Thyroglobulin may affect telomerase activity in thyroid follicular cells

Telomerase (TA) activity is known to be present in malignant tumor cells, but not in most somatic differentiated cells. TA shows relatively high activity in thyroid cancer cells, but reports vary. This fact prompted us to elucidate whether cell component inhibitors of TA in the thyroid follicles can modulate its activity. The activity of TA extracted from Hela cells was inhibited by mixing with the supernatant fraction of human thyroid tissue extract. To examine the effect of iodine, thyroid hormones (?-T3 and ?-T4) and human thyroglobulin (hTg) contained in the thyroid follicles, ?-T3, ?-T4 and hTg were added to the TRAP assay system in vitro, using TA from Hela cells. Iodine, ?-T3 and ?-T4 did not affect TA activity, but hTg inhibited the TA activity in a dose-dependent manner (IC₅₀ of hTg: ca 0.45 μM: inhibiting concentration of hTg was from 0.15 μM to 3.0 μM). The hTg inhibition was not evident in the RT-PCR system, suggesting no effect of hTg on Taq DNA polymerase activity. The hTg inhibition of TA activity was attenuated by dNTP but not significantly by TS primer. These data suggest that hTg contained in thyroid follicular cells of various thyroid diseases may affect the TA activity measured in biopsied thyroid specimens, and that the reduction of the TA activity by hTg may induce slow progression and growth, and low grade malignancy of thyroid cancer, particularly differentiated carcinoma.     

Inhibition of thyroid-restricted genes by follicular thyroglobulin involves iodinated degree

Follicular thyroglobulin (TG) reflects the storage of both iodine and thyroid hormone. This is because it is a macromolecular precursor of thyroid hormone and organic iodinated compound in follicular lumen. Thus, it may have an important feedback role in thyroid function. In this study, monolayer cells were cultured and follicles were reconstituted with primary pig thyroid cells in vitro. Reconstituted follicles were treated with iodine and methimazole (MMI), a drug that blocks iodine organification and reduces the degree of TG iodination in follicular lumen. The high degree of iodinated TG in follicular lumen was observed to inhibit thyroid-restricted gene expression. To confirm this finding, monolayer thyroid cells were treated with a different degree of TG iodination at the same concentration. These iodinated TG were extracted from reconstituted follicles of different groups. In this manner, this study provides firsthand evidence suggesting that follicular TG inhibits the expressions of thyroid-restricted genes NIS, TPO, TG, and TSHr.     

Thyroid autoregulation. Inhibitory effects of iodinated derivatives of arachidonic acid on iodine metabolism

Thyroid autoregulation has been linked to an organified iodocompound. Since several iodolipids are produced by the gland their possible role in thyroid autoregulation was examined. The following pure synthetic compounds were prepared: 1) 14-iodo-15-hydroxy-5,8,11-eicosatrienoic acid (I-OH-A); 2) its omega lactone (IL-ω);3)5-hydroxy-6-iodo-8,11,14-eicosatrienoic acid delta lactone (IL-δ). Their action on iodine metabolism was studied. Iodine uptake was measured in calf thyroid slices. At 10-⁴M I-OH-A caused a 64% decrease in the T/M ratio while IL-ω inhibited it by 36% and IL-δ was without effect. At 10⁻⁵M the inhibition was 44% for I-OH-A and 19% for IL-ω, while T₃ was without action. A possible isotopic dilution effect was excluded, and no change in iodine efflux was observed. The inhibition by I-OH-A of iodine uptake was observed after only 15 min preincubation. This compound alse decreased ¹²⁵I accumulation in rats.In calf thyroid slice, I-OH-A at 10⁻⁴M, inhibited PB¹²⁵I formation by 80%, IL-ω 62% and IL-ω by 37% and arachidonic acid were without action. I-OH-A also caused a dose-dependent inhibition of TSH-stimulated iodide organification.The present results demonstrate, for the first time, that iodinated derivatives of arachidonic acid inhibition thyroid function and mimic the effect of iodine on thyroid autoregulation.     

Molecular and cellular mechanisms of transepithelial iodide transport in the thyroid

Thyroid hormone is an essential regulator of developmental growth and metabolism in vertebrates. Iodine is a necessary constituent of thyroid hormone. Due to the scarcity and uneven distribution of iodine on the Earth's crust, the structure of the thyroid gland is adjusted to collect and store this element in order to secure a continuous supply of thyroid hormone throughout life. Still, disease resulting from hypothyroidism due to iodine deficiency is a global health problem, illustrating the great biological significance that iodine saving mechanisms have evolved. Iodide is accumulated together with prohormone (thyroglobulin) in the lumen of the thyroid follicles. The rate-limiting step of this transport is the sodium/iodide symporter located in the basolateral plasma membrane of the thyroid follicular cells. Iodide is also transferred across the apical plasma membrane into the lumen where hormonogenesis takes place. In this review, recent progress in the understanding of transepithelial iodide transport in the thyroid is summarized.     

The effect of long-term external ionizing radiation on the functional activity of rat thyroid under enhanced potassium iodide consumption

The study was devoted to the effect of long-term (20 days) external ionizing radiation at a dose of 0.5 Gy on the iodide metabolism in the rat thyroid under supplementation of high iodine doses (10 daily KI doses). It was found that the potassium iodide administration partially prevented the effects of a post radiation decrease of serum thyroid hormone levels (the level of T4 was normal and that of T3 was 77.4% of the controls). After the supplementation of 10 daily iodide doses, the rat thyroid tissue showed the most pronounced increase in the levels of total, free and protein-bound iodide compared to the groups of animals consuming normal and elevated KI doses. Pronounced inhibition of thyroid peroxidase activity (3.1-fold) was noted in the same group. The data obtained indicate a radiation-induced activation of iodide uptake during its enhanced supplementation and disturbed iodide enzymatic oxidation and organification.     

Polarity reversal of inside-out thyroid follicles cultured within collagen gel: reexpression of specific functions

Isolated porcine thyroid cells cultured in suspension in Eagle Minimum Essential Medium supplemented with calf serum (5-20%) reorganize to form vesicles, i.e. closed structures in which all cells have an inverted polarity as compared to that found in follicles: the apical membranes are bathed by the culture medium. Under these conditions, cells neither concentrate iodide nor respond to acute thyrotropin (TSH) stimulation. When embedded in collagen gel, these vesicles undergo polarity reversal to form follicles. We describe here the change in the orientation of cell polarity and the subsequent reappearance of specific thyroid functions. Six hr after embedding, membrane areas in contact with collagen fibers show basal characteristics. At this time, cells begin to concentrate iodide and to respond to acute TSH stimulation (iodide efflux and increased cAMP levels). Most cells form follicles 24 hr after embedding, but 48 hr are required for the transformation of all vesicles into follicles. This occurs without opening of the tight junctions. Iodide organification is detected 24 hr after embedding, when periodic acid-Schiff positive material, identified as thyroglobulin by immunofluorescence, accumulates in the lumen. Iodide concentration and organification, as well as response to TSH stimulation reach maximal levels after 3 days in the collagen matrix. After a 5-day culture in the collagen matrix in the absence of TSH, cell activity can be stimulated by chronic treatment with low hormone concentrations (10-100 microU/ml). As shown with thyroid cells grown in monolayer on permeable substrates (Chambard M., et al., 1983, J. Cell Biol. 96, 1172-1177), iodide uptake and cAMP-mediated TSH responses are expressed when the halogen and the hormone have direct access to the basal membrane. Organification, on the contrary, requires a closed apical compartment.     

Generation of oxygen free radicals in thyroid cells and inhibition of thyroid peroxidase

We examined whether superoxide (O(2)(-)) is produced as a precursor of hydrogen peroxide (H(2)O(2)) in cultured thyroid cells using the cytochrome c method and the electron paramagnetic resonance (EPR) method. No O(2)(-) or its related radicals was detected in thyroid cells under the physiological condition. The presence of quinone, 2,3-dimethoxy-l-naphthoquinone (DMNQ), or 2-methyl-1, 4-naphthoquinone (menadione), in the medium produced O(2)(-) and hydroxyl radicals (OH*); the amount of H(2)O(2) generation was also increased. Incubation of follicles with DMNQ or menadione inhibited iodine organification (a step of thyroid hormone formation) and its catalytic enzyme, thyroid peroxidase (TPO). This inhibition should be caused by reactive oxygen species because the two quinones, particularly DMNQ, exert their effect through the generation of reactive oxygen species. It is speculated that the site-specific inactivation of TPO might have occurred at the heme-linked histidine residue of the TPO molecule, a critical amino acid for enzyme activity because OH* (vicious free radicals) can be formed at the iron-linked amino acid. TPO mRNA level and electrophoretic mobility of TPO were not inhibited by quinones. Our study suggests that thyroid H(2)O(2) is produced by divalent reduction of oxygen without O(2)(-) generation. If thyroid cells happen to be exposed to significant amount of reactive oxygen species, TPO and subsequent thyroid hormone formation are inhibited.     

Effect of potassium iodide and perchlorate on the process of thyroid hormone secretion]

The influence of potassium iodide and perchlorate on the parameters characterizing the thypoid hormones secretion, such as the cAMP level in the gland tissue and the number of intracellular colloid droplets under condition of stimulation by thyrotropic hormone was studied. It was shown that the abovementioned parameters were depressed by an excess of iodide, but perchlorate administration prevented the inhibitory effect of iodide. The results obtained favour the conception on the sensitivity of the thyroid adenylate cyclase system to the organic iodine concentration. Apparently and excess of iodide depressed the capacity of perchlorate to influence its concentration in the gland, and thereby the process of iodine organification and of the thyroid hormone secretion maintained at the optimal leve.     

Thyroid autoregulation. Inhibitory effects of iodinated derivatives of arachidonic acid on iodine metabolism

Thyroid autoregulation has been linked to an organified iodocompound. Since several iodolipids are produced by the gland their possible role in thyroid autoregulation was examined. The following pure synthetic compounds were prepared: 1) 14-iodo-15-hydroxy-5,8,11-eicosatrienoic acid (I-OH-A); 2) its omega lactone (IL-omega); 3) 5-hydroxy-6-iodo-8,11,14-eicosatrienoic acid delta lactone (IL-delta). Their action on iodine metabolism was studied. Iodine uptake was measured in calf thyroid slices. At 10(-4)M I-OH-A caused a 64% decrease in the T/M ratio, while IL-omega inhibited it by 36% and IL-delta was without effect. At 10(-5)M the inhibition was 44% for I-OH-A and 19% for IL-omega, while T3 was without action. A possible isotopic dilution effect was excluded, and no change in iodine efflux was observed. The inhibition by I-OH-A of iodide uptake was observed after only 15 min preincubation. This compound also decreased 125I accumulation in rats. In calf thyroid slices, I-OH-A at 10(-4)M, inhibited PB125I formation by 80%, IL-omega by 62% and IL-delta by 37%. T3 and arachidonic acid were without action. I-OH-A also caused a dose-dependent inhibition of TSH-stimulated iodide organification. The present results demonstrate, for the first time, that iodinated derivatives of arachidonic acid inhibit thyroid function and mimic the effect of iodide on thyroid autoregulation.     

Effects of forskolin on adenylate cyclase,cyclic AMP,protein kinase and intermediary metabolism of the thyroid gland

Forskolin (40 μM) stimulated adenylate cyclase activities of bovine thyroid plasma membranes without pthe addition of guanine nucleotides. GDP had little effect on the forskolin-stimulated adenylate cyclase activity while Gpp[NH]p (0.1–1.0 μM) decreased it. In the presence of TSH (10 mU/0.11), Gpp[NH]p no longer caused inhibition. Forskolin did not affect phosphodiesterase activities of thyroid homogenates. Forskolin (10 μM) rapidly increased cAMP levels in bovine thyroid slices both in the absence and presence of a phosphodiesterase inhibitor. The effect of TSH (50 mU/ml) on cAMP levels was additive or greater than additive to that of forskolin. An initial 2-h incubation of slices with forskolin did not decrease their subsequent cAMP responses to either forskolin and/or TSH while similar treatment of slices with TSH induced desensitization of the cAMP response to TSH, but not to forskolin. Forskolin (10 μM) as well as TSH (50 mU/ml) activated cAMP-dependent protein kinase of slices in the absence of a phosphodiesterase inhibitor. Although forskolin activated the adenylate cyclase cAMP system, it did not stimulate iodide organification or glucose oxidation, effects which have been attributed to cAMP. In fact, forskolin inhibited these parameters and ³²P incorporation into phospholipids as well as their stimulation by TSH. These results indicate that an increase in cAMP levels and cAMP-dependent protein kinase activity in thyroid slices may not necessarily reproduce the effects of TSH on the thyroid.     

Thyroid hormones stimulate Na+–Pi transport activity in rat renal brush-border membranes: Role of membrane lipid composition and fluidity

Antecedent studies have suggested that lipid composition and fluidity of cellular membranes of various organs are altered in response to thyroid hormone status. To date, the effects of thyroid hormone status on these parameters have not been examined in rat renal apical membrane in regard to sodium-dependent phosphate transport. In the present study, we determined the potential role of alterations in cortical brush-border membrane lipid composition and fluidity in modulation of Na⁺–P_i transport activity in response to thyroid hormone status. Thyroid hormone status influences the fractional excretion of Pi, which is associated with alteration in renal brush-border membrane phosphate transport. The increment in Na⁺–P_i transport in renal BBMV isolated from Hyper-T rats is manifested as an increase in the maximal velocity (V_max) of Na⁺–P_i transport. Further, the cholesterol content was significantly increased in renal BBM of Hypo-T rats and decreased in Hyper-T rats as compared to the Eu-T rats. The molar ratio of cholesterol/phospholipids was also higher in renal BBM from hypo-T rats. Subsequently, fluorescence anisotropy of diphenyl hexatriene (rDPH) and microviscosity were significantly decreased in the renal BBM of the Hyper-T rats and increased in the Hypo-T rats as compared to Eu-T rats. The result of this study, therefore, suggest that alteration in renal BBM cholesterol, cholesterol/phospholipid molar ratio, and membrane fluidity play an important role in the modulation of renal BBM Na⁺–P_i transport in response to thyroid hormone status of animals. (Mol Cell Biochem 268: 75–82, 2005)     

Dose-related influence of sodium selenite on apoptosis in human thyroid follicles in vitro induced by iodine, EGF, TGF-β, and H2O2

Apoptosis of thyroid follicular cells is induced by high doses of iodide, epidermal growth factor (EGF), transforming growth factor-β (TGF-β), as well as H₂O₂ and might be attenuated by antioxidants. Therefore, we examined the apoptotic index induced by these substances in selenium-treated vs untreated human thyroid follicular cells. Reconstituted human thyroid follicles were incubated with sodium selenite (10 or 100 nM) for 72 h; controls received none. The follicles were then distributed to 24-well plates and incubated with potassium iodide (5, 10, or 20 nM), EGF (5 ng/mL), TGF-β (5 ng/mL), or H₂O₂ (100 μM). Apoptosis was determined by a mitochondrial potential assay and the number of apoptotic cells counted by two independent, experienced technicians and the glutathione peroxidase (GPx) activity was determined. A significant increase of apoptic cells was obtained in control thyroid follicles treated with iodine (5, 10, or 20 μM), thyroid-stimulating hormone (TSH) 1, or 10 mU/mL in combination with 5 and 10 μM iodine, EGF (5 ng/mL) and TGF-β (5 ng/mL), or H₂O₂ (100 μM) (p<0.001). In contrast, in thyroid follicles preincubated with 10 or 100 nM sodium selenite, the apoptototic index was identical to the basal rate. In H₂O₂-treated follicles, the apoptotic index was still significantly elevated but 50% lower compared to control cells. The GPx activity increased from 1.4±0.2 to 2.25±0.4 mU/μg DNA with 10 nM selenite and 2.6+0.4 mU/μg DNA with 100 nM selenite. Sodium selenite might increase the antioxidative potential in human thyroid follicles in vitro and therefore diminish the apoptosis induced by TGF-β, EGF, iodide, and even H₂O₂     

Quantitative modulation of thyroid iodide peroxidase by thyroid stimulating hormone

The activity of rat thyroid iodide peroxidase fell to 8% of the normal value 48 hours after hypophysectomy. Rats given injections of thyroid stimulating hormone manifested an enzyme activity indistinguishable from that of the sham-operated animals. Cycloheximide prevented the thyroid stimulating hormone-induced restoration of the enzyme activity. The incorporation of ¹⁴C-leucine into the thyroid gland decreased gradually and reached two thirds of the sham-operated group by 48 hours after hypophysectomy. Thyroid stimulating hormone administration prevented this decrease, as observed for iodide peroxidase activity. Thyroidal RNA contents decreased also in hypophysectomized rats, thyroid stimulating hormone treatment prevented the reduction of RNA contents and no significant change was observed in thyroidal DNA contents. These data are consistent with the idea that protein biosynthesis is involved in thyroid stimulating hormone regulation of thyroidal iodide peroxidase and that the life span of the peroxidase is less than 48 hours.     

Role of iodine in antioxidant defence in thyroid and breast disease

The role played in thyroid hormonogenesis by iodide oxidation to iodine (organification) is well established. Iodine deficiency may produce conditions of oxidative stress with high TSH producing a level of H_2O_2, which because of lack of iodide is not being used to form thyroid hormones. The cytotoxic actions of excess iodide in thyroid cells may depend on the formation of free radicals and can be attributed to both necrotic and apoptotic mechanisms with necrosis predominating in goiter development and apoptosis during iodide induced involution. These cytotoxic effects appear to depend on the status of antioxidative enzymes and may only be evident in conditions of selenium deficiency where the activity of selenium containing antioxidative enzymes is impaired. Less compelling evidence exists of a role for iodide as an antioxidant in the breast. However the Japanese experience may indicate a protective effect against breast cancer for an iodine rich seaweed containing diet. Similarly thyroid autoimmunity may also be associated with improved prognosis. Whether this phenomenon is breast specific and its possible relationship to iodine or selenium status awaits resolution.     

Inhibition of iodothyronine transport into rat liver cells by a monoclonal antibody

The role of the rat liver plasma membrane in the regulation of uptake and subsequent deiodination of thyroxine (T4) or the biologically active thyroid hormone 3,3',5-triiodothyronine (T3) was investigated. Here we report on the production of monoclonal antibodies raised against rat hepatocytes. Two antibodies were selected. Antibody ER-22 did bind to a Mr 52,000 membrane protein and inhibited the 1- and 5-min uptake of both T4 and T3 by primary cultured rat hepatocytes in a dose-dependent fashion. As the uptake of T4 and T3 depends on the presence of a sodium gradient over the plasma membrane, the inhibitory potency of ER-22 on the Na+,K+-ATPase activity was investigated. No inhibition of the uptake of 86Rb+ could be determined, indicating that antibody ER-22 is not directed against the Na+,K+-ATPase but probably the carrier protein itself. Clearance of T3 from the medium and concomitant iodide production by cultured rat hepatocytes during a 20-h incubation in the presence of ER-22 were both inhibited by 50% with respect to a control incubation in the absence of monoclonal antibody, pointing to the importance of carrier-mediated transport in cellular uptake and metabolism of T3. A second monoclonal antibody did bind to two other plasma membrane proteins but did not inhibit transport of thyroid hormone.     

Regulation of thyroid peroxidase activity by thyrotropin, epidermal growth factor and phorbol ester in porcine thyroid follicles cultured in suspension

The activity of thyroid peroxidase (TPO) in porcine follicles cultured for 96 h in suspension with five hormones (5H) still attained over 50% of that in the freshly isolated follicles. On the other hand, the activity in those cultured with 5H + TSH (6H) was several times higher than that cultured with 5H after 96 h, although an initial decrease of TPO activity during the first 24 h of culture was observed in both conditions. The ability of follicles to metabolize iodide (uptake and organification) when cultured with 6H for 96 h was also several times higher than that of those cultured with 5H. The half-maximal dose of TSH for stimulation of TPO activity and iodide metabolism was 0.03-0.04 mU/ml and the effect was mediated by cAMP. These results indicate that in porcine thyroid follicles in primary suspension culture, TPO activity as well as the ability of iodide metabolism is induced by chronic TSH stimulation. In addition, epidermal growth factor (EGF, 10(-9)M) and phorbol 12-myristate 13-acetate (PMA, 10(-8) M) completely inhibited TSH stimulation on both activities and also basal (5H) activity of iodide metabolism.     

Glucocorticoid effects on gastric peroxidase activity

The peroxidase activity in rat gastric mucosa is inhibited after administration of glucocorticoids. The synthetic steroid dexamethasone is more potent than the naturally occurring steroids, such as cortisone or corticosterone. Almost complete inhibition of the enzyme occurs after 24 h with a single dose of 100 μg dexamethasone/120 g body weight. Other mitochondrial enzyme activities, like monoamine oxidase, succinic dehydrogenase and Mg²⁺-ATPase, remain unaltered under the same experimental condition. Submaxillary peroxidase and thyroid peroxidase activity are not inhibited by dexamethasone. Gastric peroxidase activity is increased 200–250% on the 6th day after adrenalectomy. This effect is blocked by the administration of dexamethasone. In fact, the enzyme becomes more sensitive to dexamethasone after adrenalectomy, since it is inhibited by more than 90% at the dose of 25 μg/120 g body weight. The inhibition by dexamethasone in normal animals is reversible. The enzyme is also inhibited after the administration of a single dose of ACTH. The apparent K_m of the enzyme for H₂O₂ is not altered after dexamethasone treatment or after adrenalectomy. The increase in enzyme activity following adrenalectomy is not blocked by actinomycin D or by α-amanitin, but is prevented by puromycin or cycloheximide. After administration of dexamethasone, the iodide concentration process in the gastric mucosa is not affected, but the organification of iodide is significantly diminished.     

Hydrogen Peroxide-Dependent Uptake of Iodine by Marine Flavobacteriaceae Bacterium Strain C-21

The cells of the marine bacterium strain C-21, which is phylogenetically closely related to Arenibacter troitsensis, accumulate iodine in the presence of glucose and iodide (I⁻). In this study, the detailed mechanism of iodine uptake by C-21 was determined using a radioactive iodide tracer, ¹²⁵I⁻. In addition to glucose, oxygen and calcium ions were also required for the uptake of iodine. The uptake was not inhibited or was only partially inhibited by various metabolic inhibitors, whereas reducing agents and catalase strongly inhibited the uptake. When exogenous glucose oxidase was added to the cell suspension, enhanced uptake of iodine was observed. The uptake occurred even in the absence of glucose and oxygen if hydrogen peroxide was added to the cell suspension. Significant activity of glucose oxidase was found in the crude extracts of C-21, and it was located mainly in the membrane fraction. These findings indicate that hydrogen peroxide produced by glucose oxidase plays a key role in the uptake of iodine. Furthermore, enzymatic oxidation of iodide strongly stimulated iodine uptake in the absence of glucose. Based on these results, the mechanism was considered to consist of oxidation of iodide to hypoiodous acid by hydrogen peroxide, followed by passive translocation of this uncharged iodine species across the cell membrane. Interestingly, such a mechanism of iodine uptake is similar to that observed in iodine-accumulating marine algae.     

Effect of methyl thiouracil on radioiodine thyroidal retention in rats

Summary Some goitrogens like methyl thiouracil (MTU) because of their thynamide grouping act as antithyroid drugs inhibiting the organification of iodide, but do not alter the iodide transport. Their administration to an intact animal, therefore, might alter the thyroidal iodine kinetics. Here an attempt has been made to study the effect of MTU on thyroidal iodine kinetics in rats as well as to find out whether any difference in kinetics could be detected between different radioiodines, viz.,¹³¹I,¹²⁵I, and¹²³I. Cumulated thyroidal activity which is a time integral of the activity has been taken as the parameter to represent the sum effect of thyroidal iodine kinetics over a specific time period of interest.From the in vivo thyroidal activity measurements, carried out over extended periods of time, the cumulated activity was calculated for both MTU treated and normal rats that received¹³¹I,¹²⁵I, or¹²³I at different times before the MTU start. Within a day of the start of the MTU there is a rapid loss of thyroidal iodine. The severity of the loss depended upon the time that elapsed between the start of the MTU schedule and the particular radioiodine administered. The absence of isotopic effect on the uptake as well as on the rate of uptake for the three different radioiodine isotopes studied has been brought out.Alexander von Humboldt Fellow, on leave from Institute of Nuclear Medicine and Allied Sciences, Delhi -7, India