Differential regulation of the production of reactive oxygen species in Th1 cytokine-treated thyroid cells.

BACKGROUND: Th1 cytokines exert pleiotropic effects in Hashimoto's thyroiditis. Previous studies reported a downregulation of thyroperoxidase and dual oxidase (DUOX) protein and mRNA expression in thyroid cells treated with Th1 cytokines. Although this effect is partially mediated by intracellular reactive oxygen species (ROS) and reactive nitrogen species, the nature and the source of the ROS involved are currently unknown. The aim of this study was to examine further the nature and source of the ROS produced in response to Th1 cytokines. METHODS: Two rat thyroid cell lines (PCCL3 and FRTL-5) and human thyrocytes were incubated with Th1 cytokines (interleukin [IL]-1α and interferon-γ) in the presence or absence of the Th2 cytokine IL-4, the nitric oxide synthase inhibitor N-nitroso-L-arginine methyl ester (L-NAME), or the synthetic antioxidant N-acetylcysteine. The nature and source of the intracellular and extracellular ROS produced were determined. RESULTS: A rapid increase in intracellular ROS was observed in cells incubated with Th1 cytokines. This increase was not caused by extracellular hydrogen peroxide (H2O2) produced by DUOX because both DUOX expression and extracellular H2O2 synthesis were decreased by Th1 cytokines. Confocal colocalization experiments showed that the Th1 cytokine-triggered ROS were not produced from mitochondria. Electron paramagnetic resonance investigations of PCCL3 cells indicated that the highly reactive hydroxyl radical was not involved in the response to Th1 cytokines. NOX2 mRNA expression was significantly increased in PCCL3 cells incubated with Th1 cytokines, as was the expression of the protein in the thyroid of Hashimoto's thyroiditis patients. NOX4 expression was by contrast unaffected. These results suggest that at least superoxide could be produced after exposure of thyroid cells to Th1 cytokines. The effects of L-NAME and IL-4, both of which partially or totally reverse Th1 cytokine-induced effects, on ROS release were also analyzed. L-NAME and IL-4 significantly reduced the Th1 cytokine-induced surge of intracellular ROS in PCCL3 and human thyroid cells. CONCLUSION: The data presented here reinforce the idea that ROS, other than extracellular H2O2 produced by DUOX, are released from NOX2 after exposure of thyroid cells to Th1 cytokines. ROS/reactive nitrogen species act as important, but as further explained, not exclusive intracellular mediators of Th1 cytokine-induced effects in thyroid cells.

The interactions of apamin and tetraethylammonium are differentially affected by single mutations in the pore mouth of small conductance calcium-activated potassium (SK) channels.

Valine residues in the pore region of SK2 (V366) and SK3 (V520) were replaced by either an alanine or a phenylalanine to evaluate the impact on the interactions with the allosteric blocker apamin. Unlike TEA which showed high sensitivity to phenylalanine mutated channels, the binding affinity of apamin to the phenylalanine mutants was strongly reduced. In addition, currents from phenylalanine mutants were largely resistant to block by apamin. On the other hand, when the valine residue was replaced by an alanine residue, an increase of the binding affinity and the amount of block by apamin was observed for alanine mutated SK2 channels, but not for mutated SK3 channels. Interestingly, the VA mutation reduced the sensitivity to TEA. In silico data confirmed these experimental results. Therefore, such mutations in the pore region of SK channels show that the three-dimensional structure of the SK tetramers can be disorganized in the outer pore region leading to reduced interaction of apamin with its target.

Iodine-deficiency-induced long lasting angiogenic reaction in thyroid cancers occurs via a vascular endothelial growth factor-hypoxia inducible factor-1-dependent, but not a reactive oxygen species-dependent, pathway.

BACKGROUND: In the thyroid, iodine deficiency (ID) induces angiogenesis via a tightly controlled reactive oxygen species (ROS)-hypoxia inducible factor-1 (HIF-1)-vascular endothelial growth factor (VEGF) dependent pathway (ROS-HIF-VEGF). Deficient iodine intake may be associated with increased thyroid cancer incidence. The hypothesis of this work is to test whether ID affects the angiogenic processes in thyroid malignant cells by altering the ROS-HIF-VEGF pathway. METHODS: Goiters were obtained in RET/PTC3 transgenic and wild-type (wt) mice and ID was induced in three thyroid carcinoma cell lines (TPC-1, 8305c, and R082-w1). Thyroid blood flow, VEGF mRNA and protein, and HIF-1α protein expression were measured. The role of HIF-1 and of ROS was assessed using echinomycin and N-acetylcysteine (NAC), respectively. RESULTS: The goitrogen treatment increased the thyroid blood flow in wt and RET/PTC3 mice. Compared with wt mice, basal VEGF expression was higher in RET/PTC3 mice and increased with goitrogen treatment. In the three cell lines, ID induced marked increases in VEGF mRNA, and moderate increases in HIF-1α protein expression that were not transient as in normal cells. ID-induced VEGF mRNA expression was fully (8305c), partially (TPC-1), or not (R082-w1) blocked by echinomycin. NAC had no effect on ID-induced VEGF mRNA and HIF-1α protein expression in the three cell lines. CONCLUSIONS: ID induces a long lasting angiogenic phenotype in thyroid cancer cells that occurs through VEGF induction via a pathway partially mediated by HIF-1, but not by ROS. These results suggest that, in contrast with normal cells, ID-induced angiogenesis in cancer cells occurs via alternative and likely less controlled routes, thereby leading to uncontrolled growth.

[Physiology, pharmacology and modelling of potassium channels: focus on SK channels]. / Physiologie, pharmacologie et modélisation de canaux potassiques: zoom sur les canaux SK.

Various types of ion channels are involved in the control of neuronal activity. Among them, SK channels represent an interesting therapeutic target. Indeed, they underlie medium duration after hyperpolarizations in many types of neurons, thus inhibiting cell excitability. A thorough knowledge of the physiology of these channels and the discovery of non-peptidic selective modulators able to cross the blood-brain barrier are essential in view of developing future drugs for brain diseases such as those related to a dysfunction of dopaminergic and serotonergic systems.

Synthesis and radioligand binding studies of bis-(8-isopropyl-isoquinolinium) derivatives as ligands for apamin-sensitive sites on cloned SK2 and SK3 channels.

A structure-activity relationship study of N-methyl-laudanosine, a SK channel blocker, has indicated that the 6,7-dimethoxy group could be successfully replaced by a hydrophobic moiety such as an isopropyl substituent in position 8 of the isoquinoline ring. In the present study, bis-(8-isopropyl-isoquinolinium) derivatives (2a-e) were synthesized and tested for their affinity for cloned SK2 and SK3 channels in comparison with their 6,7-dimethoxy analogues (4a-f). Several ligands were investigated, both in flexible (propyl, butyl and pentyl) and rigid (m- or p-xylyl) series, the m-xylyl derivative (2d) having the best profile in terms of affinity and selectivity for SK3/SK2 channels. Molecular studies showed that the optimal conformation of compound 2d fits well with our SK pharmacophore model.

N-acetylcysteine and 15 deoxy-{delta}12,14-prostaglandin J2 exert a protective effect against autoimmune thyroid destruction in vivo but not against interleukin-1{alpha}/interferon {gamma}-induced inhibitory effects in thyrocytes in vitro.

Reactive oxygen species (ROS) are crucial for thyroid hormonogenesis, and their production is kept under tight control. Oxidative stress (OS) is toxic for thyrocytes in an inflammatory context. In vitro, Th1 pro-inflammatory cytokines have already been shown to decrease thyroid-specific protein expression. In the present study, OS level and its impact on thyroid function were analyzed in vitro in Th1 cytokine (interleukin [IL]-1alpha/interferon [IFN] gamma)-incubated thyrocytes (rat and human), as well as in vivo in thyroids from nonobese diabetic mice, a model of spontaneous autoimmune thyroiditis. N-acetylcysteine (NAC) and prostaglandin, 15 deoxy-(Delta12,14)-prostaglandinJ2 (15dPGJ2), were used for their antioxidant and anti-inflammatory properties, respectively. ROS production and OS were increased in IL-1alpha/IFNgamma-incubated thyrocytes and in destructive thyroiditis. In vitro, NAC not only reduced ROS production below control levels, but further decreased the expression of thyroid-specific proteins in addition to IL-1alpha/IFNgamma-inhibitory effects. Thus, besides ROS, other intracellular intermediaries likely mediate Th1 cytokine effects. In vivo, NAC and 15dPGJ2 reduced OS and the immune infiltration, thereby leading to a restoration of thyroid morphology. It is therefore likely that NAC and 15dPGJ2 mainly exert their protective effects by acting on infiltrating inflammatory cells rather than directly on thyrocytes.

Oxidative stress: a required condition for thyroid cell proliferation.

Goiter is associated with increased oxidative stress (OS). We studied the effects of an anti-inflammatory agent, 15 deoxy-Delta12,14-prostaglandin J2 (15dPGJ2) and an antioxidant, N-acetylcysteine (NAC), on OS, thyroid function, and goiter expansion in a model of goiter induced by propylthiouracil (PTU) or perchlorate. OS was assessed by the immunodetection of 4-hydroxynonenal, thyroid function by measuring thyroxin (T4) and thyrotropin (TSH) plasma levels and detecting T4-rich thyroglobulin (Tg-I), and goiter expansion by weighing the thyroids and measuring cell proliferation (PCNA and cyclin D1 immunodetection). In both PTU and perchlorate-induced goiters, OS, TSH plasma levels, thyroid weight, and cell proliferation were strongly enhanced, whereas Tg-I expression was negative. All these parameters were reversed by NAC and 15dPGJ2 in PTU-goiters. In perchlorate-goiters, TSH plasma levels remained elevated and Tg-I-negative after NAC or 15dPGJ2 treatment. OS was reduced by NAC, but not by 15dPGJ2. In addition, NAC reduced PCNA and cyclin D1 immunostainings, as well as thyroid weight, whereas 15dPGJ2 influenced neither thyroid weight nor cell proliferation. In conclusion, NAC and 15dPGJ2 overcome PTU- but not perchlorate-induced effects. The retrieval of hormonal synthesis may result from direct chemical interactions between PTU and NAC/15dPGJ2. Although 15dPGJ2 has no effect in perchlorate-goiters, the reduction of OS by NAC is associated with altered goiter development, making OS a required condition for the growth of the thyroid gland.

Role of caveolin-1 in thyroid phenotype, cell homeostasis, and hormone synthesis: in vivo study of caveolin-1 knockout mice.

In human thyroid, caveolin-1 is localized at the apex of thyrocytes, but its role there remains unknown. Using immunohistochemistry, (127)I imaging, transmission electron microscopy, immunogold electron microscopy, and quantification of H(2)O(2), we found that in caveolin-1 knockout mice thyroid cell homeostasis was disrupted, with evidence of oxidative stress, cell damage, and apoptosis. An even more striking phenotype was the absence of thyroglobulin and iodine in one-half of the follicular lumina and their presence in the cytosol, suggesting that the iodide organification and binding to thyroglobulin were intracellular rather than at the apical membrane/extracellular colloid interface. The latter abnormality may be secondary to the observed mislocalization of the thyroid hormone synthesis machinery (dual oxidases, thyroperoxidase) in the cytosol. Nevertheless, the overall uptake of radioiodide, its organification, and secretion as thyroid hormones were comparable to those of wild-type mice, suggesting adequate compensation by the normal TSH retrocontrol. Accordingly, the levels of free thyroxine and TSH were normal. Only the levels of free triiodothyronine showed a slight decrease in caveolin-1 knockout mice. However, when TSH levels were increased through low-iodine chow and sodium perchlorate, the induced goiter was more prominent in caveolin-1 knockout mice. We conclude that caveolin-1 plays a role in proper thyroid hormone synthesis as well as in cell number homeostasis. Our study demonstrates for the first time a physiological function of caveolin-1 in the thyroid gland. Because the expression and subcellular localization of caveolin-1 were similar between normal human and murine thyroids, our findings in caveolin-1 knockout mice may have direct relevance to the human counterpart.

Iodide deficiency-induced angiogenic stimulus in the thyroid occurs via HIF- and ROS-dependent VEGF-A secretion from thyrocytes.

Vascular supply is an obvious requirement for all organs. In addition to oxygen and nutrients, blood flow also transports essential trace elements. Iodine, which is a key element in thyroid hormone synthesis, is one of them. An inverse relationship exists between the expansion of the thyroid microvasculature and the local availability of iodine. This microvascular trace element-dependent regulation is unique and contributes to keep steady the iodide delivery to the thyroid. Signals involved in this regulation, such as VEGF-A, originate from thyrocytes as early TSH-independent responses to iodide scarcity. The question raised in this paper is how thyrocytes, facing an acute drop in intracellular stores of iodine, generate angiogenic signals acting on adjacent capillaries. Using in vitro models of rat and human thyroid cells, we show for the first time that the deficit in iodine is related to the release of VEGF-A via a reactive oxygen species/hypoxia-inducible factor-1-dependent pathway.

Minimal oxidative load: a prerequisite for thyroid cell function.

In addition to reactive oxygen species (ROS) produced by mitochondria during aerobic respiration, thyrocytes are continuously producing H(2)O(2), a key element for hormonogenesis. Because nothing is known about ROS implication in normal non-stimulated cells, we studied their possible involvement in thyrocytes incubated with a potent antioxidant, N-acetylcysteine (NAC). NAC, which blocked the production of intracellular ROS, also decreased dual oxidases, thyroperoxidase, pendrin, and thyroglobulin protein and/or gene expression. By contrast, Na(+)/I(-) symporter mRNA expression was unaffected. Among antioxidant systems, peroxiredoxin (PRDX) five expression was reduced by NAC, whereas peroxiredoxin three increased and catalase remained unchanged. In vivo, the expression of both dual oxidases and peroxiredoxin five proteins was also decreased by NAC. In conclusion, when intracellular ROS levels drop below a basal threshold, the expression of proteins involved in thyroid cell function is hampered. This suggests that keeping ROS at a minimal level is required for safeguarding thyrocyte function.

Iodine deficiency induces a thyroid stimulating hormone-independent early phase of microvascular reshaping in the thyroid.

Expansion of the thyroid microvasculature is the earliest event during goiter formation, always occurring before thyrocyte proliferation; however, the precise mechanisms governing this physiological angiogenesis are not well understood. Using reverse transcriptase-polymerase chain reaction and immunohistochemistry to measure gene expression and laser Doppler to measure blood flow in an animal model of goitrogenesis, we show that thyroid angiogenesis occurred into two successive phases. The first phase lasted a week and involved vascular activation; this process was thyroid-stimulating hormone (TSH)-independent and was directly triggered by expression of vascular endothelial growth factor (VEGF) by thyrocytes as soon as the intracellular iodine content decreased. This early reaction was followed by an increase in thyroid blood flow and endothelial cell proliferation, both of which were mediated by VEGF and inhibited by VEGF-blocking antibodies. The second, angiogenic, phase was TSH-dependent and was activated as TSH levels increased. This phase involved substantial up-regulation of the major proangiogenic factors VEGF-A, fibroblast growth factor-2, angiopoietin 1, and NG2 as well as their receptors Flk-1/VEGFR2, Flt-1/VEGFR1, and Tie-2. In conclusion, goiter-associated angiogenesis promotes thyroid adaptation to iodine deficiency. Specifically, as soon as the iodine supply is limited, thyrocytes produce proangiogenic signals that elicit early TSH-independent microvascular activation; if iodine deficiency persists, TSH plasma levels increase, triggering the second angiogenic phase that supports thyrocyte proliferation.

Differential interactions between Th1/Th2, Th1/Th3, and Th2/Th3 cytokines in the regulation of thyroperoxidase and dual oxidase expression, and of thyroglobulin secretion in thyrocytes in vitro.

Hypothyroidism, together with glandular atrophy, is the usual outcome of destructive autoimmune thyroiditis. The impairment in the thyroid function results either from cell destruction or from Th1 cytokine-induced alteration in hormonogenesis. Here, we investigated the impact of the local immune context on the thyroid function. We used two rat thyroid cell lines (PCCL3 and FRTL-5) and human thyrocytes incubated with IL-1alpha/interferon (IFN) gamma together with IL-4, a Th2 cytokine, or with TGF-beta, or IL-10, two Th3 cytokines. We first observed that IL-4 totally blocked IL-1alpha/interferon gamma-induced alteration in dual oxidase and thyroperoxidase expression, and in thyroglobulin secretion. By contrast, TGF-beta and IL-10 had no such effect. They rather repressed thyrocyte function as do Th1 cytokines. In addition, IL-4 blocked IL-10-induced repression of thyrocyte function, but not that induced by TGF-beta. In conclusion, Th1 cytokine- and IL-10-induced local inhibitory actions on thyroid function can be totally overturned by Th2 cytokines. These data provide new clues about the influence of the immune context on thyrocyte function.

Oxidative stress in the thyroid gland: from harmlessness to hazard depending on the iodine content.

In basal conditions, thyroid epithelial cells produce moderate amounts of reactive oxygen species (ROS) that are physiologically required for thyroid hormone synthesis. They are not necessarily toxic because they are continuously detoxified either in the process of hormone synthesis or by endogenous antioxidant systems. Using a rat model of goiter formation and iodine-induced involution, we found that compared with control thyroids, the oxidative stress, assessed by the detection of 4-hydroxynonenal, was strongly enhanced both in hyperplastic and involuting glands. The level of antioxidant defenses (glutathione peroxidases and peroxiredoxins) was also up-regulated in both groups, although somewhat less in the latter. Of note, increased oxidative stress came along with an inflammatory reaction, but only in involuting glands, suggesting that although antioxidant systems can adequately buffer a heavy load of ROS in goiter, it is not necessarily the case in involuting glands. The effects of 15-deoxy-Delta(12,14)-prostaglandin J2 (15dPGJ2), an endogenous ligand of peroxisome proliferated-activated receptor gamma (PPARgamma) with antiinflammatory properties, were then investigated in involuting glands. This drug strongly reduced both 4-hydroxynonenal staining and the inflammatory reaction, indicating that it can block iodine-induced cytotoxicity. When experiments were carried out with the PPARgamma antagonist, bisphenol A diglycidyl ether, 15dPGJ2-induced effects remained unchanged, suggesting that these effects were not mediated by PPARgamma. In conclusion, thyroid epithelial cells are well adapted to endogenously produced ROS in basal and goitrous conditions. In iodine-induced goiter involution, the increased oxidative stress is accompanied by inflammation that can be blocked by 15dPGJ2 through PPARgamma-independent protective effects.

Proteomic profiling of cold thyroid nodules.

Cold thyroid nodules (CTNs) represent a frequent endocrine disorder accounting for up to 85% of thyroid nodules in a population living in an iodine-deficient area. Benign CTNs need to be distinguished from thyroid cancer, which is relatively rare. The molecular etiology of benign CTNs is unresolved. To obtain novel insights into their pathogenesis, protein expression profiling was performed in a series of 27 solitary CTNs (10 follicular adenoma and 20 adenomatous nodules) and surrounding normal thyroid tissues using two-dimensional gel electrophoresis combined with mass spectrometry analysis, Western blotting, and immunohistochemistry. The proteome analysis revealed a specific fingerprint of CTNs with up-regulation of three functional systems: 1) thyroid cell proliferation, 2) turnover of thyroglobulin, and 3) H2O2 detoxification. Western blot analysis and immunohistochemistry confirmed the proteome data and showed that CTNs exhibit significant up-regulation of proteins involved in thyroid hormone synthesis yet are deficient in T4-containing thyroglobulin. This is consequential to intranodular iodide deficiency, mainly due to cytoplasmic sodium iodide symporter localization, and portrays the CTN as an activated proliferating lesion with an intranodular hypothyroid milieu. Furthermore, we provide preliminary evidence that up-regulation of H2O2 generation in CTNs could override the antioxidative system resulting in oxidative stress, which is suggested by the finding of raised 8-oxo-guanidine DNA adduct formation in CTNs.