Mechanisms and pathogenesis of thyroid cancer in animals and man

The transformation of the normal fully differentiated thyroid follicular cell to the rapidly growing undifferentiated anaplastic thyroid carcinoma cell involves a number of stages which have been defined morphologically and are now being related to various growth pathways and to molecular biological defects. The two main factors involved in this transformation are growth stimulation and mutagenesis. Growth stimulation alone, through elevated TSH, can lead to the development of thyroid tumours, usually benign, and retaining TSH dependency in some cases. Mutagens alone, if growth is suppressed, do not produce tumours, the combination of mutagens and increased growth is a potent carcinogenic regime. Non-genotoxic carcinogenesis in the thyroid involves growth, without mutagenesis the agent often causes this through affecting one component of thyroid hormone synthesis or metabolism, leading to a fall in thyroid hormone levels and a rise in TSH. Growth stimulation increases the rate of cell division, and therefore increases the chance of a mutation. Continued growth increases the change of subsequent events, in particular loss of heterozygosity in a tumour suppressor gene. The main oncogenes involved in human thyroid carcinogens are ras in the follicular tumour pathway, and ret in the papillary carcinoma pathway. p53 is involved in the progression of either papillary or follicular adenoma to an undifferentiated carcinoma. In experimental thyroid carcinogenesis, ras is again involved, with a link between the mutagenic agent used and the type of ras gene showing mutation. Analysis of the involvement of different growth factors and oncogenes in thyroid carcinogenesis suggests that genes related to the two receptors concerned with normal TSH stimulated growth, TSH receptor and the IGF1 recpptor may be involved in the progression of thyroid tumours of follicular pathology. Several tyrosine kinase receptors with unknown ligands or of uncertain physiological function are linked to papillary carcinoma. The recent large increase in papillary carcinoma of the thyroid in children exposed to fallout from the Chernobyl nuclear accident underlines the importance of understanding the pathobiology of thyroid neoplasia.     

Evidence for a functional role of cholecystokinin receptors in the rat thyroid gland.

The aim of the present study was to examine the role of cholecystokinin (CCK) and/or cholecystokinin receptors subtypes (CCK1R and CCK2R) in the regulation of the thyroid gland structure and function. Animals were autopsied after 6 days of treatment with CCK or CCK receptor-specific antagonists (CCK1a--PD 140,548 or CCK2a--PD 135,158) solely or in combination with CCK. Results suggest that CCK exerts a stimulatory effect on follicular thyroid cells manifested by increased epithelium/colloid volume fraction ratio (E/C). Application of selective antagonists of CCK receptor subtypes has demonstrated that CCK acts through the CCK1 receptor subtype at the level of pituitary TSH. The model of endogenous hormone action reveals that thyroid CCK1 is responsible for the thyroid growth. It can be concluded that the physiological activity of CCK1 receptor plays a significant role in a complex interrelationship between TSH, vagal system and CCK1-dependent function of the thyroid gland.     

Thyroid autoantigens and their relevance in the pathogenesis of thyroid autoimmunity

Humoral and cellular immune responses are both involved in autoimmune disorders of the thyroid gland. In the last five years, new substantial data have been obtained on the nature and the expression of thyroid cell surface autoantigens and on the demonstration of the functional heterogeneity of autoantibodies to the thyroid stimulating hormone (TSH) receptor. In the present report, attention will be mainly focused on recent studies carried out in our laboratory. The main autoantigens so far identified include the 'microsomal' antigen, thyroglobulin and the TSH receptor. For many years the 'microsomal' antigen (M) was considered a poorly characterized constituent of the cytoplasm of the thyroid cell. In the last five years, several lines of evidence were provided indicating that M is also well represented on the surface of the follicular cell and is identical to thyroid peroxidase (TPO). The use of anti-TPO monoclonal antibodies, presently available, have confirmed this antigenic identity. Microsomal (anti-TPO) antibodies are very useful markers of autoimmune thyroid disorders and are generally present in Hashimoto's thyroiditis, idiopathic myxedema and Graves' disease. TSH receptor antibodies (TRAb) are present in the sera of patients with Graves' disease. TRAb are able to stimulate thyroid adenylate cyclase and also to mimic TSH in its thyroid growth stimulation. Thus, these antibodies may have a pathogenetic role in goiter formation and in thyroid hyperfunction in Graves' disease. TRAb were also shown to inhibit both TSH binding to its receptor and TSH-stimulated adenylate cyclase activity. Recently TRAb, which inhibited TSH-stimulated adenylate cyclase activity, were found in idiopathic myxedema patients and may be responsible for impairment of thyroid function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative high-throughput screening using a live-cell cAMP assay identifies small-molecule agonists of the TSH receptor

The thyroid-stimulating hormone (TSH; thyrotropin) receptor belongs to the glycoprotein hormone receptor subfamily of 7-transmembrane spanning receptors. TSH receptor (TSHR) is expressed mainly in thyroid follicular cells and is activated by TSH, which regulates the growth and function of thyroid follicular cells. Recombinant TSH is used in diagnostic screens for thyroid cancer, especially in patients after thyroid cancer surgery. Currently, no selective small-molecule agonists of the TSHR are available. To screen for novel TSHR agonists, the authors miniaturized a commercially available cell-based cyclic adenosine 3',5' monophosphate (cAMP) assay into a 1536-well plate format. This assay uses an HEK293 cell line stably transfected with the TSHR coupled to a cyclic nucleotide gated ion channel as a biosensor. From a quantitative high-throughput screen of 73,180 compounds in parallel with a parental cell line (without the TSHR), 276 primary active compounds were identified. The activities of the selected active compounds were further confirmed in an orthogonal homogeneous time-resolved fluorescence cAMP-based assay. Forty-nine compounds in several structural classes have been confirmed as the small-molecule TSHR agonists that will serve as a starting point for chemical optimization and studies of thyroid physiology in health and disease.     

Evidence for a role of the cytoskeleton in the in vitro folliculogenesis of the thyroid gland of the fetal rat

Summary Thyrotropic hormone (TSH) or cAMP accelerate the formation of follicular cavities in the explanted thyroid gland of the 15-day-old rat fetus. Cytochalasin B or vinblastine and nocodazole or colchicine, which disorganize microfilamental and microtubular structures respectively, inhibit or completely block in vitro-induced folliculogenesis. Exposure of the thyroid tissue to lumicolchicine, a structural isomer of colchicine deprived of antimicrotubular activity, does not inhibit the activation of folliculogenesis induced by TSH. These results are strong evidence for the supposition that microfilaments and microtubules are involved in the TSH-stimulated mechanisms resulting in thyroid folliculogenesis. Folliculogenesis requires the integrity of both microfilaments and microtubules.     

Investigation of putative control mechanisms for thyroid growth

We have previously shown that the growth response of the rat thyroid to a sustained elevation of the serum level of TSH, induced by goitrogen administration, is self-limiting. This study investigated the possibility that this limitation of growth is due to the inhibitory action of a chalone secreted by the thyroid follicular cells, the serum concentration of which increases as the gland grows. Twenty-seven adult rats were treated with the goitrogen aminotriazole for 5 months to reach a 'plateau of growth'. One group of 9 rats was then subjected to hemithyroidectomy, another to a sham operation, while a third acted as unoperated controls. Four weeks later there was no significant difference between the groups in thyroid weight, follicular cell number or serum TSH. The absence of regeneration following hemithyroidectomy indicates that a systemically-circulating chalone does not play a role in the regulation of growth in the goitrous thyroid. Other mechanisms including the possible role of a 'local' chalone are briefly discussed.     

TSH is a negative regulator of skeletal remodeling

The established function of thyroid stimulating hormone (TSH) is to promote thyroid follicle development and hormone secretion. The osteoporosis associated with hyperthyroidism is traditionally viewed as a secondary consequence of altered thyroid function. We provide evidence for direct effects of TSH on both components of skeletal remodeling, osteoblastic bone formation, and osteoclastic bone resorption, mediated via the TSH receptor (TSHR) found on osteoblast and osteoclast precursors. Even a 50% reduction in TSHR expression produces profound osteoporosis (bone loss) together with focal osteosclerosis (localized bone formation). TSH inhibits osteoclast formation and survival by attenuating JNK/c-jun and NFkappaB signaling triggered in response to RANK-L and TNFalpha. TSH also inhibits osteoblast differentiation and type 1 collagen expression in a Runx-2- and osterix-independent manner by downregulating Wnt (LRP-5) and VEGF (Flk) signaling. These studies define a role for TSH as a single molecular switch in the independent control of both bone formation and resorption.     

Thyrotropin-releasing hormone receptor expression in thyroid follicular cells: a new paracrine role of C-cells?

Thyrotropin-releasing hormone (TRH) synthesized in the hypothalamus has the capability of inducing the release of thyroid-stimulating hormone (TSH) from the anterior pituitary, which in turn stimulates the production of thyroid hormones in the thyroid gland. Immunoreactivity for TRH and TRH-like peptides has been found in some tissues outside the nervous system, including thyroid. It has been demonstrated that thyroid C-cells express authentic TRH, affecting thyroid hormone secretion by follicular cells. Therefore, C-cells could have a paracrine role in thyroid homeostasis. If this hypothesis is true, follicular cells should express TRH receptors (TRH-Rs) for the paracrine modulation carried out by C-cells. In order to elucidate whether or not C-cell TRH production could act over follicular cells modulating thyroid function, we studied TRH-Rs expression in PC C13 follicular cells from rat thyroid, by means of immunofluorescence technique and RT-PCR analysis. We also investigated the possibility that C-cells present TRH-Rs for the autocrine control of its own TRH production. Our results showed consistent expression for both receptors, TRH-R1 and TRH-R2, in 6-23 C-cells, and only for TRH-R2 in PC C13 follicular cells. Our data provide new evidence for a novel intrathyroidal regulatory pathway of thyroid hormone secretion via paracrine/autocrine TRH signaling.     

Thyroid dysfunction in adult long-term survivors after hemapoeitic stem-cell transplantation (HSCT).

Thyroid function was evaluated in 72 adult survivors (41 females and 31 males) at 16 to 56 years of age, 1.5 years mean time (range 0.2 - 9.8) after hemapoeitic stem cell transplantation (HSCT) with no known prior history of thyroid dysfunction. Thyroid stimulating hormone (TSH) and free thyroxin levels (FT4) were determined before and after stimulation with thyrotropin releasing hormone (TRH). Conditioning regimens for HSCT did not include TBI. Overt hypothyroidism (basal TSH > 8 microIU/ml, FT4 < 0.8 ng/dl) was observed in 6% of male patients and 5% of female patients; subclinical hypothyroidism (basal TSH 4 - 8 microIU/ml, low normal FT4 0.8 - 1.9 ng/dl) was observed in 13% of males and 5% of females. A significant number of euthyroid patients (40% males and 54% females) with normal basal TSH and FT4 levels overresponded to TRH stimulation; the finding being statistically significant (p < 0.005). A heavy TSH response after TRH stimulation indicates compensated subclinical dysfunction of the thyroid gland. Chemotherapy-only conditioning regimens may have an adverse effect on thyroid gland function not always detected by determination of basal TSH and FT4 levels. This finding warrants long-term evaluation of thyroid function in HSCT patients.     

Thyroid follicular morphogenesis mechanism: organ culture of the fetal gland as an experimental approach

The morphological and physiological changes induced by organ culture and thyroid-stimulating hormone (TSH) stimulation in the rat fetal thyroid gland were studied. Organ culture increased Golgi activity which was further enhanced by TSH, subsequently facilitating the formation of intracellular lumina. TSH also raised the intracellular cAMP level. The intracellular lumina observed during follicular morphogenesis are structurally comparable to typical intracellular cavities formed in adult thyroid cells, which are considered as being the result of increased Golgi activity. The intracellular lumen, therefore, is probably not a physiologically significant step in thyroid morphogenesis.     

LOCALIZATION OF ESTERASE AND ACID PHOSPHATASE IN GRANULES AND COLLOID DROPLETS IN RAT THYROID EPITHELIUM

Droplets which stain like colloid occur in the cytoplasm of the thyroid follicular epithelium of the rat following stimulation of the gland by thyroid-stimulating hormone (TSH). The occurrence of droplets was remarkably reduced when the lumen became depleted of colloid. Acid phosphatase and esterase were localized in the thyroid droplets and, in addition, in granules largely around the nucleus. Stimulation by TSH resulted in an increase in the number of droplets containing enzyme. Twenty-four hours after hypophysectomy, enzyme-associated granules were localized at the basal end of the cell and droplets were absent. Intravenous injection of TSH resulted in formation of droplets at the apical end of the cell and migration of enzyme-associated granules toward the apical end of the cell. The droplets were first observed approximately 10 minutes after TSH administration and at this time did not appear to contain enzyme. Within 15 minutes many droplets contained enzyme. The granules were largely localized near the nucleus on its apical side 30 minutes after a dose of 25 milliunits of TSH, but were less well localized following one-tenth this dose. These results indicate that the epithelial cell of the thyroid gland contains preformed hydrolytic enzymes associated with granules (lysosomes). When the gland is stimulated by TSH, droplets are formed from colloid derived from the lumen (phagosomes), and hydrolytic enzymes are transferred from granules to the droplets. The droplets may be intracellular organelles for hydrolysis of colloid and liberation of thyroxine prior to the release of thyroxine into the blood.     

Can neonatal treatment with a related hormone adapt the receptor for itself?

Treatment of rats with endotoxin immediately after birth caused destruction of the cell membrane, resulting in depression of the thyroxin level and of the response to thyrotropin in adulthood. The thyroid gland of the rats treated neonatally with endotoxin failed to differentiate TSH from gonadotropin. Neonatal treatment (imprinting) with thyrotropin or gonadotropin after preexposure to the endotoxin improved the adult response to the exogenous hormone presented for imprinting after endotoxin. It appears that during the reconstruction stage following upon membrane perturbation in the critical period of receptor maturation, the adequate hormone or a related molecule can equally adapt the receptor for itself, but neither can fully compensate the perinatal membrane injury, nor the consequent diminution of receptor activity.     

Influence of experimentally induced endotoxemias on the thyroid function of rats

The general membrane-damaging effect of endotoxins (LPS) may be also demonstrated on the follicular cells of thyroid gland. Serum T4 level significantly decreased and the response of thyroid gland to exogenous THS was markedly inhibited in experimental endotoxin and other so-called enteroendotoxemic shocks (e.g. intestinal ischemia, tourniquet shock, intestinal syndrome of radiation disease). A single subtoxic dose of LPS given to newborn rats decreased the T4 level, the response of thyroid to TSH in adulthood and caused a somatic retardation. The radio-detoxified endotoxin (TOLERIN) did not inhibit the thyroid response to TSH. TOLERIN pretreatment protected the rats against LPS and other enteroendotoxemic shocks.     

Localization and role of leptin in the thyroid gland of the lizard Podarcis sicula (Reptilia, Lacertidae)

Leptin, the product of the ob gene, is a hormone secreted by adipocytes that regulates food intake and energy expenditure. The hypothalamus-pituitary-thyroid axis is markedly influenced by the metabolism status, being suppressed during food deprivation. The present study was designed to ascertain whether (1) lizard thyroid gland expresses the long form of leptin receptor (Ob-Rb) and (2) the leptin administration affects the thyroid gland activity in this species (and to verify whether leptin plays a similar role in reptiles as observed in the other vertebrates). The presence of leptin receptor in the thyroid gland of Podarcis sicula was demonstrated by immunohistochemical technique (avidin-biotin-peroxidase complex--ABC method). The role of leptin in the control of thyroid gland activity was studied in vivo using light microscopy (LM) technique coupled to a specific radioimmunoassay for thyroid-stimulating hormone (TSH) and thyroid hormones (T4 and T3). Leptin (0.1 mg/100 g body wt)/day increased T4 and T3 release for 3 days but decreased the plasma concentration of TSH; using LM clear signs of stimulation in the thyroid gland were observed. These findings suggest that systemic administration of leptin stimulates the morphophysiology of the thyroid gland in the lizard through a direct mechanism involving Ob-Rb.     

Detection and characterization of RANK ligand and osteoprotegerin in the thyroid gland

Receptor activator of NF-kappaB (RANK) ligand (RANKL) and osteoprotegerin (OPG) play essential roles in bone metabolism and immune responses. RANKL activates RANK, which is expressed by osteoclasts and dendritic cells (DC), whereas OPG acts as its decoy receptor. The role of RANKL and OPG in thyroid physiology is unclear. Northern analysis revealed pronounced OPG mRNA levels in normal human thyroid. By contrast, RANKL mRNA levels were most abundant in lymph node and appendix, and low in the thyroid. In the human thyroid follicular cell line XTC and in primary human thyroid follicular cells, OPG mRNA levels and protein secretion were upregulated by interleukin (IL)-1beta (33-fold), tumor necrosis factor (TNF)-alpha (eightfold), and thyrotropin (TSH) (threefold). RANKL mRNA was stimulated in XTC by IL-1beta and TNF-alpha, but inhibited by TSH. Conditioned medium harvested from IL-1beta-treated XTC (containing high concentrations of OPG) inhibited RANKL-induced CD40 upregulation and cluster formation of DC. OPG mRNA levels were three times more abundant in surgical thyroid specimens of Graves' disease as compared to other thyroid diseases. Our data suggest that RANKL and OPG are produced in the thyroid gland by thyroid follicular cells, are regulated by cytokines and TSH, and are capable of modulating dendritic cell functions. Thus, these cytokines may represent important local immunoregulatory factors involved in the pathogenesis of autoimmune thyroid diseases.     

Defective thyroglobulin storage in LDL receptor-associated protein-deficient mice

The molecular chaperone receptor-associated protein (RAP) is required for biosynthesis of megalin, an endocytic receptor for follicular thyroglobulin (Tg), the thyroid hormone precursor. RAP also binds to Tg itself, suggesting that it may affect Tg trafficking in various manners. To elucidate RAP function, we have studied the thyroid phenotype in RAP-knockout (RAP-KO) mice and found a reduction of Tg aggregates into thyroid follicles. Serum Tg levels were significantly increased compared with those of wild-type (WT) mice, suggesting a directional alteration of Tg secretion. In spite of these abnormalities, hormone secretion was maintained as indicated by normal serum thyroxine levels. Because Tg in thyroid extracts from RAP-KO mice contained thyroxine residues as in WT mice, we concluded that in RAP-KO mice, follicular Tg, although reduced, was nevertheless sufficient to provide normal hormone secretion. Serum TSH was increased in RAP-KO mice, and although no thyroid enlargement was observed, some histological features resembling early goiter were present. Megalin was decreased in RAP-KO mice, but this did not affect thyroid function, probably because of the concomitant reduction of follicular Tg. In conclusion, RAP is required for the establishment of Tg reservoirs, but its absence does not affect hormone secretion. low-density lipoprotein; knockout mice     

Concomitant association of thyroid sarcoidosis and Graves' disease

OBJECTIVE: Graves' disease (GD) with sarcoid involvement of the thyroid gland has rarely been reported. METHOD: We report a case of GD with thyroid sarcoidosis in a 28-year-old woman. Thyroid function was assessed by triiodothyronine (T(3)), thyroxine (T(4)), thyroid-stimulating hormone (TSH) and TSH receptor antibodies (TSH-R Ab). Thyroid scintigraphy, ultrasound and fine-needle aspiration biopsy were performed. The patient underwent surgery. RESULT: The patient had a nodular goiter. Serum T(3), T(4) and TSH-R Ab levels were elevated with suppressed TSH level. Scintigraphy showed diffuse activity as seen in GD, and ultrasound revealed that parenchyma was heterogenous. Sarcoidosis was discovered on routine chest X-ray. Although no sarcoid involvement was found on specimen, the thyroid gland showed non-caseating granulomas on histology. CONCLUSION: Since sarcoid involvement of the thyroid gland can cause hypofunction, we report the uncommon infiltration of sarcoidosis with hyperthyroidism.