Functional Toll-like Receptor 4 Overexpression in Papillary Thyroid Cancer by MAPK/ERK-Induced ETS1 Transcriptional Activity.

Emerging evidence suggests that unregulated Toll-like receptor (TLR) signaling promotes tumor survival signals, thus favoring tumor progression. Here, the mechanism underlying TLR4 overexpression in papillary thyroid carcinomas (PTC) mainly harboring the BRAFV600E mutation was studied. TLR4 was overexpressed in PTC compared with nonneoplastic thyroid tissue. Moreover, paired clinical specimens of primary PTC and its lymph node metastasis showed a significant upregulation of TLR4 levels in the metastatic tissues. In agreement, conditional BRAFV600E expression in normal rat thyroid cells and mouse thyroid tissue upregulated TLR4 expression levels. Furthermore, functional TLR4 expression was demonstrated in PTC cells by increased NF-κB transcriptional activity in response to the exogenous TLR4-agonist lipopolysaccharide. Of note, The Cancer Genome Atlas data analysis revealed that BRAFV600E-positive tumors with high TLR4 expression were associated with shorter disease-free survival. Transcriptomic data analysis indicated a positive correlation between TLR4 expression levels and MAPK/ERK signaling activation. Consistently, chemical blockade of MAPK/ERK signaling abrogated BRAFV600E-induced TLR4 expression. A detailed study of the TLR4 promoter revealed a critical MAPK/ERK-sensitive Ets-binding site involved in BRAFV600E responsiveness. Subsequent investigation revealed that the Ets-binding factor ETS1 is critical for BRAFV600E-induced MAPK/ERK signaling-dependent TLR4 gene expression. Together, these data indicate that functional TLR4 overexpression in PTCs is a consequence of thyroid tumor-oncogenic driver dysregulation of MAPK/ERK/ETS1 signaling.Implications: Considering the participation of aberrant NF-κB signaling activation in the promotion of thyroid tumor growth and the association of high TLR4 expression with more aggressive tumors, this study suggests a prooncogenic potential of TLR4 downstream signaling in thyroid tumorigenesis. Mol Cancer Res; 16(5); 833-45. ©2018 AACR.

Dissecting thyroid hormone transport and metabolism in dendritic cells.

We reported thyroid hormone (TH) receptor expression in murine dendritic cells (DCs) and 3,5,3'-triiodothyronine (T3)-dependent stimulation of DC maturation and ability to develop a Th1-type adaptive response. Moreover, an increased DC capacity to promote antigen-specific cytotoxic T-cell activity, exploited in a DC-based antitumor vaccination protocol, was revealed. However, putative effects of the main circulating TH, l-thyroxine (T4) and the mechanisms of TH transport and metabolism at DC level, crucial events for TH action at target cell level, were not known. Herein, we show that T4 did not reproduce those registered T3-dependent effects, finding that may reflect a homoeostatic control to prevent unspecific systemic activation of DCs. Besides, DCs express MCT10 and LAT2 TH transporters, and these cells mainly transport T3 with a favored involvement of MCT10 as its inhibition almost prevented T3 saturable uptake mechanism and reduced T3-induced IL-12 production. In turn, DCs express iodothyronine deiodonases type 2 and 3 (D2, D3) and exhibit both enzymatic activities with a prevalence towards TH inactivation. Moreover, T3 increased MCT10 and LAT2 expression and T3 efflux from DCs but not T3 uptake, whereas it induced a robust induction of D3 with a parallel slight reduction in D2. These findings disclose pivotal events involved in the mechanism of action of THs on DCs, providing valuable tools for manipulating the immunogenic potential of these cells. Furthermore, they broaden the knowledge of the TH mechanism of action at the immune system network.

Effects of 2-iodohexadecanal in the physiology of thyroid cells.

Iodide has direct effects on thyroid function. Several iodinated lipids are biosynthesized by the thyroid and they were postulated as intermediaries in the action of iodide. Among them, 2-iodohexadecanal (2-IHDA) has been identified and proposed to play a role in thyroid autoregulation. The aim of this study was to compare the effect of iodide and 2-IHDA on thyroid cell physiology. For this purpose, FRTL-5 thyroid cells were incubated with the two compounds during 24 or 48 h and several thyroid parameters were evaluated such as: iodide uptake, intracellular calcium and H2O2 levels. To further explore the molecular mechanism involved in 2-IHDA action, transcript and protein levels of genes involved in thyroid hormone biosynthesis, as well as the transcriptional expression of these genes were evaluated in the presence of iodide and 2-IHDA. The results obtained indicate that 2-IHDA reproduces the action of excess iodide on the "Wolff-Chaikoff" effect as well as on thyroid specific genes transcription supporting its role in thyroid autoregulation.

Antitumor Responses Stimulated by Dendritic Cells Are Improved by Triiodothyronine Binding to the Thyroid Hormone Receptor ß.

Bidirectional cross-talk between the neuroendocrine and immune systems orchestrates immune responses in both physiologic and pathologic settings. In this study, we provide in vivo evidence of a critical role for the thyroid hormone triiodothyronine (T3) in controlling the maturation and antitumor functions of dendritic cells (DC). We used a thyroid hormone receptor (TR) ß mutant mouse (TRßPV) to establish the relevance of the T3-TRß system in vivo. In this model, TRß signaling endowed DCs with the ability to stimulate antigen-specific cytotoxic T-cell responses during tumor development. T3 binding to TRß increased DC viability and augmented DC migration to lymph nodes. Moreover, T3 stimulated the ability of DCs to cross-present antigens and to stimulate cytotoxic T-cell responses. In a B16-OVA mouse model of melanoma, vaccination with T3-stimulated DCs inhibited tumor growth and prolonged host survival, in part by promoting the generation of IFNγ-producing CD8(+) T cells. Overall, our results establish an adjuvant effect of T3-TRß signaling in DCs, suggesting an immediately translatable method to empower DC vaccination approaches for cancer immunotherapy.

Dexamethasone counteracts the immunostimulatory effects of triiodothyronine (T3) on dendritic cells.

Glucocorticoids (GCs) are widely used as anti-inflammatory and immunosuppressive agents. Several studies have indicated the important role of dendritic cells (DCs), highly specialized antigen-presenting and immunomodulatory cells, in GC-mediated suppression of adaptive immune responses. Recently, we demonstrated that triiodothyronine (T3) has potent immunostimulatory effects on bone marrow-derived mouse DCs through a mechanism involving T3 binding to cytosolic thyroid hormone receptor (TR) ß1, rapid and sustained Akt activation and IL-12 production. Here we explored the impact of GCs on T3-mediated DC maturation and function and the intracellular events underlying these effects. Dexamethasone (Dex), a synthetic GC, potently inhibited T3-induced stimulation of DCs by preventing the augmented expression of maturation markers and the enhanced IL-12 secretion through mechanisms involving the GC receptor. These effects were accompanied by increased IL-10 levels following exposure of T3-conditioned DCs to Dex. Accordingly, Dex inhibited the immunostimulatory capacity of T3-matured DCs on naive T-cell proliferation and IFN-γ production while increased IL-10 synthesis by allogeneic T cell cultures. A mechanistic analysis revealed the ability of Dex to dampen T3 responses through modulation of Akt phosphorylation and cytoplasmic-nuclear shuttling of nuclear factor-κB (NF-κB). In addition, Dex decreased TRß1 expression in both immature and T3-maturated DCs through mechanisms involving the GC receptor. Thus GCs, which are increased during the resolution of inflammatory responses, counteract the immunostimulatory effects of T3 on DCs and their ability to polarize adaptive immune responses toward a T helper (Th)-1-type through mechanisms involving, at least in part, NF-κB- and TRß1-dependent pathways. Our data provide an alternative mechanism for the anti-inflammatory effects of GCs with critical implications in immunopathology at the cross-roads of the immune-endocrine circuits.

Growth hormone treatment in children with idiopathic short stature: correlation of growth response with peripheral thyroid hormone action.

OBJECTIVE: Idiopathic short stature (ISS) describes short children with normal GH secretion. Although GH treatment increases their heights, growth response to the therapy differs among patients. Thyroid hormones (TH) are essential for longitudinal growth acting mainly through TH receptors (TR) α and ß. We have previously reported that GH treatment reduced peripheral TH action in Turner Syndrome by TR down-regulation. The aims of the study were to assess the effect of GH treatment to ISS on peripheral TH action and the correlation between thyroid status and growth response to the therapy. SUBJECTS, DESIGN AND MEASUREMENTS: Eighteen normal (control) and twenty-five ISS children were enrolled and evaluated before and after 12 months of life time (control) or 12 months of GH therapy (ISS). Fasting blood was used for serum biochemical evaluations, peripheral blood mononuclear cells for TR mRNA determination by QRT-PCR and growth parameters by standard methods. RESULTS: GH treatment modified neither TR mRNA levels nor serum markers of TH action in ISS evaluated as a whole group. However, the individual change in TRß mRNA levels correlated to the change in sex hormone-binding globulin (SHBG) levels after GH therapy. The growth response to GH correlated positively with the change in TRα mRNA level and negatively with that in TRß mRNA, TSH and SHBG levels. The change in each TR mRNA isoform after GH treatment correlated negatively with its own basal level. CONCLUSIONS: GH therapy induced individual changes in TR expression in ISS that correlated with their growth response. The basal TR mRNA level could predetermine the change in TR expression and therefore the sensitivity to GH treatment.

Nuclear factor (NF)-kappaB-dependent thyroid hormone receptor beta1 expression controls dendritic cell function via Akt signaling.

Despite considerable progress in our understanding of the interplay between immune and endocrine systems, the role of thyroid hormones and their receptors in the control of adaptive immunity is still uncertain. Here, we investigated the role of thyroid hormone receptor (TR) beta(1) signaling in modulating dendritic cell (DC) physiology and the intracellular mechanisms underlying these immunoregulatory effects. Exposure of DCs to triiodothyronine (T(3)) resulted in a rapid and sustained increase in Akt phosphorylation independently of phosphatidylinositol 3-kinase activation, which was essential for supporting T(3)-induced DC maturation and interleukin (IL)-12 production. This effect was dependent on intact TR beta(1) signaling as small interfering RNA-mediated silencing of TR beta(1) expression prevented T(3)-induced DC maturation and IL-12 secretion as well as Akt activation and I kappaB-epsilon degradation. In turn, T(3) up-regulated TR beta(1) expression through mechanisms involving NF-kappaB, suggesting an autocrine regulatory loop to control hormone-dependent TR beta(1) signaling. These findings were confirmed by chromatin immunoprecipitation analysis, which disclosed a new functional NF-kappaB consensus site in the promoter region of the TRB1 gene. Thus, a T(3)-induced NF-kappaB-dependent mechanism controls TR beta(1) expression, which in turn signals DCs to promote maturation and function via an Akt-dependent but PI3K-independent pathway. These results underscore a novel unrecognized target that regulates DC maturation and function with critical implications in immunopathology at the cross-roads of the immune-endocrine circuits.

Nitric oxide mediates the suppressive effect of testosterone on cell proliferative response to myelin basic protein.

This study assessed whether the in vitro effect of testosterone on the proliferative response of mononuclear cells to myelin basic protein (MBP) could be mediated by nitric oxide (NO). Testosterone but not cholesterol supplementation specifically suppressed the proliferative response of rat mononuclear cells to MBP and in parallel increased the NO level. NG-monomethyl 1-l-arginine, an inhibitor of NO synthesis, reverted the suppression of the testosterone-induced proliferative response to MBP. These results indicate that changes in the production of NO by testosterone are able to alter the specific T cell proliferation induced by the encephalitogenic MBP and in this way; it could be one of the molecular mechanisms that modulate the development of experimental autoimmune encephalomyelitis (EAE).

Control of dendritic cell maturation and function by triiodothyronine.

Accumulating evidence indicates a functional crosstalk between immune and endocrine mechanisms in the modulation of innate and adaptive immunity. However, the impact of thyroid hormones (THs) in the initiation of adaptive immune responses has not yet been examined. Here we investigated the presence of thyroid hormone receptors (TRs) and the impact of THs in the physiology of mouse dendritic cells (DCs), specialized antigen-presenting cells with the unique capacity to fully activate naive T cells and orchestrate adaptive immunity. Both immature and lipopolysaccharide-matured bone marrow-derived DCs expressed TRs at mRNA and protein levels, showing a preferential cytoplasmic localization. Remarkably, physiological levels of triiodothyronine (T3) stimulated the expression of DC maturation markers (major histocompatibility complex II, CD80, CD86, and CD40), markedly increased the secretion of interleukin-12, and stimulated the ability of DCs to induce naive T cell proliferation and IFN-gamma production in allogeneic T cell cultures. Analysis of the mechanisms involved in these effects revealed the ability of T3 to influence the cytoplasmic-nuclear shuttling of nuclear factor-kappaB on primed DCs. Our study provides the first evidence for the presence of TRs on bone marrow-derived DCs and the ability of THs to regulate DC maturation and function. These results have profound implications in immunopathology, including cancer and autoimmune manifestations of the thyroid gland at the crossroads of the immune and endocrine systems.

Endogenous thyrocyte-produced nitric oxide inhibits iodide uptake and thyroid-specific gene expression in FRTL-5 thyroid cells.

Nitric oxide (NO) is a free radical that mediates a wide array of cell functions. It is generated from l-arginine by NO-synthase (NOS). Expression of NOS isoforms has been demonstrated in thyroid cells. Previous reports indicated that NO donors induce dedifferentiation in thyrocytes. However, the functional significance of endogenous thyrocyte-produced NO has not been explored. This work aimed to study the influence of endogenous NO on parameters of thyroid cell function and differentiation in FRTL-5 cells. We observed that treatment with the NOS inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME), increased the TSH-stimulated iodide uptake. The TSH-induced sodium iodide symporter (NIS) and thyroglobulin (TG) mRNA expressions were increased after incubation with L-NAME. In transient transfection assays, TSH-stimulated transcriptional activities of NIS and TG promoters were increased by L-NAME. An increment of the TSH-stimulated cell proliferation was observed after NOS inhibition. Similar results were obtained when the action of another NOS inhibitor, N(g)-monomethyl-L-arginine, was analysed for most of these studies. The production of NO, which was not detectable in basal conditions, was increased by TSH. Our data provide strong evidence that endogenous NO could act as a negative signal for TSH-stimulated iodide uptake and thyroid-specific gene expression as well as proliferation in thyrocytes. These findings reveal a possible new inhibitory pathway in the regulation of thyroid cell function.

Bacterial lipopolysaccharide stimulates the thyrotropin-dependent thyroglobulin gene expression at the transcriptional level by involving the transcription factors thyroid transcription factor-1 and paired box domain transcription factor 8.

The bacterial lipopolysaccharide (LPS) is a biological activator that induces expression of multiple genes in several cell types. LPS has been proposed as an etiopathogenic agent in autoimmune diseases. However, whether LPS affects the expression of autoantigens has not been explored. Thyroglobulin (TG) is a key protein in thyroid hormonogenesis and one of the major thyroid autoantigens. This study aimed to analyze the action of LPS on TG gene expression in Fisher rat thyroid cell line FRTL-5 thyroid cells. We demonstrate that LPS increases the TSH-induced TG protein and mRNA level. Evidence that the effect of LPS is exerted at the transcriptional level was obtained by transfecting the minimal TG promoter. The C element of the TG promoter, which contains sequences for paired box domain transcription factor 8 (Pax8) and thyroid transcription factor (TTF)-1 binding, is essential for full TG promoter expression under TSH stimulation. The transcriptional activity of a construct containing five tandem repeats of the C site is increased by LPS, indicating a possible involvement of the C site in the LPS-induced TG gene transcription. We demonstrate that the TG promoter mutated at the Pax8 or TTF-1 binding element in the C site does not respond to LPS. In band shift assays, binding of Pax8 and TTF-1 to the C site is increased by LPS. The Pax8 and TTF-1 mRNA and protein levels are augmented by LPS. The half-lives of TG, Pax8, and TTF-1 are increased in endotoxin-treated cells. Our results reveal the ability of LPS to stimulate the expression of TG, a finding of potential pathophysiological implication.

Ultrastructural localization of thyroid peroxidase, hydrogen peroxide-generating sites, and monoamine oxidase in benign and malignant thyroid diseases.

Despite thyroid tissue heterogeneity, biochemical and morphological features have been associated with certain thyroid diseases. We analyzed the ultracytochemical localization of thyroperoxidase (TPO), TPO-associated hydrogen peroxide-generating sites (H(2)O(2) sites), and monoamine oxidase (MAO) in terms of morphology and biochemical TPO activity in abnormal thyroids. We examined 11 cases of nontoxic multinodular goiter, 5 cases of Hashimoto's thyroiditis, 1 case of oncocytic (Hürthle or oxyphilic cell) adenoma, 5 cases of Graves' disease, 4 cases of papillary carcinoma, and 4 cases of perinodular normal tissue. In the perinodular tissue, TPO was detected mainly in the nuclear envelope, rough endoplasmic reticulum (RER), and subapical vesicles, but not in the apical surface. In multinodular goiter, heterogeneous TPO reactivity ranging from almost null to strongly positive was detected in similar locations as in the perinodular tissue, and was absent in the microvilli. Follicular cells from Hashimoto's thyroiditis displayed TPO in the nuclear envelope and the scarce RER. Remarkably, oncocytic cells from both Hashimoto's thyroiditis and oncocytic adenoma, typically packed with mitochondria, displayed evident TPO reaction exclusively in mitochondrial cristae. In Graves' disease, the nuclear envelope, enlarged RER, and apical vesicles were strongly TPO positive, and microvilli also exhibited TPO activity. Papillary carcinoma cells were negative for TPO. The localization and characteristics of TPO activity in the H(2)O(2) sites were similar to that of TPO in all tissues. MAO was positive in mitochondria of perinodular tissues, multinodular goiter, and oncocytes and negative in Hashimoto's thyroiditis and Graves' disease. Interestingly, MAO was intensely positive in the nuclear envelope of papillary carcinoma but unreactive in mitochondria. Biochemical TPO activity was increased in multinodular goiter and Graves' disease. In conclusion, several changes in ultracytochemical characteristics of TPO, H(2)O(2) sites, and MAO were associated with thyroid disease. Nonmalignant oncocytic cells exhibited an unusual mitochondrial location of TPO and H(2)O(2) sites. The distribution of MAO in nuclear envelope of papillary carcinoma cells could be a further feature of malignancy.