretreated cells with atropine and almost abolished by a low concentration of 4-DAMP and HHSD. Removal of extracellular Ca 2+ from the incubation medium reduced the initial transient peak and completely abolished the plateau phase, while the transient phase was markedly reduced by the phospholipase C inhibitor U73122. These data indicate that [Ca 2+] i results from both Ca 2+ influx across Ca 2+ channels and mobilization of Ca 2+ from intracellular Ca 2+ stores. The present data showed the presence of the M 3 muscarinic acetylcholine receptor subtype in plasma membrane of FRT cells, which may influence cellular function via modulation of [Ca 2+] i.