Comparative proteomic analysis to dissect differences in signal transduction in activating TSH receptor mutations in the thyroid

In the thyroid, cAMP controls both thyroid growth and function. Gain-of-function mutations in the thyroid-stimulating hormone receptor (TSHR) lead to constitutive cAMP formation and are a major cause of autonomous thyroid adenomas. The impact of activating TSHR mutations on the signal transduction network of the thyrocyte is not fully understood. To gain more insights into constitutive TSHR signaling, rat thyrocytes (FRTL-5 cells) with stable expression of three activating TSHR mutants (mutTSHR: A623I, L629F and Del613-621), which differ in their functional characteristics in vitro, were analyzed by a quantitative proteomic approach and compared to the wild-type TSHR (WT-TSHR). This study revealed (1) differences in the expression of Rab proteins suggesting an increased TSHR internalization in mutTSHR but not in the WT-TSHR; (2) differential stimulation of PI3K/Akt signaling in mutTSHR vs. WT-TSHR cells, (3) activation of Epac, impairing short-time Akt phosphorylation in both, mutTSHR and WT-TSHR cells. Based on the analysis of global changes in protein expression patterns, our findings underline the complexity of gain-of-function TSHR signaling in thyrocytes, which extends beyond pure cAMP and/or IP formation. Moreover, evidence for augmented endocytosis in the mutTSHR, adds to a new concept of TSHR signaling in thyroid autonomy. Further studies are required to clarify whether the observed differences in Rab, PI3K and Epac signaling may contribute to differences in the phenotypic presentation, i.e. stimulation of function and growth of thyroid autonomy in vivo.     

Effect of iodoarachidonates on thyroid FRTL-5 cells growth.

Excess iodide inhibits several thyroid parameters, by a putative organic iodocompound. Different iodolipids, including iodinated derivatives of arachidonic acid (IAs), are produced by rat, calf and pig thyroid. The action of two iodolactones, one bearing the iodine atom at the position 6 (IL-d) and the other at position 14 (IL-w) on growth of FRTL-5 cells was studied. KI, IL-w and IL-d exert a dose-related inhibition on FRTL-5 cell proliferation. The first two compounds caused inhibition at 1 microM while IL-d was effective at 10 microM. This inhibitory action of iodolactones (ILs) was not altered by 1 mM methyl-mercaptoimidazol (MMI), indicating that they exert their effect per se. The action of ILw on cell growth was reversible. The growth-stimulating effect of 10 microM forskolin was inhibited by IAs, showing that one possible site of action lies at the cAMP pathway. The present results give further support to our hypothesis about the role of IAs in thyroid growth autoregulation.     

Fluorescence quantitation of thyrocyte iodide accumulation with the yellow fluorescent protein variant YFP-H148Q/I152L

The thyroid gland accumulates iodide for the synthesis of thyroid hormones. The aim of the current study was to quantify iodide accumulation in cultured thyroid cells by live cell imaging using the halide-sensitive yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In vivo calibrations were performed in FRTL-5 thyrocytes to determine the sensitivity of YFP-H148Q/I152L to iodide. In the presence of ion-selective ionophores, YFP-H148Q/I152L fluorescence was suppressed by halides in a pH-dependent manner with 20-fold selectivity for iodide versus chloride and competition between the two halides. At a physiological pH of 7 and a chloride concentration of 15mM, the affinity constant of YFP-H148Q/I152L for iodide was 3.5mM. In intact FRTL-5 cells, iodide induced a reversible decrease in YFP-H148Q/I152L fluorescence. FRTL-5 cells concentrated iodide to 60 times the extracellular concentration. Iodide influx exhibited saturation kinetics with respect to extracellular iodide with a K(m) of 35 microM and a V(max) of 55 microM/s. Iodide efflux exhibited saturation kinetics with respect to intracellular iodide concentration with a K(m) of 2.2mM and a V(max) of 43 microM/s. The results of this study demonstrate the utility of YFP-H148Q/I152L as a sensitive and selective biosensor for the quantification of iodide accumulation in thyroid cells.     

Resveratrol Inhibits Sodium/Iodide Symporter Gene Expression and Function in Rat Thyroid Cells

Resveratrol is a polyphenol found in grapes and berries that has antioxidant, antiproliferative and anti-inflammatory properties. For these reasons, it is available as a dietary supplement, and it is under investigation in several clinical trials. Few data are available regarding the effects of resveratrol on thyroid function. A previous study showed that resveratrol transiently increases iodide influx in FRTL-5 rat thyroid cells. Indeed, this increase arises after short treatment times (6–12 h), and no further effects are seen after 24 h. The aim of the present study was to investigate the effects of resveratrol on iodide uptake and sodium/iodide symporter expression in thyroid cells after longer times of treatment. For this purpose, the effects of resveratrol were evaluated both in vitro and in vivo using the rat thyroid FRTL-5 cell line and Sprague-Dawley rats, respectively. In FRTL-5 cells, resveratrol decreased the sodium/iodide symporter RNA and protein expression as a function of time. Furthermore, resveratrol decreased cellular iodide uptake after 48 h of treatment. The inhibitory effect of resveratrol on iodide uptake was confirmed in vivo in Sprague-Dawley rats. This study demonstrates that with longer-term treatment, resveratrol is an inhibitor of sodium/iodide symporter gene expression and function in the thyroid. These data suggest that resveratrol can act as a thyroid disruptor, which indicates the need for caution as a supplement and in therapeutic use.     

Thyrotropin receptor trafficking relies on the hScrib-betaPIX-GIT1-ARF6 pathway

G protein-coupled receptors are regulated by ligand stimulation, endocytosis, degradation of recycling to the cell surface. Little information is available on the molecular mechanisms underlying G protein-coupled receptors recycling. We have investigated recycling of the G protein-coupled thyroid stimulating hormone receptor (TSHR) and found that it relies on hScrib, a membrane-associated PDZ protein. hScrib directly binds to TSHR, inhibits basal receptor endocytosis and promotes recycling, and thus TSHR signalling, at the cell membrane. We previously demonstrated that hScrib is associated with a betaPIX-GIT1 complex comprised of a guanine nucleotide exchange factor and a GTPase-activating protein for ADP ribosylation factors that is involved in vesicle trafficking. We used dominant-negative constructs and small interfering RNA to show that TSHR recycling is regulated by the interaction between hScrib and betaPIX, and by the activity of GIT1. In addition, ARF6, a major target for GIT1, is activated during TSH stimulation of HEK293 and FRTL-5 thyroid cells, and plays a key role in TSHR recycling. Thus, we have uncovered an hScrib-betaPIX-GIT1-ARF6 pathway devoted to TSHR trafficking and function.     

1,25-Dihydroxycholecalciferol attenuates thyrotropin stimulated iodide accumulation in rat thyroid follicular FRTL-5 cells by reducing iodide porter number.

We have recently shown that rat thyroid follicular FRTL-5 cells have functional receptors for 1,25-dihydroxycholecalciferol (1,25- (OH)2D3) and that 1,25-(OH)2D3 attenuates the thyrotropin (TSH) induced iodide uptake. Here we show that the dibutyrylcyclic AMP induced iodide uptake was significantly reduced by 1,25-(OH)2D3, indicating that 1,25-(OH)2D3 affects the cAMP signal pathway beyond cAMP generation. The Vmax of the iodide porter was significantly reduced in 1,25-(OH)2D3 treated cells as compared to cells treated with TSH alone, indicating that 1,25-(OH)2D3 reduces the effective number of iodide porters in FRTL-5 cells.     

Differential effects of growth factors on [3H]thymidine incorporation and [125I]iodine uptake in FRTL-5 rat thyroid cells

We studied the effect of several growth factors on DNA synthesis and function of FRTL-5 rat thyroid cells by simultaneous measurement of [3H]thymidine incorporation and [125I]iodide uptake. Endothelial cell growth factor, fibroblast growth factor, platelet-derived growth factor, and insulin-like growth factor I stimulated thymidine incorporation in a dose-dependent manner without the parallel increase of [125I]iodide uptake. These growth factors had an additive effect with thyroid-stimulating hormone (TSH) on thymidine incorporation, but they inhibited TSH-stimulated iodide uptake. Bombesin stimulated thymidine incorporation and inhibited TSH-stimulated iodide uptake; epidermal growth factor and gastrin-releasing peptide 10 had neither effect. None of the growth factors studied affected iodide uptake in the absence of TSH. Of the growth factors tested, endothelial cell growth factor, fibroblast growth factor, insulin-like growth factor bombesin, and platelet-derived growth factor all share similar differential effects on FRTL-5 cells: stimulation of DNA synthesis, potentiation of the effects of TSH on DNA synthesis, and attenuation of the effects of TSH on cell function. The data suggest that these growth factors may play important roles in regulation of thyroid function.     

Phagocytic Activity of FRTL-5 Rat Thyroid Follicular Cells as Measured by Ingestion of Fluorescent Latex Beads

A rat thyroid cell line (FRTL-5) was used to study the phagocytic activity of thyroid follicular cells using fluorescent latex beads and flow cytometric analysis. Morphologic studies demonstrated that latex beads were engulfed and located within cytoplasmic vacuoles of thyrocytes. Flow cytometric evaluation of cell suspensions revealed high levels of fluorescence in cells engulfing latex beads. Using thyrotropin (TSH) as a stimulator of thyroid function and human interleukin-1β as an inhibitor, protocols were established for measuring the effects of these substances on either basal or TSH-induced phagocytosis. Cells exposed to latex beads over time in basal (0H) or TSH-containing medium had an increase in time-dependent phagocytic activity which was maximal after 24 or 8 h, respectively. Treatment of FRTL-5 cells with either a stimulator or an inhibitor revealed maximal change in phagocytic activity after 72 h as measured by the percentage of phagocytic cells as well as the mean fluorescence intensity. Phagocytic activity and iodide trapping by FRTL-5 cells were qualitatively similar in both sensitivity and magnitude of change in the assays used in this study. Phagocytosis of fluorescent latex beads represents a sensitive nonradioactive assay of thyrocyte function whose regulation is similar to iodide trapping.     

Role of pertussis toxin sensitive G proteins in the alpha 1 adrenergic receptor but not in the thyrotropin receptor mediated activation of membrane phospholipases and iodide fluxes in FRTL-5 thyroid cells

The regulation of thyroid hormone formation by thyrotropin and norepinephrine involves the activation of both phospholipases C and A2. When FRTL-5 cells are incubated with 10(-10)M pertussis toxin for 4 to 20 h, the stimulation of iodide efflux by norepinephrine is inhibited by 50 to 70%. At the same toxin concentration the norepinephrine induced increase in cytosolic Ca2+ is unaffected; however upon 20 h pretreatment with 10(-9)M pertussis toxin a 30% inhibition is observed. By contrast, the pertussis toxin treatment had no effect on the increase in iodide efflux or in cytosolic Ca2+ levels induced by thyrotropin. Our data suggest that two GTP binding proteins sensitive to pertussis toxin are involved in the alpha 1 adrenergic but not in the thyrotropin induced activation of the signal transduction mechanisms leading to iodide efflux in FRTL-5 cells.     

Vitamin D: a hormonal regulator of the cAMP signaling pathway

In the rat thyroid FRTL-5 cell line calcitriol, the biologically most active of the naturally occurring vitamin D metabolites, attenuates both TSH-stimulated cAMP production and the effects of cAMP. Calcitriol treatment abolishes the upregulation of the TSHR number occurring in cells cultivated in the absence of TSH. In addition, the level of G(i-2)alpha increases, which may further attenuate the transmembrane signaling of TSH and facilitate the effects of IGFs. The effect of cAMP on PKAI stimulation is inhibited by increasing the level of the PKA subunit RIIbeta. Regulation of TSHR, G(i-2)alpha and RIIbeta is associated with altered cell proliferation and differentiation in several cells and tissues. Effects of calcitriol on these proteins indicate how the vitamin D endocrine system may regulate cAMP signaling in both classical and nonclassical target tissues.     

Iodinated phospholipids and the iodination of proteins of dog thyroid gland in vitro

Slices of dog thyroid gland were incubated with liposomes consisting of (125)I-labelled phosphatidylcholine (the iodine was covalently linked to unsaturated fatty acyl chains). The (125)I label of (125)I-labelled liposomes was incorporated into thyroid protein and/or thyroglobulin at a higher rate than was the (131)I label of either Na(131)I or (131)I(2). The iodine was shown to be protein-bound by the co-migration of the labelled iodine with protein under conditions where free iodine, iodide and lipid-bound iodine were removed from protein. The uptake of iodine from the iodinated phospholipid was probably due to phospholipid exchange between the iodinated liposomes and the thyroid cell membrane, since (a) (14)C-labelled phospholipid was metabolized to (14)CO(2) and (b) many lipids in the tissue slice became (14)C-labelled. A very strong inhibition of iodide ;uptake' from Na(131)I, caused by thiosulphate, produced only a minor inhibition of the incorporation of (125)I from (125)I-labelled liposomes into thyroid protein and/or thyroglobulin. This implies that free iodide may not necessarily be formed from the iodinated phospholipids before their entrance or utilization in the cell. Synthetic polytyrosine polypeptide suspensions showed some iodination by (131)I-labelled liposomes. In tissues with low tyrosine contents, such as liver and kidney, only a trace uptake was observed. Salivary gland showed some uptake. Endoplasmic reticulum of thyroid gland showed a higher iodine uptake than that of the corresponding plasma membranes. These experiments, together with the demonstration of the diet-dependent presence of iodinated phospholipids in dog thyroid, leads us to suggest that iodination of the membrane phospholipids of thyroid cells may be directly or indirectly involved at some stage in the synthesis of thyroglobulin, or exists as a scavenger mechanism, to re-utilize and/or recover released iodine from unstable compounds inside the thyroid cell.     

Regulation of sodium iodide symporter gene expression by Rac1/p38β mitogen-activated protein kinase signaling pathway in MCF-7 breast cancer cells

Activation of p38 MAPK is a key pathway for cell proliferation and differentiation in breast cancer and thyroid cells. The sodium/iodide symporter (NIS) concentrates iodide in the thyroid and lactating breast. All-trans-retinoic acid (tRA) markedly induces NIS activity in some breast cancer cell lines and promotes uptake of β-emitting radioiodide (131)I sufficient for targeted cytotoxicity. To identify a signal transduction pathway that selectively stimulates NIS expression, we investigated regulation by the Rac1-p38 signaling pathway in MCF-7 breast cancer cells and compared it with regulation in FRTL-5 rat thyroid cells. Loss of function experiments with pharmacologic inhibitors and small interfering RNA, as well as RT-PCR analysis of p38 isoforms, demonstrated the requirement of Rac1, MAPK kinase 3B, and p38β for the full expression of NIS in MCF-7 cells. In contrast, p38α was critical for NIS expression in FRTL-5 cells. Treatment with tRA or overexpression of Rac1 induced the phosphorylation of p38 isoforms, including p38β. A dominant negative mutant of Rac1 abolished tRA-induced phosphorylation in MCF-7 cells. Overexpression of p38β or Rac1 significantly enhanced (1.9- and 3.9-fold, respectively), the tRA-stimulated NIS expression in MCF-7 cells. This study demonstrates differential regulation of NIS by distinct p38 isoforms in breast cancer cells and thyroid cells. Targeting isoform-selective activation of p38 may enhance NIS induction, resulting in higher efficacy of (131)I concentration and treatment of breast cancer.     

Cassava is not a goitrogen in mice

To examine the effect of cassava on the thyroid function of mice, we fed fresh cassava root to mice and compared this diet with low iodine diet and Purina. Cassava provided a low iodine intake and increased urine thiocyanate excretion and serum thiocyanate levels. Mice on cassava lost weight. The thyroid glands of mice on cassava were not enlarged, even when normalized for body weight. The 4- and 24-hr thyroid uptakes of mice on cassava were similar to those of mice on low iodine diets. Protein-bound [125I]iodine at 24 hr was high in mice on either the cassava or low iodine diets. The thyroid iodide trap (T/M) was similar in mice on cassava and low iodine diets. When thiocyanate was added in vitro to the incubation medium, T/M was reduced in all groups of mice; under these conditions, thiocyanate caused a dose-related inhibition of T/M. The serum thyroxine (T4) and triiodothyronine (T3) concentrations of mice on cassava were reduced compared with mice on Purina diet. Thyroid T4 and T3 contents of mice on cassava were relatively low compared with mice on Purina diet. Hepatic T3 content and T4 5'-monodeiodination in liver homogenates were reduced in mice on cassava compared with other groups. The data show that cassava does not cause goiter in mice. The thiocyanate formed from ingestation of cassava is insufficient to inhibit thyroid iodide transport or organification of iodide. The cassava diet leads to rapid turnover of hormonal iodine because it is a low iodine diet. It also impairs 5'-monodeiodination of T4 which may be related to nutritional deficiency. These data in mice do not support the concept that cassava per se has goitrogenic action in man.     

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.     

Iodine-Induced Thyroid Blockade: Role of Selenium and Iodine in the Thyroid and Pituitary Glands

The purpose of this study was to determine the content of iodine and selenium in the thyroid and pituitary glands of rats under iodine-induced blockade of the thyroid gland. Electron probe microanalysis, wavelength-dispersive spectrometry, and point analysis were used in this investigation. We also determined the expression of sodium iodide symporter and caspase 32 in the thyroid and pituitary glands and the expression of thyroid-stimulating hormone in the pituitary. The samples for iodine analysis must be thoroughly dehydrated, and for this purpose, we developed a method that produced samples of constant mass with minimal loss of substrate (human thyroid gland was used for the investigation). Normal levels of iodine and selenium were found in the thyroid, pituitary, ovaries, testes hypothalamus, and pancreas of healthy rats. The levels of iodine and selenium in I- or Se-positive points and the percentage of positive points in most of these organs were similar to those of controls (basal level), except for the level of iodine in the thyroid gland and testes. Blockade of the thyroid gland changed the iodine level in iodine-positive points of the thyroid and the pituitary glands. On the sixth day of blockage, the iodine level in iodine-positive points of the thyroid gradually decreased to the basal level followed by an abrupt increase on the seventh day, implying a rebound effect. The opposite was found in the pituitary, in which the level of iodine in iodine-positive points increased during the first 6 days and then abruptly decreased on the seventh day. Expression of the thyroid-stimulating hormone in the pituitary decreased during the first 5 days but sharply increased on the sixth day, with a minimum level of iodine in the thyroid and maximum in the pituitary, before normalization of the iodine level in both glands preceding the rebound effect. The expression of sodium iodide symporter increased during the first 4 days of blockage and then decreased in both glands. The fluctuations of the thyroid-stimulating hormone in the pituitary gland reflected the changes of iodine in the thyroid gland more precisely than the changes of sodium iodide symporter. The selenium level in the selenium-positive points changed only in the pituitary, dropping to zero on the second and fifth day of the blockade. Simultaneously, the maximum induction of caspase 32 was observed in the pituitary gland. We believe that these results may help to clarify a role of the pituitary gland in the thyroid blockade.