TSH-induced Thyroid Research Articles

The Transcription Factor NF-κB Mediates Thyrotropin-Stimulated Expression of Thyroid Differentiation Markers.

Background: The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor is a key regulator of cell survival, proliferation, and gene expression. Although activation of NF-κB signaling in thyroid follicular cells after thyrotropin (TSH) receptor (TSHR) engagement has been reported, the downstream signaling leading to NF-κB activation remains unexplored. Here, we sought to elucidate the mechanisms that regulate NF-κB signaling activation in response to TSH stimulation. Methods: Fisher rat-derived thyroid cell lines and primary cultures of NF-κB essential modulator (NEMO)-deficient mice thyrocytes were used as models. Signaling pathways leading to the activation of NF-κB were investigated by using chemical inhibitors and phospho-specific antibodies. Luciferase reporter gene assays and site-directed mutagenesis were used to monitor NF-κB-dependent gene transcriptional activity and the expression of thyroid differentiation markers was assessed by reverse transcription quantitative polymerase chain reaction and Western blot, respectively. Chromatin immunoprecipitation (ChIP) was carried out to investigate NF-κB subunit p65 DNA binding, and small interfering RNA (siRNA)-mediated gene knockdown approaches were used for studying gene function. Results: Using thyroid cell lines, we observed that TSH treatment leads to protein kinase C (PKC)-mediated canonical NF-κB p65 subunit nuclear expression. Moreover, TSH stimulation phosphorylated the kinase TAK-1, and its knockdown abolished TSH-induced NF-κB transcriptional activity. TSH induced the transcriptional activity of the NF-κB subunit p65 in a protein kinase A (PKA)-dependent phosphorylation at Ser-276. In addition, p65 phosphorylation at Ser-276 induced acetyl transferase p300 recruitment, leading to its acetylation on Lys-310 and thereby enhancing its transcriptional activity. Evaluation of the role played by NF-κB in thyroid physiology demonstrated that the canonical NF-κB inhibitor BAY 11-7082 reduced TSH-induced expression of thyroid differentiation markers. The involvement of NF-κB signaling in thyroid physiology was confirmed by assessing the TSH-induced gene expression in primary cultures of NEMO-deficient mice thyrocytes. ChIP and the knockdown experiments revealed that p65 is a nuclear effector of TSH actions, inducing the transcripcional expression of thyroid differentiation markers. Conclusions: Taken together, our results point to NF-κB being a pivotal mediator in the TSH-induced thyroid follicular cell differentiation, a relevant finding with potential physiological and pathophysiological implications.

MicroRNA-22 regulates thyroid cell growth and lipid accumulation via IL6R.

Thyroid stimulating hormone (TSH) is the main regulator of thyroid cell proliferation and endocrine function. Here, we tested the effect of mir-22 in TSH induced proliferation and lipid metabolism of thyroid cells. TSH significantly down-regulated miR-22-3p, promoted proliferation and inhibited apoptosis of thyroid epithelial cells line, FRTL-5; effects that were opposed by overexpression of miR-22-3p. Overexpression of miR-22-3p significantly inhibited TSH- induced expression of lipid metabolic marker genes and increased the expression of lipid catabolism markers and IL6R and led to accumulation of intracellular lipid. IL6R overexpression also led to excessive proliferation, restrained apoptosis and promoted lipid accumulation. In conclusion, miR-22 regulates TSH-induced thyroid cell proliferation and lipid metabolism disorder by impacting IL6R.

Growth hormone and insulin-like growth factor 1 affect the severity of Graves' disease.

Graves’ disease, the most common form of hyperthyroidism in iodine-replete countries, is associated with the presence of immunoglobulins G (IgGs) that are responsible for thyroid growth and hyperfunction. In this article, we report the unusual case of a patient with acromegaly and a severe form of Graves’ disease. Here, we address the issue concerning the role of growth hormone (GH) and insulin-like growth factor 1 (IGF1) in influencing thyroid function. Severity of Graves’ disease is exacerbated by coexistent acromegaly and both activity indexes and symptoms and signs of Graves’ disease improve after the surgical remission of acromegaly. We also discuss by which signaling pathways GH and IGF1 may play an integrating role in regulating the function of the immune system in Graves’ disease and synergize the stimulatory activity of Graves’ IgGs.Learning points:Clinical observations have demonstrated an increased prevalence of euthyroid and hyperthyroid goiters in patients with acromegaly.The coexistence of acromegaly and Graves’ disease is a very unusual event, the prevalence being <1%.Previous in vitro studies have showed that IGF1 synergizes the TSH-induced thyroid cell growth-activating pathways independent of TSH/cAMP/PKA cascade.We report the first case of a severe form of Graves’ disease associated with acromegaly and show that surgical remission of acromegaly leads to a better control of symptoms of Graves’ disease.

Nitric oxide-repressed Forkhead factor FoxE1 expression is involved in the inhibition of TSH-induced thyroid peroxidase levels

Thyroid peroxidase (TPO) is essential for thyroid hormone synthesis mediating the covalent incorporation of iodine into tyrosine residues of thyroglobulin process known as organification. Thyroid-stimulating hormone (TSH) via cAMP signaling is the main hormonal regulator of TPO gene expression. In thyroid cells, TSH-stimulated nitric oxide (NO) production inhibits TSH-induced thyroid-specific gene expression, suggesting a potential autocrine role of NO in modulating thyroid function. Indeed, NO donors downregulate TSH-induced iodide accumulation and organification in thyroid cells. Here, using FRTL-5 thyroid cells as model, we obtained insights into the molecular mechanism underlying the inhibitory effects of NO on iodide organification. We demonstrated that NO donors inhibited TSH-stimulated TPO expression by inducing a cyclic guanosine monophosphate-dependent protein kinase-mediated transcriptional repression of the TPO gene. Moreover, we characterized the FoxE1 binding site Z as mediator of the NO-inhibited TPO expression. Mechanistically, we demonstrated that NO decreases TSH-induced FoxE1 expression, thus repressing the transcripcional activation of TPO gene. Taken together, we provide novel evidence reinforcing the inhibitory role of NO on thyroid cell function, an observation of potential pathophysiological relevance associated with human thyroid pathologies that come along with changes in the NO production.

Abstract 119: MiR-1 downregulation plays a critical role in thyroid cell proliferation

Abstract MiR-1 downregulation plays a critical role in thyroid cell proliferation V. Leone, D. D'Angelo, PL. Pallante, A. Ferraro. Istituto di Endocrinologia ed Oncologia Sperimentale del CNR c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facoltà di Medicina e Chirurgia di Napoli, Università degli Studi di Napoli “Federico II”, Naples, Italy. MicroRNAs have emerged as an important class of short endogenous RNAs that act as post-transcriptional regulators of gene expression, and their expression is constantly deregulated in human cancer. MiR-1 has been found downregulated in lung, colon and prostate cancer. The aim of our work has been to investigate the possible role of miR-1 in thyroid carcinogenesis. Therefore, we have analysed miR-1 expression in a panel of thyroid neoplasias including benign and malignant lesions. Our results show that miR-1 expression is drastically downregulated in thyroid adenomas and carcinomas in comparison with normal thyroid tissue. Interestingly, miR-1 downregulation was also found in thyroid hyperproliferative non neoplastic lesions such as goiters. These results suggested a general role of miR-1 downregulation in the control of thyroid cell proliferation. This suggestion was then confirmed by the drastic decrease in miR-1 expression following exposure of rat thyroid cells to thyrotropin (TSH), the physiological thyroid stimulator. Searching for miR-1 targets, we identified CREB1 transcriptional factor, that is the main effector of the TSH-induced thyroid cell growth, as miR-1 target, suggesting the presence of a regulatory synergistic loop including TSH, CREB1 and the miR-1. Moreover, we have demonstrated that also the CXCR4 protein and its ligand SDF1α, that are involved in cell migration and are constantly overexpressed in human thyroid carcinoma, are targets of miR-1. Functional studies demonstrate that miR-1 is able to inhibit thyroid carcinoma cell proliferation and migration validating a critical role of miR-1 downregulation in thyroid carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 119. doi:10.1158/1538-7445.AM2011-119

Amiodarone Reversibly Decreases Sodium-Iodide Symporter mRNA Expression at Therapeutic Concentrations and Induces Antioxidant Responses at Supraphysiological Concentrations in Cultured Human Thyroid Follicles

Amiodarone, a potent antiarrhythmic, iodine-containing agent, is a highly active oxidant exerting cytotoxic effects on thyrocytes at pharmacological concentrations. Patients receiving amiodarone usually remain euthyroid, but occasionally develop thyroid dysfunction. Although there is a general consensus that amiodarone-associated hypothyroidism is iodine induced, the destructive mechanism of thyroid follicles in amiodarone-induced thyrotoxicosis remains unknown. To elucidate the mechanism by which amiodarone elicits thyroid dysfunction. Human thyroid follicles were cultured with thyroid-stimulating hormone (TSH) and amiodarone at therapeutic (1-2 microM) and pharmacological (10-20 microM) concentrations, and the drug-induced effect on whole human gene expression was analyzed by cDNA microarray. Microarray data were confirmed by real-time PCR and Western blot. Amiodarone at 1-2 muM decreased the expression level of the sodium-iodide symporter (NIS) to nearly half, but did not affect genes participating in thyroid hormonogenesis (thyroid peroxidase, thyroglobulin, pendrin, and NADPH oxidase). Higher concentrations (10-20 microM) decreased the expression of all these genes, accompanied by increased expression of antioxidant proteins such as heme oxygenase 1 and ferritin. When thyroid follicles obtained from a patient with Graves' disease who had been treated with amiodarone were cultured in amiodarone-free medium, TSH-induced thyroid function was intact, suggesting that amiodarone at a maintenance dose did not elicit any cytotoxic effect on thyrocytes. The ultrastructural features of cultured thyroid follicles were compatible with these in vitro findings. These in vitro and ex vivo findings suggest that patients taking maintenance doses of amiodarone usually remain euthyroid, probably due to escape from the Wolff-Chaikoff effect mediated by decreased expression of NIS mRNA. Further, amiodarone is not cytotoxic for thyrocytes at therapeutic concentrations but elicits cytotoxicity through oxidant activity at supraphysiological concentrations. We speculate that when amiodarone-induced prooxidant activity somehow exceeds the endogenous antioxidant capacity, the thyroid follicles will be destroyed and amiodarone-induced destructive thyrotoxicosis may develop.

Suppression of Iodide Uptake and Thyroid Hormone Synthesis with Stimulation of the Type I Interferon System by Double-Stranded Ribonucleic Acid in Cultured Human Thyroid Follicles

Although viral infection is thought to be associated with subacute thyroiditis and probably with autoimmune thyroid disease, possible changes in thyroid function during the prodromal period of infection or subclinical infection remain largely unknown. Recently, it was shown that pathogen-associated molecular patterns stimulate Toll-like receptors (TLR) and activate innate immune responses by producing type I interferons (IFN). Using a human thyroid follicle culture system, in which de novo synthesized thyroid hormones are released into the culture medium under physiological concentrations of human TSH, we studied the effects of polyinosinic-polycytidylic acid [Poly(I:C)], a chemical analog of viral double-stranded RNA (dsRNA), on TSH-induced thyroid function. Thyrocytes expressed ligands for dsRNA (TLR 3, CD14, and retinoic-acid-inducible protein-1) comparable with the TSH receptor. DNA microarray and real-time PCR analyses revealed that dsRNA increased the expression of mRNA for TLR3, IFN-beta, IFN-regulating factors, proinflammatory cytokines, and class I major histocompatibility complex (MHC), whereas genes associated with thyroid hormonogenesis (sodium/iodide symporter, peroxidase, deiodinases) were suppressed. In accordance to these data, Poly(I:C) suppressed TSH-induced 125I uptake and hormone synthesis dose dependently, accompanied by a decrease in the ratio of 125I-T3/125I-T4 released into the culture medium, whereas peptidoglycan, lipopolysaccharides, or unmethylated CpG DNA, ligands for TLR2, TLR4, and TLR9, respectively, had no significant effect. These inhibitory effects of Poly(I:C) were not blocked by a neutralizing antibody against TLR3 and an anti-IFN alpha/beta receptor antibody. These in vitro findings suggest that when thyrocytes are infected with certain viruses, dsRNA formed intracellularly in thyrocytes may be a cause for thyroid dysfunction, leading to development of autoimmune thyroiditis.

Identification of differentially expressed genes in thyrotropin stimulated dog thyroid cells by the cDNA-AFLP technique

In dog thyrocytes in primary culture, thyrotropin (TSH), through cAMP, positively controls proliferation and differentiation. As until now, the key events and the genes involved in the action of TSH remain largely uncharacterized, our goal was to identify new differentially expressed genes in TSH-induced thyroid proliferation. Using cDNA-AFLP, we vizualized 105 different transcripts showing significant differential expression during the stimulation of dog thyrocytes with TSH for different times, in the presence of insulin. Northern blot and RT-PCR analyses confirmed the pattern expression of 5 clones encoding known proteins: thrombospondine 1, TNFr1, RhoE, RalB, and annexin A2. These regulations provide molecular counterparts of in vivo physiological effects of TSH: angiogenesis (decreased thrombospondin 1), decreased apoptosis (decreased TNFR1) and actin filament disruption, macropinocytosis and thyroid hormone secretion (decreased RhoE).

Leptin regulation of the thyroids: negative regulation on thyroid hormone levels in euthyroid subjects and inhibitory effects on iodide uptake and Na+/I- symporter mRNA expression in rat FRTL-5 cells.

Leptin receptors are distributed throughout the body and leptin has been shown to have various effects. As we have recently demonstrated a positive correlation between serum leptin levels and TSH in euthyroid subjects, we investigated the effect of leptin on the thyroids. It was observed that serum leptin levels were negatively correlated with free thyroxine/TSH ratios in the serum of euthyroid female subjects. This suggests that leptin may modulate TSH effects. RT-PCR for leptin receptor expression revealed that FRTL-5 cells possess the gene transcript to the long cytoplasmic form of the receptor. Leptin actually appeared to induce an increase in c-fos mRNA expression. However, it inhibited iodide uptake typically induced by both TSH and dibutyryl cAMP, while leptin did not inhibit TSH-induced cAMP production or TSH-stimulated DNA synthesis in 4H medium (in the absence of insulin and TSH). Leptin also was observed to inhibit TSH- and dibutyryl cAMP-induced Na+/I- symporter and thyroglobulin mRNA expression. Lastly, leptin was seen to inhibit TSH-stimulated thymidine incorporation in 5H medium. Taken together, these results suggest that leptin suppresses TSH-induced thyroid function. Therefore, we hypothesized that leptin may be one of the regulators of thyroid function in obese patients.

Sex steroids regulate TSH-induced thyroid growth during sexual maturation in Wistar rats.

Sex steroids have been reported to influence thyroid pathogenesis in human and experimental animals. However, there is no much report on the effect of sex steroids in the growth of normal thyroid. The present study is an attempt to know TSH-induced thyroid growth during sexual maturation in Wistar rats. Wistar rats of day 1 to day 150 postpartum (pp) were used in the present study. Thyroid growth indices such as absolute and relative thyroid weights, concentration of DNA in the thyroid, mitotic index and numerical density of thyrocytes, hormonal profiles in the serum and thyroid tissue such as thyrotropin (TSH), thyroid hormones, testosterone and estradiol, and receptors for TSH, androgen and estrogen in the thyroid were estimated. A gender-specific shift in the pattern of serum TSH and TSH-R was observed during 30-45 days of postnatal life, the period at which steroidogenesis by testes or ovaries of rats are reactivated. Testosterone in males and estradiol in females modulate thyroid growth through TSH. It is concluded from the present study that changes in the sex steroid status between male and female rats have a definite influence on the pattern of thyroid growth and TSH.

Interleukin-6 (IL-6) inhibits thyroid function in the presence of soluble IL-6 receptor in cultured human thyroid follicles.

Interleukin-6 (IL-6), a pleiotropic cytokine, is postulated to be involved in the pathogenesis of sick euthyroid syndrome, although the direct in vitro effects of IL-6 on human thyroid function are controversial. Because IL-6 signal can be transduced when the complex of IL-6 and soluble IL-6 receptor (sIL-6R) binds to gp 130, an IL-6 signal transducer, we studied the effects of IL-6 and sIL-6R on thyroid function, using human thyroid follicles obtained from patients with Graves' disease. IL-6 alone had no inhibitory effect on TSH-induced thyroid function (125I incorporation and organic 125I release), even at supraphysiological concentrations. However, in the presence of physiological concentrations of sIL-6R (100 ng/ml), IL-6 inhibited thyroid function dose dependently and completely, accompanied with the decreased ratio of 125I-T3/125I-T4 not only in the thyroid follicles but also in the culture medium. Thyroid follicles did not secrete sIL-6R but produced IL-6 constitutively. Consistent with these findings, sIL-6R inhibited thyroid function slightly at high concentrations. Furthermore, RT-PCR analyses revealed that human thyroid follicles expressed the messenger RNAs for IL-6 and gp130 but scarcely messenger RNA for IL-6R. These in vitro findings suggest that IL-6 alone hardly affects thyroid function in thyroid follicles in which IL-6R gene is scarcely expressed. However, because sIL-6R is present abundantly in serum, IL-6 in vivo would be capable of inhibiting the synthesis and release of T4 and, to a greater extent, T3 from the thyroid gland. These in vitro findings are at least partly related to the development of sick euthyroid syndrome.

Thyrotropin-induced hydrogen peroxide production in FRTL-5 thyroid cells is mediated not by adenosine 3',5'-monophosphate, but by Ca2+ signaling followed by phospholipase-A2 activation and potentiated by an adenosine derivative.

The production of hydrogen peroxide (H2O2) as an essential process for iodide organification is a key reaction in TSH-induced thyroid hormone synthesis. Here we characterize the signal transduction pathway involved in TSH-induced H2O2 production in FRTL-5 thyroid cells. At higher than 1 nM TSH, N6-(L-2-phenylisopropyl)adenosine (PIA), an adenosine receptor agonist having, by itself, no influence on H2O2 generation, potentiated this TSH action, whereas the TSH increase and PIA addition reduced cAMP accumulation. RO 20-1724, a phosphodiesterase inhibitor, amplified the TSH-induced cAMP accumulation, but did not change H2O2 generation in the whole range of TSH used. Ca(2+)-mobilizing agonists, GTP and ATP, also induced H2O2 production without stimulating cAMP accumulation. Chelation of intracellular Ca2+ markedly inhibited the TSH action, but intracellular Ca2+ increases by either thapsigargin or ionomycin mimicking it. All of the findings show the participation of Ca2+, but not cAMP, in the action of TSH. Desensitization of protein kinase-C (PKC) did not influence the receptor-mediated H2O2 production, suggesting the reduced importance of PKC activation compared to Ca2+ signaling to the reaction. A rise in intracellular Ca2+ independent of receptor activation also induced H2O2 production as well as arachidonate release, and both were potentiated by PIA. In addition, inhibitors of phospholipase-A2 and the arachidonate metabolic pathway depressed H2O2 generation, suggesting the participation of an arachidonate cascade in the Ca(2+)-dependent H2O2 production. Lipoxygenase inhibitors depressed the Ca2+ action without influencing arachidonate release, suggesting the involvement of a lipoxygenase product(s) of arachidonate in the Ca(2+)-signaling mechanism. In conclusion, in FRTL-5 cells, TSH-induced H2O2 production is mediated not by cAMP, but by the phospholipase-C/Ca2+ cascade, possibly followed by the Ca(2+)-dependent phospholipase-A2/arachidonate cascade. PIA amplifies TSH-induced H2O2 production at the steps of phospholipase-C and phospholipase-A2 activation in a pertussis toxin-sensitive manner.

Basic Fibroblast Growth Factor (Basic FGF) in Isolated Ovine Thyroid Follicles: Thyrotropin Stimulation and Effects of Basic FGF on DNA Synthesis, Iodine Uptake and Organification, and the Release of Insulin-Like Growth Factors (IGFs) and IGF-Binding Proteins

We examined the effects of thyroid-stimulating hormone (TSH) on basic fibroblast growth factor (basic FGF) expression in isolated ovine thyroid follicles in vitro, and the effects of exogenous basic FGF on thyroid growth and function, to elucidate the significance of increased basic FGF expression during TSH-induced rat thyroid hyperplasia in vivo. Primary cultures of ovine thyroid follicles were maintained in serum-free Ham's modified F-12M medium containing transferrin, somatostatin, and glycyl-histidyl-lysine (designated 3H) with or without basic FGF alone, or in combination with TSH (100 microU/mL) and cortisol (10 nM). Following 48 h incubation, cells were harvested and total RNA prepared for the detection of basic FGF mRNA using Northern blot analysis and ribonuclease protection assay. Basic FGF in the cytoplasm and extracellular matrix fractions was quantified by radioimmunoassay. Basic FGF mRNA transcripts of 3.7, 3.0, and 2.2 kb, respectively, were found in thyroid follicles cultured in 3H medium, and the abundance of each increased between 2- and 3-fold following incubation with 10-50 microU/mL TSH, although higher concentrations of TSH were less effective. Similar results were seen using a more sensitive ribonuclease protection assay. Cells cultured in control, 3H medium contained 2.4 +/- 0.5 fmol immunoreactive basic FGF/micrograms cell DNA within the cytoplasm and 21.1 +/- 1.5 fmol/micrograms DNA within the extracellular matrix (mean +/- SD, n = 6). A significant increase (p < 0.05) in basic FGF content was seen in both cell compartments following incubation with 50 or 100 microU/mL TSH, while 250 microU/mL was less effective.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversible inhibition by interferons alpha and beta of 125I incorporation and thyroid hormone release by human thyroid follicles in vitro.

When interferons (IFN-alpha-2a, IFN-alpha-2b, natural IFN-alpha and IFN-beta) were cultured with human thyroid follicles, each IFN inhibited TSH-induced thyroid function (125I incorporation and release of 125I-T4) in a concentration-dependent manner. The minimal inhibitory effect was exerted at 1-10 U/ml. However, the inhibitory effect was reversible, and no inhibitory effect was detected when IFNs were removed from the medium within 12 h. These in vitro findings suggest that each IFN directly inhibits human thyroid function at clinically attainable levels. However, since the inhibitory effect was reversible, IFN therapy would provoke only subtle thyroid dysfunction in a majority of patients without preexisting thyroid autoimmunity.

Regulatory peptides in the thyroid gland — a review on their localization and function

It has been demonstrated that nerve fibres storing immunoreactivity of vasoactive intestinal polypeptide, peptide histidine iso-leucine, neuropeptide Y, substance P, calcitonin gene-related peptide, galanin, and cholecystokinin exists in the thyroid, though the content of these neuropeptides is lower in the thyroid than in other organs, like in the gut. Furthermore, the parafollicular C-cells have been shown to harbour several different peptides: calcitonin, somatostatin, calcitonin gene-related peptide, gastrin-releasing peptide, katacalcin and helodermin. In addition, other regulatory peptides like atrial natriuretic hormone, growth factors, and cytokines are also produced in the thyroid. This review summarizes today's knowledge on the effects of these peptides on thyroid hormone secretion and their possible role in thyroid physiology. So far, functional studies have failed to establish any convincing effect of substance P, calcitonin gene-related peptide, galanin and cholecystokinin on basal or TSH-stimulated thyroid hormone secretion. In contrast, vasoactive intestinal peptide has convincingly been demonstrated to stimulate thyroid hormone secretion, and neuropeptide Y to potentiate the inhibitory action of noradrenaline on TSH-induced thyroid hormone secretion. This suggests that these two neuropeptides are involved in the intrathyroidal neural regulation of thyroid function. Moreover, the C-cell peptides somatostatin, calcitonin, calcitonin gene-related peptide, and katacalcin seem to be involved as inhibitors of thyroid hormone secretion, whereas both gastrin-releasing peptide and helodermin stimulate thyroid hormone secretion. Atrial natriuretic hormone and growth factors, and cytokines seem to inhibit thyroid hormone secretion. Hence, studies undertaken so far suggest a local intrathyroidal peptidergic regulatory concept, the exact role of which remains to be established.

Interleukin 6 inhibits human thyroid peroxidase gene expression

It has been reported that cytokines, especially interleukin 1 and interferon-gamma, inhibit the thyroid hormone secretion and the gene expression of human thyroid peroxidase and thyroglobulin. Interleukin 6 has recently been found to be an important cytokine for the regulation of immunoendocrine interaction and intrathyroidal production of interleukin 6 has been reported. Therefore, we investigated the regulation of thyroid hormone secretion and thyroid peroxidase messenger RNA by interleukin 6 in human thyrocytes to clarify further the functional role of interleukin 6 in thyroid glands. Thyrocytes dispersed from Graves' thyroid tissues were incubated with TSH with or without interleukin 6. TSH (5 U/l) stimulated the expression of thyroid peroxidase mRNA transcripts (4.0, 3.2, 2.1, and 1.7 kb, respectively), although unstimulated thyrocytes contained the low level of 3.2 kb thyroid peroxidase mRNA transcript. Interleukin 6 (10(4)-10(5) U/l) inhibited TSH-induced thyroid peroxidase mRNA in a dose-dependent manner, although the basal level of thyroid peroxidase mRNA expression was not suppressed by interleukin 6. Interleukin 6 also inhibited 8-bromo-cyclic adenosine monophosphate-induced thyroid peroxidase mRNA levels. In contrast, the gamma-action mRNA hybridization signal was not altered in control or treated cells. Subsequently, interleukin 6 inhibited TSH-induced T3 secretion in a dose-dependent manner after 72 h treatment. However, interleukin 6 did not affect DNA synthesis. Pretreatment with specific antibody against interleukin 6 selectively restored the inhibitory effect of interleukin 6 on thyroid peroxidase gene expression. Our results suggest that interleukin 6 plays an inhibitory role in the thyroid gland, in addition to interleukin 1 and interferon gamma.

Inhibition of human thyroid peroxidase gene expression by interleukin 1.

We have already demonstrated the inhibitory effect of interleukin 1 on thyroglobulin gene expression. Recent availability of thyroid peroxidase cDNA has allowed us to investigate the regulation of thyroid peroxidase gene. Therefore, the regulation of thyroid peroxidase mRNA by interleukin 1 in cultured human thyrocytes was investigated. Thyrocytes dispersed from thyroid tissues from patients with Graves' disease were incubated with TSH with or without recombinant human interleukin 1. Unstimulated human thyrocytes did not contain any detectable thyroid peroxidase mRNA, however, TSH-stimulated thyrocytes expressed four thyroid peroxidase mRNA transcripts (4.0, 3.2, 2.1 and 1.7 kb, respectively). Both interleukin 1 alpha and beta inhibited TSH-induced thyroid peroxidase mRNA in a dose responsive manner; 10(3) U/1 interleukin 1 caused maximal suppression of TSH-induced thyroid peroxidase mRNA level to nearly basal levels. Interleukin 1 also inhibited cAMP analogue 8-bromo-cyclic AMP induced thyroid peroxidase mRNA level. In contrast the gamma-actin mRNA hybridization signal was not altered in control or treated cells. These results demonstrate that interleukin 1 directly inhibits TSH-induced thyroid peroxidase gene expression and provide further evidence for a paracrine role of interleukin 1 as a local inhibitor of thyroid hormone synthesis.

Effects of calcitonin, katacalcin, and calcitonin gene-related peptide on basal and TSH-stimulated thyroid hormone secretion in the mouse.

It is known that transcripts of the calcitonin gene in the thyroid C cells are processed to mRNAs generating precursors for the three peptides, calcitonin, katacalcin, and calcitonin gene-related peptide (CGRP), and that these peptides are produced and secreted from the C cells. The present study investigated the effects of these three peptides on basal and TSH-stimulated thyroid hormone secretion in the mouse, according to the McKenzie technique. Mice were pre-treated with 125I and thyroxine. Calcitonin (3 nmol animal-1) reduced the basal blood radioiodine levels to 72 +/- 5% compared to 95 +/- 6% in controls (P less than 0.01). This effect is likely to be renal, since calcitonin significantly increased the renal excretion of a concomitantly injected 125I load. In contrast, katacalcin and CGRP were without effect on basal blood radioiodine levels. All three peptides (1 or 3 nmol animal-1) were without effect on TSH (70 microU animal-1)-induced increase in blood radiodine levels. However, when the peptides were given together (either two and two concomitantly, or all three together), the TSH-induced increase in blood radioiodine levels was inhibited. This inhibition of TSH-induced thyroid hormone secretion might represent an intrathyroidal regulatory mechanism of the follicular cell activity.

Mechanism for the inhibitory action of 2-deoxy-glucose on thyroid hormone secretion in the mouse.

The thyroid gland is richly innervated but the effects of activation of these nerves on thyroid hormone secretion are not yet established. In the present study, intravenous injection to mice of 2-deoxy-glucose (2-DG; 60 mumol/animal) was used to activate the autonomic nerves. The nerve activation occurs through the neuroglycopenia. We found that 2-DG inhibited the thyroid-stimulating hormone (TSH; 70 microU/animal)-induced thyroid hormone secretion, measured as release of radioiodine bound to anti-T4 in radioiodine-pretreated mice. This inhibition by 2-DG was completely reversed by the alpha-adrenoceptor antagonist phentolamine but not affected by the beta-adrenoceptor antagonist L-propranolol or the muscarinic receptor antagonist methyl-atropine. It is therefore concluded that neuroglycopenia-induced activation of the autonomic nerves inhibits TSH-induced thyroid hormone secretion by a mechanism that is reversed by phentolamine. It is suggested that it is mainly the adrenergic nerves that are involved in this action, and, consequently, that the function of the adrenergic nerves in thyroid physiology is to restrain the stimulatory action of TSH.

Coexistence of thyroid-stimulating and thyroid-blocking antibodies in a patient with graves' disease who had transient hypothyroidism

T hyrotropin (TSH)-binding inhibitor immunoglobulins (TBIIs), detected by the ability of patients’ immunoglobulins to inhibit the binding of radiolabeled TSH to its receptor, were first demonstrated in patients with Graves’ disease [l-3] and later in some patients with primary myxoedema of Hashimoto’s disease [4]. The TBIIs include antibodies against the TSH receptor, which stimulate thyroid function or block TSH-induced thyroid stimulation. Thyroid-stimulating antibodies (TSAb), measured as biologic activities to stimulate the thyroid, are considered to be the major cause of hyperthyroidism in Graves’ disease [5-81. Thyroid-stimulation-blocking antibodies (TSBAb), measured by inhibition of TSHinduced CAMP increase, may play a pathogenic role in some patients with primary myxoedema [9]. Herein we report the coexistence of TSAb and TSBAb in a patient with Graves’ disease in whom hypothyroidism developed shortly after antithyroid drug treatment and who experienced a relapse during L-thyroxine replacement therapy.