Accumulation Of Thyroglobulin Research Articles

Microcystin-LR induced transgenerational effects of thyroid disruption in zebrafish offspring by endoplasmic reticulum stress-mediated thyroglobulin accumulation and apoptosis

MC-LR can interfere with thyroid function in fish, but the underlying mechanism is still unclear. Current study focuses to study the intergenerational inheritance of MC-LR-induced thyroid toxicity in zebrafish and in rat thyroid cells. In vivo experiments, adult female zebrafish (F0) were exposed to MC-LR (0, 5, and 25 μg/L) for 90 days and mated with male zebrafish without MC-LR exposure to generate F1 generation. F1 embryos were allowed to develop normally to 7 days post-fertilization (dpf) in clear water. In the F0 generation, MC-LR induced disturbance of the hypothalamic-pituitary-thyroid (HPT) axis, leading to a decrease in the production of thyroid hormones. Maternal MC-LR exposure also induced growth inhibition by altering thyroid hormones (THs) homeostasis and interfering with thyroid metabolism and development in F1 offspring. Mechanistically, MC-LR caused excessive accumulation of ROS and induced ER stress that further lead to activation of UPR in the F0 and F1 offspring of zebrafish. Interestingly, our findings suggested that MC-LR exposure hampered thyroglobulin turnover by triggering IRE1 and PERK pathway in zebrafish and FRTL-5 thyroid cells, thus disturbing the thyroid endocrine system and contributing to the thyroid toxicity from maternal to its F1 offspring of zebrafish. Particularly, inhibition of the IRE1 pathway by siRNA could alleviate thyroid development injury induced by MC-LR in FRTL-5 cells. In addition, MC-LR induced thyroid cell apoptosis by triggering ER stress. Taken together, our results demonstrated that maternal MC-LR exposure causes thyroid endocrine disruption by ER stress contributing to transgenerational effects in zebrafish offspring.

Treatment of rat thyrocytes in vitro with cathepsin B and L inhibitors results in disruption of primary cilia leading to redistribution of the trace amine associated receptor 1 to the endoplasmic reticulum

Taar1 is a G protein-coupled receptor (GPCR) confined to primary cilia of rodent thyroid epithelial cells. Taar1-deficient mouse thyroid follicles feature luminal accumulation of thyroglobulin suggesting that Taar1 acts as a regulator of extra- and pericellular thyroglobulin processing, which is mediated by cysteine cathepsin proteases present at the apical plasma membrane of rodent thyrocytes.Here, by immunostaining and confocal laser scanning microscopy, we demonstrated co-localization of cathepsin L, but only little cathepsin B, with Taar1 at primary cilia of rat thyrocytes, the FRT cells. Because proteases were shown to affect half-lives of certain receptors, we determined the effect of cathepsin activity inhibition on sub-cellular localization of Taar1 in FRT cells, whereupon Taar1 localization altered such that it was retained in compartments of the secretory pathway. Since the same effect on Taar1 localization was observed in both cathepsin B and L inhibitor-treated cells, the interaction of cathepsin activities and sub-cellular localization of Taar1 was thought to be indirect. Indeed, we observed that cathepsin inhibition resulted in a lack of primary cilia from FRT cells. Next, we proved that primary cilia are a necessity for Taar1 trafficking to reach the plasma membrane of FRT cells, since the disruption of primary cilia by treatment with β-cyclodextrin resulted in Taar1 retention in compartments of the secretory pathway. Furthermore, in less well-polarized rat thyrocytes, namely in FRTL-5 cells lacking primary cilia, Taar1 was mainly confined to the compartments of the secretory pathway.We conclude that Taar1 localization in polarized thyroid epithelial cells requires the presence of primary cilia, which is dependent on the proteolytic activity of cysteine cathepsins B and L.

Role of iodine in pathogenesis of thyroid disease - is induction of apoptosis consequence of iodine cytotoxicity?

Iodine is one of the best-characterized environmental factors associated with autoimmune thyroid disease (ATD). Epidemiological studies have shown that ATD incidence has increased following the introduction of salt iodination in the 1920s; in addition, ATD patients can improve upon iodine restriction. In animal models such as BioBreeding/Worcester and Buffalo rats, obese chicken strain, and non-obese diabetic H-2h4 mice, excess iodine is associated with autoimmunity. Analyses of Hashimoto thyroiditis (HT) have shown enlarged number of apoptotic follicular cells, and the destruction is an effect of death receptormediated apoptosis. Excess of iodine induces rapid apoptosis of goitrogen Wistar pretreated rats, possibly connected with inhibition of polyamine synthesis, inhibitors of DNA fragmentation. Percentage of apoptotic cells was statistically higher in patients with HT than in those with euthyroid goiter, with significant increase of caspase 32. Genes for Bcl-2 and Bax proteins are under the transcriptional control of p53. In TAD-2 cell cultures, apoptosis is p53-independed, suggesting that DNA damage is not primarily evoked by potassium iodide (KI). High concentrations of NaI increase the proportion of apoptotic cells in FTRL5 thyroid cell line. Iodide cytotoxicity is inhibited by a TPO inhibitor and is relieved with an anti-oxidant agent. Chronic iodine excess induces apoptosis and necrosis of thyroid follicular and endothelial cells, leading to thyroglobulin accumulation in connective tissue. Iodide excess requires peroxidase enzymatic activity to induce apoptosis. Ionic iodide is not directly toxic, whereas its molecular form I2 mediates the apoptotic effect of KI.

Morphological and functional changes in pituitary-thyroid axis following prolonged exposure of female rats to constant light.

Light regulates numerous physiological functions and synchronizes them with the environment, in part by adjusting secretion of different hormones. We hypothesized that constant light (CL) would disturb pituitary-thyroid axis. Our aim was to determine morphological and functional changes in this endocrine system in such extreme conditions and, based on the obtained results, to propose the underlying mechanism(s). Starting from the thirtieth postnatal day, female Wistar rats were exposed to CL (600 lx) for the following 95 days. The controls were maintained under the regular laboratory lighting conditions. After decapitation, pituitaries and thyroids were prepared for further histomorphometric, immunohistochemical, and immunofluorescence examinations. Concentration of thyroid stimulating hormone (TSH), total T4 and T3 (TH) were determined. Thyroid tissue of light-treated rats was characterized by microfollicular structure. We detected no change in total thyroid volume, localization and accumulation of thyroglobulin, thyroid peroxidase, and sodium-iodide symporter in the follicular epithelium of CL rats. The volume of follicular epithelium and activation index were increased, while volume of the colloid and serum levels of TH decreased. In the pituitary, the relative intensity of TSH β-immunofluorescence signal within the cytoplasm of thyrotrophs increased, but their average cell volume and the relative volume density decreased. Serum TSH was unaltered. We conclude that exposure of female rats to CL induced alterations in pituitary-thyroid axis. Thyroid tissue was characterized by microfollicular structure. Serum TH levels were reduced without accompanying increase in serum TSH. We hypothesize that increased secretion and clearance of TH together with unchanged or even decreased hormonal synthesis, resulted in decreased serum TH levels in CL group. We assume this decrease consequently led to increased synthesis and/or accumulation of pituitary TSH. However, decreased average TSH cell volume and relative volume density, together with unchanged serum TSH, point to additional, negative regulation of thyrotrophs.

Thyroglobulin gene is associated with premature ovarian failure

Variants of the thyroglobulin gene were significantly associated with premature ovarian failure in a Korean population. Three single nucleotide polymorphisms and one haplotype were found to be associated with a significant increase in the risk for premature ovarian failure.

Mucin-producing signet ring cell adenoma of the thyroid

Signet ring cell adenoma of the thyroid, though rare, is well documented. This change is chiefly due to intracellular accumulation of thyroglobulin that appears mucinous. Awareness of this entity is important as it may closely simulate a metastatic mucin-secreting signet ring cell carcinoma. Although the mucinous material in signet ring cells has been reported to stain positive with thyroglobulin, in some cases it may not be so. We herein describe a rare case of a 46-year-old man who was hypothyroid and the mass removed from the thyroid showed a mucin-producing signet ring cell adenoma of the thyroid.

Role of thyroglobulin in the pathogenesis of Graves' ophthalmopathy: the hypothesis of Kriss revisited.

One of the hypothesis to explain the pathogenesis of Graves' ophthalmopathy (GO) was formulated by Joseph P. Kriss in the early 1970s. He postulated that the initiating event in the pathogenesis of GO is the deposition and accumulation of thyroglobulin (Tg) in orbital tissues, followed by an autoimmune reaction against Tg. In the last 30 yrs several studies have addressed this hypothesis, through various, different experimental approaches, raising results that are both in favor and against the possibility that Tg plays a role in the pathogenesis of GO. The finding that intact Tg is present in orbital tissues of GO patients supports Kriss' hypothesis, although the role of Tg as an autoantigen seems to be unlikely, as GO is not significantly associated with serum TgAb and mice immunized with Tg do not develop GO. Whether Tg is indeed involved in the pathogenesis of GO remains to be established. Our current view is that, provided that Tg plays a role, it is unlikely the only factor involved and Tg in orbital tissues may rather reinforce or worsen a damage initiated by other mechanisms.

Isolation of a normal human thyroid cell line: hormonal requirement for thyroglobulin regulation.

The long-term culture of functional follicular cells from normal adult human thyroid tissue has been obtained. They were expanded using a 1:2 split ratio until passage 28 (present status) in Click-RPMI medium enhanced with 5% fetal calf serum and diverse associations of hormones or components including porcine insulin and bovine thyrotropin. At passages 10 and 20, chromosome countings showed a normal diploid number and a normal karyotype. In calf serum containing media, cells are epithelial in the presence of thyrotropin (TSH) but present a slight elongated form in the absence of TSH. In serum-free media, 30 minutes after TSH stimulation, both epithelial and elongated cells changed in morphology to stellate-shaped, arborized forms, indicating the presence of functional TSH-receptors even in long term (18 months) TSH-free cultures. Cells produce thyroglobulin constitutively and large amounts of thyroglobulin are easily recovered in TSH-supplemented media, especially in the presence of insulin. Thyroglobulin production was increased versus days under TSH or insulin stimulation. Combination of the two hormones clearly resulted in a synergistic and not an additive effect. The other hormones present in the 6H components (transferrin, glycylhistidyl-lysine, somatostatin, and hydrocortisone) had no positive effect on thyroglobulin accumulation in media in our experimental conditions. Addition of TSH to hormone-free cultures or to insulin-, insulin plus hydrocortisone-, or 5H-containing cultures resulted in a clear increase in thyroglobulin production. Withdrawal of TSH from 6H cultures resulted in a decrease in thyroglobulin accumulation in media. Six months were required to select fibroblast-free cultures and to get passage 6. But only 17 months separated passage 6 to passage 28, indicating that the proliferative rate is increasing with in vitro cell adaptation. Such normal adult thyroid cells, thyroglobulin-producing, TSH, and insulin-sensitive, represent a new normal human thyroid cell line allowing comparative studies with cells originating from pathologic thyroid tissues.

Increased major histocompatibility complex (MHC) expression in nontoxic goiters is associated with iodide depletion, enhanced ability of the follicular thyroglobulin to increase MHC gene expression, and thyroid autoantibodies.

Recent studies suggest that thyroglobulin (TG) accumulated in the follicular lumen of colloid nodular goiters can increase major histocompatibility complex (MHC) class I gene expression in FRTL-5 thyrocytes. Iodide deficiency, also present in these patients, was separately suggested to enhance thyroidal MHC class I and class II gene expression in vivo and in vitro. To test the clinical relevance of these observations, we examined 41 nontoxic goiters surgically removed from patients who had compression problems. Northern analysis revealed that there was a mean 3.9-fold increase in MHC class I expression and a 8.3-fold increase in class II expression by comparison to 9 normal glands. In situ hybridization showed that thyrocytes were the main source of class I and class II transcripts; histological examination revealed that lymphocytic infiltration was minimal to non-existent. The iodine content of the 41 nontoxic goiters was significantly lower than in normal glands, consistent with increased MHC class I and class II. There is also a profound accumulation of TG in the follicles of the nontoxic goiters, and TG purified from the follicles of these glands increased MHC class I gene expression in FRTL-5 thyroid cells significantly more than TG from normal glands per mg protein. Nearly all patients with nontoxic goiter had low, but significantly elevated, levels of antibodies against thyroid peroxidase and/or against TG in their sera compared with those in normal individuals. Moreover, there was a positive correlation between the titer of the serum antibodies against thyroid peroxidase and against TG and MHC class I and class II expression in the thyroid. The data support the possibility that the TG accumulated in the follicular lumen of nontoxic goiters together with relative iodine deficiency contributes to increased MHC expression in thyroid cells in vivo and that increased MHC gene expression contributes to the ability of thyroid antigens to trigger an autoimmune reaction.

Follicular Thyroglobulin Suppresses Iodide Uptake by Suppressing Expression of the Sodium/Iodide Symporter Gene

A major function of the thyrocyte is to take up and concentrate iodide. This is needed for thyroid hormone synthesis and is accomplished by the sodium iodide symporter (NIS), whose expression and activity are up-regulated by TSH. Recently, we reported that follicular thyroglobulin (TG) is a potent suppressor of thyroid-specific gene expression and can overcome TSH-increased gene expression. We suggested this might be a negative feedback, autoregulatory mechanism that counterbalanced TSH stimulation of follicular function. In this report, we support this hypothesis by coordinately evaluating TG regulation of NIS gene expression and iodide transport. We show that physiological concentrations of TG similarly and significantly suppress TSH-increased NIS promoter activity, NIS protein, and NIS-dependent iodide uptake as well as RNA levels. We show, in vivo, that TG accumulation at the apical membrane of a thyrocyte facing the follicular lumen is associated with decreased uptake of radioiodide. It is likely, therefore, that TG suppresses NIS-dependent iodide uptake and NIS gene expression in vivo, as is the case in vitro. RNA levels of NIS and vascular endothelial growth factor/vascular permeability factor, which has been reported to be TSH regulated and possibly associated with TSH-increased iodide uptake, are coordinately decreased by follicular TG as a function of concentration and time. Also, removal of follicular TG from the medium, but not TSH, coordinately returns NIS and vascular endothelial growth factor/vascular permeability factor RNA levels to their TSH-stimulated state. TG accumulated in the follicular lumen appears, therefore, to be a negative feedback regulator of critical TSH-increased follicular functions, iodide uptake, and vascular permeability.

Follicular thyroglobulin suppresses iodide uptake by suppressing expression of the sodium/iodide symporter gene.

A major function of the thyrocyte is to take up and concentrate iodide. This is needed for thyroid hormone synthesis and is accomplished by the sodium iodide symporter (NIS), whose expression and activity are up-regulated by TSH. Recently, we reported that follicular thyroglobulin (TG) is a potent suppressor ofthyroid-specific gene expression and can overcome TSH-increased gene expression. We suggested this might be a negative feedback, autoregulatory mechanism that counterbalanced TSH stimulation of follicular function. In this report, we support this hypothesis by coordinately evaluating TG regulation of NIS gene expression and iodide transport. We show that physiological concentrations of TG similarly and significantly suppress TSH-increased NIS promoter activity, NIS protein, and NIS-dependent iodide uptake as well as RNA levels. We show, in vivo, that TG accumulation at the apical membrane of a thyrocyte facing the follicular lumen is associated with decreased uptake ofradioiodide. It is likely, therefore, that TG suppresses NIS-dependent iodide uptake and NIS gene expression in vivo, as is the case in vitro. RNA levels of NIS and vascular endothelial growth factor/vascular permeability factor, which has been reported to be TSH regulated and possibly associated with TSH-increased iodide uptake, are coordinately decreased by follicular TG as a function of concentration and time. Also, removal of follicular TG from the medium, but not TSH, coordinately returns NIS and vascular endothelial growth factor/vascular permeability factor RNA levels to their TSH-stimulated state. TG accumulated in the follicular lumen appears, therefore, to be a negative feedback regulator of critical TSH-increased follicular functions, iodide uptake, and vascular permeability.

Inhibition of iodine organification and regulation of follicular size in rat thyroid tissue in vitro.

The factors mediating the accumulation of thyroglobulin are of great importance to the understanding of the pathogenesis of human and experimentally induced colloid goiters. To elucidate further the underlying cellular mechanism, thyroid fragments from newborn rats were incorporated into semisolid alginate beads and were cultured as three-dimensional organoids for up to 21 d. In five parallel cultures, the medium contained either no supplements (group A), Nal (group B), thyroid-stimulating hormone (TSH) (group C), Nal plus TSH in the same concentrations as B and C (group D), or Nal and TSH (as in group D) plus methimazole (MMI, group E). The thyroid organoids maintained morphological integrity, functional activity, and ability to proliferate in vitro. Addition of iodine to the cultures significantly increased mean (+/-SEM) follicular diameters from 19.5 +/- 0.7 microm in controls to 33.9 +/- 2.2 microm (p < 0.0001) when NaI was added alone (group B), and 30.4 +/- 1.7 microm (p < 0.0001) when combined with TSH (group D). The effect of NaI on follicular size was abolished by MMI (group E, follicular diameter 23.5 +/- 1.3 microm). The results presented support the recent finding, using a rat colloid goiter model, that not only TSH but also iodine organification or its inhibition are important factors in modulating follicular morphology.

Methimazole increases thyroid-specific mRNA concentration in human thyroid cells and FRTL-5 cells

Methimazole (1-methyl-2-mercaptoimidazole; MMI) increases thyroglobulin mRNA and thyroid peroxidase mRNA concentration in human thyroid cells and in FRTL-5 cells. MMI (1–10,000 μM) gives a dose-dependent increase of thyroglobulin concentration in the medium of human thyroid cells and FRTL-5 cells. The stimulation by MMI has no effect on the TSH-induced cAMP production and occurs in the presence or absence of thyrotropin (TSH). TSH increases the thyroglobulin and thyroid peroxidase mRNA synthesis in human thyroid cells and FRTL-5 cells. The accumulation of thyroglobulin in the medium has an optimum at 100 μU TSH/ml in FRTL-5 cells. This optimum can also be found in most human thyroid cell cultures.

Long-term iodination of thyroglobulin by porcine thyroid cells cultured in porous-bottomed culture chambers: regulation by thyrotrophin

Thyroid cells cultured as monolayers on the porous bottom of culture chambers have been shown to express some specific functions of thyroid follicles. This system, which allows independent access to apical and basal media, is suitable for the long-term study of polarized processes, as the cells maintain their polarized organization. Iodination of thyroglobulin has been investigated under different culture conditions in the presence or absence of TSH. Apical thyroglobulin accumulation, apical iodide concentration and thyroglobulin iodination have been followed simultaneously. Iodide (0.5 mumol/l) was added to basal medium at various stages: only once for 4-day incubations and at each medium change or daily for longer experiments. TSH increased the amount of thyroglobulin secreted into the apical medium by five- to sixfold, whereas high basal iodide concentrations (greater than 5 mumol/l) inhibited thyroglobulin secretion by TSH-stimulated cells. TSH increased iodide uptake giving an iodide concentration ratio between apical and basal media of about 5. Thyroglobulin iodination was dependent upon TSH. Thyroglobulin was iodinated only in the apical compartment. Secretion and iodination of thyroglobulin were polarized phenomena, but the polarity of iodination was total whereas the polarity of secretion was only partial (10% basal secretion). This functional asymmetry was maintained for up to 29 days. The maximal incorporation of iodine into thyroglobulin obtained was never higher than 3.5 atoms/mol. Apical iodide concentrations from 1 to 15 mumol/l, depending on culture conditions, did not increase this value. These results suggest that cells cultured in this culture system are able to reproduce several steps of thyroidal iodide metabolism although there may be unknown factors which could interfere and reduce the efficiency of thyroglobulin iodination.

Thyroglobulin-rich colloid goitres: a result of the combined action of lithium and methimazole on the rat thyroid.

Naturally occurring euthyroid goitres in man and goitres produced in experimental animals by iodine deficiency or goitrogen feeding both have in common a thyroglobulin of low iodine content. The latter experimental goitres are always depleted of colloid and thyroglobulin. In contrast, natural goitres often contain excessive amounts of colloid which may accumulate because of endocytosis becoming refractory to TSH. We tested the hypothesis that minute doses of goitrogens could lower the iodine content of thyroglobulin without colloid depletion. We then examined whether such a low-dose 'classical' goitrogen could induce excessive colloid storage rather than depletion if acting in concert with lithium, a cation which blocks endocytosis. Rats on an adequate iodine intake were fed minimal doses of methimazole either alone or combined with lithium chloride. Chronic minimal-dose methimazole treatment lowered the iodine content of thyroglobulin without changing thyroglobulin content and thyroid weight. In contrast, addition of lithium to methimazole, produced goitres containing supranormal amounts of poorly iodinated thyroglobulin. We conclude that borderline doses of goitrogens can lower iodination of thyroglobulin without causing hyperplasia and colloid depletion. Thyroglobulin-rich goitres can be obtained by adding a second goitrogen which inhibits endocytosis. As an alternative to Marine's hypothesis of colloid goitre formation we suggest that inhibition of endocytosis, e g by goitrogens of the lithium type, could cause colloid and thyroglobulin accumulation in human iodine deficiency goitre.

Differentiation of the thyroid in the hypophysectomized chick embryo

The influence of hypophyseal hormones on the normal development of the thyroid gland was investigated by decapitating chick embryos at a time before the hypophysis has begun to form. Thyroids from decapitated embryos were compared with thyroids of normal embryos from shortly after decapitation to the time when the embryo should hatch. The thyroids were examined for ultrastructural differences and for accumulation of thyroglobulin. Thyroxine levels in the blood were also examined. It was concluded that the thyroids in decapitated embryos develop normally up to about 12 days of development, by which time morphogenesis and differentiation of the gland is essentially complete. The rates of thyroid growth and accumulation of thyroglobulin are diminished after 12 days of development. The amount of thyroxine in the blood is decreased by about one-half. Thyroxine was found in the yolk of unincubated eggs at levels sufficient to sustain blood thyroxine levels throughout embryonic development. We conclude that hormones that are synthesized by the embryonic hypophysis affect thyroid development only through their influence on generalized growth and metabolic activity.

Studies of thyroglobulin of hypophysectomized rats. 3. Evidence for formation and accumulation of thyroglobulin following hypophysectomy. Relationship of iodine content of thyroglobulin and sensitivity to disaggregation in alkali.

Hypophysectomy of rats affects the rate of thyroglobulin synthesis less than its rate of proteolysis. After hypophysectomy the amount of thyroglobulin protein per thyroid gland increases continually and the average iodine concentration of thyroglobulin decreases, indicating that thyroglobulin formed after hypophysectomy is less highly iodinated than the pre-existing iodoprotein which it dilutes. Thyroglobulin of hypophysectomized rats, compared to that of intact rats, when exposed to alkali exhibits an increased susceptibility to disaggregation into 12S subunits which is consistent with its lower degree of iodination. In addition, the data suggest that thyroid glands of both intact and hypophysectomized rats contain thyroglobulin molecules with a spectrum of iodine concentrations and that, in each case, when exposed to alkali, the more lightly iodinated molecules disaggregate into subunits. Thus, the iodine concentration of 12S material formed by alkaline dialysis of thyroglobulin of intact and hypophysec...