Severe Autoimmune Thyroiditis in Transgenic NOD.H2h4 Mice Expressing Interleukin-4 in the Thyroid.

Background: Hashimoto's thyroiditis is a common autoimmune thyroid disorder characterized by thyroid lymphocytic infiltrates and autoreactive antibodies against thyroglobulin (TgAbs) and thyroperoxidase. Final evolution of the disease can lead to hypothyroidism with destruction of the thyroid architecture. Interleukin-4 (IL-4) is involved in the humoral immune response and B cell activation required in autoimmune thyroiditis (AT) progression. We used our mouse model overexpressing IL-4 by thyrocytes (Thyr-IL4) to study the impact of a local IL-4 expression in AT using transgenic nonobese diabetic (NOD.H2h4) derived animals treated with iodide-supplemented water to increase the incidence of spontaneous AT (SAT). Methods: Thyr-IL4 NOD.H2h4 and nonpathogenic C57BL/6 animals aged 8 weeks were exposed to 0.05% sodium iodide (NaI) in their drinking water for 8 and 16 weeks. Circulating TgAbs and expression of intrathyroidal cytokines were quantified. Thyroid inflammation was assessed by classical histological analyses, including identification of some immune cell populations. The most sensitive parameter to evaluate the thyroid function, serum thyrotropin (TSH), was also measured at the end of the treatment. Results: Relative to wild-type (WT) animals, Thyr-IL4 NOD.H2h4 mice developed severe accelerated SAT with elevated serum TgAbs and numerous thyroid infiltrates mainly composed of CD4+/CD8+ T cells, B lymphocytes, and monocytes/macrophages. Thyroid expression of T helper (Th) Th1/Th2 cytokines was also enhanced, as well as IL-17. In contrast, excessive iodide supply did not induce TgAbs in WT and Thyr-IL4 SAT-resistant C57BL/6 animals. However, moderate leukocyte infiltrations in transgenic thyroids were evident compared to WT, but associated with a limited number of T and B cells and a different cytokine profile from Thyr-IL4 NOD.H2h4 mice. Finally, and despite their diverse immune responses, both transgenic strains presented marked thyroid enlargement and elevated serum TSH at the end of the treatment in contrast to their WT littermates. Conclusions: These findings demonstrated that ectopic expression of IL-4 from thyrocytes enhanced the severity of accelerated SAT in disease-prone Thyr-IL4 NOD.H2h4 animals and promoted thyroid leukocyte infiltration in SAT-resistant transgenic C57BL/6 mice. Moreover, impaired thyroid function emerged in both transgenic strains during the progression of the disease.

Murine Thyroid IL-4 Expression Worsens Hypothyroidism on Iodine Restriction and Mitigates Graves Disease Development.

Cytokines are known to perturb thyroid function and the role of interleukin-4 (IL-4) in the pathogenesis of Graves disease (GD) remains controversial. In our mouse model overexpressing IL-4 in thyrocytes (Thyr-IL4), we have reported that adult mice preserved normal serum thyroxine despite an iodide uptake defect. In the present work, we evaluated if iodine restriction could uncover the thyroid deficiency in Thyr-IL4 animals as well as the role of pendrin overexpression as a compensatory mechanism. Moreover, using an experimental model of GD we investigated the effect of a local expression of IL-4 on the incidence of hyperthyroidism. Thyr-IL4 mice developed more rapidly elevated serum thyrotropin under low-iodine supply with thyroid enlargement and classical histological modifications. These hallmarks of hypothyroidism were all enhanced in Thyr-IL4 mice with complete pendrin invalidation. Following immunization, a lower proportion of Thyr-IL4 animals developed hyperthyroidism. Surprisingly, immunized Thyr-IL4 animals presented numerous leukocyte infiltrates, associated with increased intrathyroidal expression of IFN-γ. We have demonstrated that thyroid deficiency in Thyr-IL4 mice is partially compensated for by the excessive iodide content of the standard chow and the overexpression of pendrin in these animals. Furthermore, we have shown that the local expression of IL-4 in the thyroid attenuates GD progression, which was associated with enhanced thyroid infiltration by immune cells that could negatively affect thyroid function.

Functional model of rat thyroid follicles cultured in Matrigel.

BACKGROUND: Long-term maintenance of functional activity of thyroid cells is an essential requirement for basic in vitro studies on the physiology and pathology of the thyroid. An important prerequisite of thyrocytes' functional activity in vivo and in vitro is their follicle organization. AIM: This study aimed at developing a method of cultivation of functionally active rat thyroid follicles in Matrigel under three-dimensional conditions. METHODS: Undamaged rat thyroid follicles were isolated by enzymatic digestion with collagenase/dispase, then embedded into Matrigel, and cultivated for 2 weeks. Thyroglobulin, thyroxine and zonula occludens-1 (ZO-1) localization were revealed by immunofluorescence analysis. Iodide organification was tested by protein-bound 125I (PBI) measurement. RESULTS: Integrity of the follicles was preserved during the whole period of cultivation and was confirmed by 3D reconstruction of ZO-1 localization. Thyroglobulin was detected in the thyrocyte cytoplasm, as well as in the intrafollicular lumen. Thyroxine was observed predominantly at the apical side of thyrocytes. Also, generated cultures were characterized by a high level of iodide organification: PB125I represented 39% of the total radioactivity in the Matrigel drop embedding the follicles; at the same time, methimazole almost totally inhibited this process (0.2% of total radioactivity). CONCLUSION: The method of rat thyrocyte cultivation in Matrigel, as described here allows to maintain the structural integrity and the functional activity of thyroid follicles in vitro and could be used for wide ranges of basic and applied researches in thyroidology.

Inhibition of the thyroid hormonogenic H2O2 production by Duox/DuoxA in zebrafish reveals VAS2870 as a new goitrogenic compound.

Thyroid hormone (TH) synthesis requires extracellular hydrogen peroxide generated by the NADPH oxidases, DUOX1 and DUOX2, with maturation factors, DUOXA1 and DUOXA2. In zebrafish, only one duox and one duoxa gene are present. Using a thyroid-specific reporter line, we investigated the role of Duox and Duoxa for TH biosynthesis in zebrafish larvae. Analysis of several zebrafish duox and duoxa mutant models consistently recovered hypothyroid phenotypes with hyperplastic goiter caused by impaired TH synthesis. Mutant larvae developed enlarged thyroids and showed increased expression of the EGFP reporter and thyroid functional markers including wild-type and mutated duox and duoxa transcripts. Treatment of zebrafish larvae with the NADPH oxidase inhibitor VAS2870 phenocopied the thyroid effects observed in duox or duoxa mutants. Additional functional in vitro assays corroborated the pharmacological inhibition of Duox activity by VAS2870. These data support the utility of this new experimental model to characterize endocrine disruptors of the thyroid function.

Dissecting the Role of Thyrotropin in the DNA Damage Response in Human Thyrocytes after 131I, γ Radiation and H2O2.

BACKGROUND: The early molecular events in human thyrocytes after 131I exposure have not yet been unravelled. Therefore, we investigated the role of TSH in the 131I-induced DNA damage response and gene expression in primary cultured human thyrocytes. METHODS: Following exposure of thyrocytes, in the presence or absence of TSH, to 131I (ß radiation), γ radiation (3 Gy), and hydrogen peroxide (H2O2), we assessed DNA damage, proliferation, and cell-cycle status. We conducted RNA sequencing to profile gene expression after each type of exposure and evaluated the influence of TSH on each transcriptomic response. RESULTS: Overall, the thyrocyte responses following exposure to ß or γ radiation and to H2O2 were similar. However, TSH increased 131I-induced DNA damage, an effect partially diminished after iodide uptake inhibition. Specifically, TSH increased the number of DNA double-strand breaks in nonexposed thyrocytes and thus predisposed them to greater damage following 131I exposure. This effect most likely occurred via Gα q cascade and a rise in intracellular reactive oxygen species (ROS) levels. ß and γ radiation prolonged thyroid cell-cycle arrest to a similar extent without sign of apoptosis. The gene expression profiles of thyrocytes exposed to ß/γ radiation or H2O2 were overlapping. Modulations in genes involved in inflammatory response, apoptosis, and proliferation were observed. TSH increased the number and intensity of modulation of differentially expressed genes after 131I exposure. CONCLUSIONS: TSH specifically increased 131I-induced DNA damage probably via a rise in ROS levels and produced a more prominent transcriptomic response after exposure to 131I.

The Dual Oxidase Duox2 stabilized with DuoxA2 in an enzymatic complex at the surface of the cell produces extracellular H2O2 able to induce DNA damage in an inducible cellular model.

Thyroid hormone synthesis requires H2O2, produced by two NADPH oxidases, Duox1 and Duox2. To be fully active at the apical pole of the thyrocytes, these enzymes need additional maturation factors DuoxA1 and DuoxA2. The proteins have been shown to be localized at the cell surface, suggesting that they could form a complex with Duox counterparts. We have generated multiple HEK293 Tet-On3G cell lines that express various combinations of DuoxA upon doxycycline induction, in association with a constitutive expression of the Duox enzyme. We compared Duox specific activity, Duox/DuoxA cell surface interactions and the cellular consequences of sustained H2O2 generation. By normalizing H2O2 extracellular production by Duox or DuoxA membrane expression, we have demonstrated that the most active enzymatic complex is Duox2/DuoxA2, compared to Duox1/DuoxA1. A direct cell surface interaction was shown between Duox1/2 and both DuoxA1 and DuoxA2 using the Duolink® technology, Duox1/DuoxA1 and Duox2/DuoxA2 membrane complexes being more stable than the unpaired ones. A significant increase in DNA damage was observed in the nuclei of Duox2/DuoxA2 expressing cells after doxycycline induction and stimulation of Duox catalytic activity. The maturation and activity of Duox2 were drastically impaired when expressed with the glycosylation-defective maturation factor DuoxA2, while the impact of the unglycosylated DuoxA1 mutant on Duox1 membrane expression and activity was rather limited. The present data demonstrate for the first time that H2O2 produced by the Duox2/DuoxA2 cell surface enzymatic complex could provoke potential mutagenic DNA damage in an inducible cellular model, and highlight the importance of the co-expressed partner in the activity and stability of Duox/DuoxA complexes.

Factors contributing to the resistance of the thyrocyte to hydrogen peroxide.

We studied the mechanism that may explain the relative resistance of thyrocytes to H2O2 compared to other cell types. Ability to degrade H2O2, glutathione peroxidase (GPx) activity, heme oxygenase-1 (HO-1) expression, cell survival and capacity to repair DNA damage after H2O2 exposure or irradiation were measured in human thyrocytes in primary culture and compared to the values obtained in human T-cells and different cell lines. Compared to other cell types, thyrocytes presented a low mortality rate after H2O2 exposure, rapidly degraded extracellular H2O2 and presented a high basal seleno-dependent GPx activity. Only in thyrocytes, H2O2 up-regulated GPx activity and expression of HO-1 mRNA. These effects were not reproduced by irradiation. DNA damage caused by H2O2 was more slowly repaired than that caused by irradiation and not repaired at all in T-cells. Our study demonstrates that the thyrocyte has specific protective mechanisms against H2O2 and its mutagenic effects.

Expression, Localization, and Regulation of the Sodium Bicarbonate Cotransporter NBCe1 in the Thyroid.

BACKGROUND: The intrafollicular space of thyroid follicles is the storage compartment for thyroid hormones. Its pH has been established at around 7.6 at least after thyrotropin (TSH) stimulation. This alkaline intrafollicular pH is thought to be critical for iodide coupling to thyroglobulin and internalization of iodinated thyroglobulin. At least in mice, this alkalinization requires the expression of pendrin (Slc26a4) within the apical membrane, and a lack of pendrin results in acidic follicular lumen pH. Yet, the mechanism importing HCO3- into the cytoplasm is unknown. This study investigated whether the rather ubiquitous sodium bicarbonate cotransporter NBCe1 (SLC4A4) might play this role. It also examined which variant was expressed and where it was localized in both rat and human thyroid tissue. Lastly, the dependence of its expression on TSH was studied. METHODS: Reverse transcription polymerase chain reaction, immunofluorescence, and Western blotting were used to test whether TSH stimulated NBCe1 protein expression in vivo. Subcellular localization of NBCe1 was performed using immunofluorescence in both rat and human thyroid. Cultured thyroid cells were also used to attempt to define how TSH affects NBCe1 expression. RESULTS: Only transcripts of the NBCe1-B variant were detected in both rat and human thyroid. Of interest, NBCe1-C was not detected in human tissues, not even in the brain. On immunofluorescence microscopy, the immunostaining of NBCe1 mainly appeared in the basolateral membrane upon stimulation with TSH. This TSH induction of basolateral membrane expression of NBCe1 protein was confirmed in vivo in rat thyroid and in vitro on human thyroid slices. CONCLUSIONS: This study demonstrates the expression of the sodium bicarbonate cotransporter NBCe1-B in rat and human thyroid. Additionally, the data suggest that TSH blocks the degradation of NBCe1 protein by trafficking it to the basolateral membrane. Hence, TSH increases NBCe1 half-life without increasing its synthesis.

Distinctive Desmoplastic 3D Morphology Associated With BRAFV600E in Papillary Thyroid Cancers.

Context: Although 60% of papillary thyroid carcinomas are BRAFV600E mutant (PTCV600E), the increased aggressiveness of these cancers is still debated. Objective: For PTCV600E we aimed to further characterize the extent of the stroma and its activation, the three-dimensional (3D) tumor-stroma interface, and the proliferation rates of tumor and stromal fibroblasts. Design: We analyzed exomes, transcriptomes, and images of 364 papillary thyroid carcinoma (PTCs) from The Cancer Genome Atlas (TCGA), including 211 PTCV600E; stained 22 independent PTCs for BRAFV600E and Ki67; sequenced the exomes and stained BRAFV600E in 5 primary tumor blocks and 4 nodal metastases from one patient with PTCV600E; and reconstructed the 3D volumes of one tumor and one metastatic block at histological resolution. Results: In TCGA, BRAFV600E was associated with higher expression of proliferation markers and lower expression of thyroid differentiation markers, independently of tumor purity. Moreover, PTCV600E, in line with their overall lower purity, also had higher expression of fibroblast- and T cell-associated genes and presented more fibrosis. Tumor cells that appeared disconnected on two-dimensional histological slices were revealed to be part of a unique tumor component in the 3D reconstructed microvolumes, and they formed a surprisingly complex connected space, infiltrating a proliferative stroma. Finally, in our PTC set, both stromal fibroblasts and tumor cells presented higher proliferation rates in PTCV600E. Conclusions: Our results support the increased aggressiveness associated with BRAFV600E in PTC and shed light on the important role of the stroma in tumor expansion. The greater and more active fibrotic component predicts better efficiency of combined targeted treatments, as previously proposed for melanomaV600E.

Roles of DUOX-mediated hydrogen peroxide in metabolism, host defense, and signaling.

SIGNIFICANCE: Among the NADPH oxidases, the dual oxidases, DUOX1 and DUOX2, constitute a distinct subfamily initially called thyroid oxidases, based on their high level of expression in thyroid tissue. Genetic alterations causing inherited hypothyroidism clearly demonstrate their physiological implication in thyroid hormonogenesis. However, a growing list of biological functions triggered by DUOX-dependent reactive oxygen species (ROS) in highly differentiated mucosae have recently emerged. RECENT ADVANCES: A role of DUOX enzymes as ROS providers for lactoperoxidase-mediated killing of invading pathogens has been well established and a role in bacteria chemorepulsion has been proposed. Control of DUOX expression and activity by inflammatory molecules and immune receptor activation consolidates their contributions to innate immune defense of mucosal surfaces. Recent studies conducted in ancestral organisms have identified effectors of DUOX redox signaling involved in wound healing including epithelium regeneration and leukocyte recruitment. Moreover, local generation of hydrogen peroxide (H2O2) by DUOX has also been suggested to constitute a positive feedback loop to promote receptor signaling activation. CRITICAL ISSUES: A correct balance between H2O2 generation and detoxification mechanisms must be properly maintained to avoid oxidative damages. Overexpression of DUOX genes has been associated with an increasing number of chronic inflammatory diseases. Furthermore, H2O2-mediated DNA damage supports a mutagenic function promoting tumor development. FUTURE DIRECTIONS: Despite the high sequence similarity shared between DUOX1 and DUOX2, the two isoforms present distinct regulations, tissue expression and catalytic functions. The phenotypic characterization of novel DUOX/DUOXA invalidated animal models will be very useful for defining their medical importance in pathological conditions.

Comparative analysis of the thyrocytes and T cells: responses to H2O2 and radiation reveals an H2O2-induced antioxidant transcriptional program in thyrocytes.

CONTEXT: Radiation is an established cause of thyroid cancer, and growing evidence supports a role for hydrogen peroxide (H2O2) in spontaneous thyroid carcinogenesis. Little is known about the molecular programs activated by these agents in thyrocytes. OBJECTIVE: The purpose of this study was to compare the responses of thyrocytes and T cells to H2O2 and radiation. METHODS: We profiled the DNA damage and cell death induced by γ-radiation (0.1-5 Gy) and H2O2 (0.0025-0.3 mM) in primary human thyrocytes and T cells. We next prepared thyroid and T-cell primary cultures from 8 donors operated for noncancerous thyroid pathological conditions and profiled their genome-wide transcriptional response 4 hours after (1) exposure to 1-Gy radiation, (2) treatment with H2O2 and (3) no treatment. Two H2O2 concentrations were investigated, calibrated in each cell type to elicit levels of single- and double-strand breaks equivalent to 1-Gy γ-radiation. RESULTS: Although thyrocytes and T cells had comparable radiation responses, 3- to 10-fold more H2O2 was needed to induce detectable DNA damage in thyrocytes. At H2O2 and radiation doses inducing double-strand breaks, cell death occurred after 24 hours in T cells but not in thyrocytes. The transcriptional responses of thyrocytes and T cells to radiation were similar, involving DNA repair and cell death genes. In addition to this transcriptional program, H2O2 also up-regulated antioxidant genes in thyrocytes, including glutathione peroxidases and heme oxygenase at the double-strand breaks-inducing concentration. In contrast, a transcriptional storm involving thousands of genes was raised in T cells. Finally, we showed that inhibiting glutathione peroxidases activity increased the DNA damaging effect of H2O2 in thyrocytes. CONCLUSION: We propose that high H2O2 production in thyrocytes is matched with specific transcriptionally regulated antioxidant protection.

The type of DUOX-dependent ROS production is dictated by defined sequences in DUOXA.

A deliberate generation of ROS is now recognized to be achieved by specific NADPH oxidases (NOX). Dual oxidases (DUOXs) are Ca(2+)-activated NOXs and operate as H(2)O(2)-generators in various tissues. A tight regulation is however required to avoid ROS overproduction that can rapidly be harmful to biological systems. DUOX activator (DUOXA) proteins act as organizing elements for surface expression and activity of the DUOX enzymes. To study DUOX activation by the maturation factors, chimeric DUOXA proteins were generated by replacing particular domains between DUOXA1 and DUOXA2. Their impact on DUOX function and membrane expression were explored in a reconstituted heterologous cell system composed of COS-7 cells. We have shown that the COOH-terminal end of DUOXA1 is responsible for DUOX1-dependent H(2)O(2) generation. The NH(2)-terminal tail of DUOXA2 is critical to specify the type of ROS released by DUOX2, hydrogen peroxide or superoxide. Native DUOXA2 would constrain DUOX2 to produce H(2)O(2). However, alterations of the DUOXA2 NH(2)-terminal domain modify DUOX2 activity triggering superoxide leaking. Our results demonstrate that specific domains of the DUOX maturation factors promote the activation of DUOXs as well as the type of ROS generated by the oxidases.

Association of duoxes with thyroid peroxidase and its regulation in thyrocytes.

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

Hydrogen peroxide induces DNA single- and double-strand breaks in thyroid cells and is therefore a potential mutagen for this organ.

DNA double-strand breaks (DSBs) are considered as one of the primary causes of cancer but their induction by hydrogen peroxide (H(2)O(2)) is still controversial. In this work, we studied whether the high levels of H(2)O(2) produced in the thyroid to oxidize iodide could induce DNA modifications. Scores of DNA damage, in terms of strand breaks, were obtained by comet assay (alkaline condition for single-strand breaks (SSBs) and neutral condition for DSBs). We demonstrated that in a rat thyroid cell line (PCCl3), non-lethal concentrations of H(2)O(2) (0.1-0.5 mmol/l) as well as irradiation (1-10 Gy) provoked a large number of SSBs ( approximately 2-3 times control DNA damage values) but also high levels of DSBs (1.2-2.3 times control DNA damage values). We confirmed the generation of DSBs in this cell line and also in human thyroid in primary culture and in pig thyroid slices by measuring phosphorylation of histone H2AX. L-Buthionine-sulfoximine, an agent that depletes cells of glutathione, decreased the threshold to observe H(2)O(2)-induced DNA damage. Moreover, we showed that DNA breaks induced by H(2)O(2) were more slowly repaired than those induced by irradiation. In conclusion, H(2)O(2) causes SSBs and DSBs in thyroid cells. DSBs are produced in amounts comparable with those observed after irradiation but with a slower repair. These data support the hypothesis that the generation of H(2)O(2) in thyroid could also play a role in mutagenesis particularly in the case of antioxidant defense deficiency.

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.

Reciprocal negative regulation between thyrotropin/3',5'-cyclic adenosine monophosphate-mediated proliferation and caveolin-1 expression in human and murine thyrocytes.

The expression of caveolins is down-regulated in tissue samples of human thyroid autonomous adenomas and in the animal model of this disease. Because several cell types present in thyroid express caveolins, it remained unclear if this down-regulation occurs in thyrocytes and which are the mechanism and role of this down-regulation in the tumor context. Here we show that prolonged stimulation of isolated human thyrocytes by TSH/cAMP/cAMP-dependent protein kinase inhibits caveolins' expression. The expression of caveolins is not down-regulated by activators of other signaling pathways relevant to thyroid growth/function. Therefore, the down-regulation of caveolins' expression in autonomous adenomas is a direct consequence of the chronic activation of the TSH/cAMP pathway in thyrocytes. The down-regulation of caveolin-1 occurs at the mRNA level, with a consequent protein decrease. TSH/cAMP induces a transcription-dependent, translation-independent destabilization of the caveolin-1 mRNA. This effect is correlated to the known proliferative role of that cascade in thyrocytes. In vivo, thyrocytes of caveolin-1 knockout mice display enhanced proliferation. This demonstrates, for the first time, the in vivo significance of the specific caveolin-1 down-regulation by one mitogenic cascade and its relation to a human disease.

Thiocyanate induces cell necrosis and fibrosis in selenium- and iodine-deficient rat thyroids: a potential experimental model for myxedematous endemic cretinism in central Africa.

Thyroid destruction leading to endemic myxoedematous cretinism is highly prevalent in central Africa, where iodine (I) and selenium (SE) deficiencies as well as thiocyanate (SCN) overload are combined. All three factors have been studied experimentally in the etiology of the disease, but they have never been studied in combination. In a model using rats, we have previously shown that combining I and SE deficiencies increases the sensitivity of the thyroid to necrosis after iodide overload, an event unlikely to occur in the African situation. To develop a model that would more closely fit with the epidemiological findings, we have determined whether an SCN overload would also result in thyroid necrosis as does the I overload. The combination of the three factors increased by 3.5 times the amount of necrotic cells, from 5.5 +/- 0.3% in the I-SE+ thyroids to 18.9 +/- 1.6% in the I-SE-SCN-overloaded ones. Methimazole administration prevented the SCN-induced necrosis. SE- thyroids evolved to fibrosis, whereas SE+ thyroids did not. TGFbeta was prominent in macrophages present in SE- glands. Thyroid destruction in central Africa might therefore originate from the interaction of three factors: I and SE deficiencies by increasing H(2)O(2) accumulation, SE deficiency by decreasing cell defense and promoting fibrosis, and SCN overload by triggering follicular cell necrosis.

Gene expression profile in thyroid of transgenic mice overexpressing the adenosine receptor 2a.

Mutations of the TSH receptor leading to constitutive activation of the cAMP cascade are responsible for the development of hot nodules, if arising in a somatic cell, and nonautoimmune hyperthyroidism, when occurring in a germinal cell. An animal model of constitutive activation of the thyroid cAMP cascade has been obtained by generating transgenic mice expressing the adenosine receptor (Tg-A2aR) under the control of the thyroglobulin promoter. These mice develop huge goiters and die prematurely due to hyperthyroidism induced cardiac failure. To identify new genes involved in the tumorigenic pathway of the thyroid, we designed a protocol using microarray technology to study the differential expression, between normal and transgenic thyroid, of +/-13,000 genes. A total of 360 genes or expressed sequence tags showed a strong modulation with background corrected values of fluorescence superior to 2-fold change. The modulated genes were classified according to their proposed gene ontology functions. Approximately half of them were up-regulated. The function of the majority of these genes in thyroid physiology is still to be determined. Some of them, like IGF-I or IGF binding protein 3 or 5, may play an important role in the development of thyroid nodules through paracrine mechanisms. This study demonstrates the feasibility of sequentially following the cascade of events leading to the formation of benign tumors such as hot thyroid nodule or hyperfunctional goiter.