Melatonin in the thyroid gland: regulation by thyroid-stimulating hormone and role in thyroglobulin gene expression.

Melatonin is an indoleamine with multiple functions in both plant and animal species. In addition to data in literature describing many other important roles for melatonin, such as antioxidant, circadian rhythm controlling, anti-aging, antiproliferative or immunomodulatory activities, our group recently reported that thyroid C-cells synthesize melatonin and suggested a paracrine role for this molecule in the regulation of thyroid activity. To discern the role played by melatonin at thyroid level and its involvement in the hypothalamic-pituitary-thyroid axis, in the present study we have analyzed the effect of thyrotropin in the regulation of the enzymatic machinery for melatonin biosynthesis in C cells as well as the effect of melatonin in the regulation of thyroid hormone biosynthesis in thyrocytes. Our results show that the key enzymes for melatonin biosynthesis (AANAT and ASMT) are regulated by thyroid-stimulating hormone. Furthermore, exogenous melatonin increases thyroglobulin expression at mRNA and protein levels on cultured thyrocytes and this effect is not strictly mediated by the upregulation of TTF1 or, noteworthy, PAX8 transcription factors. The present data show that thyroid C-cells synthesize melatonin under thyroid-stimulating hormone control and, consistently with previous data, support the hypothesis of a paracrine role for C-cell-synthesised melatonin within the thyroid gland. Additionally, in the present study we show evidence for the involvement of melatonin in thyroid function by directly-regulating thyroglobulin gene expression in follicular cells.

Comparative study of the primary cilia in thyrocytes of adult mammals.

Since their discovery in different human tissues by Zimmermann in 1898, primary cilia have been found in the vast majority of cell types in vertebrates. Primary cilia are considered to be cellular antennae that occupy an ideal cellular location for the interpretation of information both from the environment and from other cells. To date, in mammalian thyroid gland, primary cilia have been found in the thyrocytes of humans and dogs (fetuses and adults) and in rat embryos. The present study investigated whether the existence of this organelle in follicular cells is a general event in the postnatal thyroid gland of different mammals, using both immunolabeling by immunofluorescence and electron microscopy. Furthermore, we aimed to analyse the presence of primary cilia in various thyroid cell lines. According to our results, primary cilia are present in the adult thyroid gland of most mammal species we studied (human, pig, guinea pig and rabbit), usually as a single copy per follicular cell. Strikingly, they were not found in rat or mouse thyroid tissues. Similarly, cilia were also observed in all human thyroid cell lines tested, both normal and neoplastic follicular cells, but not in cultured thyrocytes of rat origin. We hypothesize that primary cilia could be involved in the regulation of normal thyroid function through specific signaling pathways. Nevertheless, further studies are needed to shed light on the permanence of these organelles in the thyroid gland of most species during postnatal life.

Ghrelin potentiates TSH-induced expression of the thyroid tissue-specific genes thyroglobulin, thyroperoxidase and sodium-iodine symporter, in rat PC-Cl3 Cells.

Ghrelin is a 28-amino-acid peptide that stimulates pituitary growth-hormone secretion and modulates food-intake and energy metabolism in mammals. It is mainly secreted by the stomach, but it is also expressed in many other tissues such as cartilage or the thyroid gland. In the present study we have analyzed by RT-PCR and using immunohistochemistry and immunofluorescence the expression and tissue distribution of ghrelin and its functional receptor (GHS-R type 1α) in thyroid cell-lines and in normal and pathological rat thyroid tissue. Additionally, by measuring the incorporation of BrdU, we have investigated if, as previously noted for FRTL-5 cells, ghrelin enhances the proliferation rate in the PC-Cl3 rat-thyrocyte cell-line. Finally, we have determined the stimulatory effect of ghrelin on TSH-induced expression of the tissue-specific key genes involved in the synthesis of thyroid hormone: thyroglobulin, thyroperoxidase and sodium-iodine symporter. Our data provide direct evidence that C-cell secreted ghrelin may be involved in the paracrine regulation of the thyroid follicular cell function.

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.

Comparative immunohistochemical study of normal, hyperplastic and neoplastic C cells of the rat thyroid gland.

In rats, the frequency of spontaneous C-cell tumours is very high and is both age and gender dependent. The three specific stages of neoplastic progression can be distinguished into diffuse C-cell hyperplasia, focal C-cell hyperplasia and bona fide C-cell tumours. Based on this hypothetical model of human medullary thyroid carcinoma (MTC), we carried out an immunohistochemical study using different markers (calcitonin, calcitonin gene-related peptide, somatostatin and chromogranin) to verify the existence of any relationship between their expression and the successive steps of tumour development. We found a characteristic immunohistochemical staining pattern, particularly for calcitonin and somatostatin, which distinguishes C-cell tumours from both normal and hyperplastic C cells, with no differences related to the gender of the animals under study. Specifically, a considerable heterogeneity in calcitonin expression was only displayed by C-cell carcinomas, being less pronounced in C-cell adenomas. As for somatostatin, this regulatory peptide was found only in a minority of calcitonin-positive cells in normal and hyperplastic glands. However, in some C-cell adenomas and most C-cell carcinomas nearly all calcitonin-positive cells also coexpressed somatostatin. We conclude that rat C-cell neoplasms constitute a very particular tumour entity which shares many but not all immunohistochemical features with human MTC.

Decrease in calcitonin and parathyroid hormone mRNA levels and hormone secretion under long-term hypervitaminosis D3 in rats.

In calcium homeostasis, vitamin D3 is a potent serum calcium-raising agent which in vivo regulates both calcitonin (CT) and parathyroid hormone (PTH) gene expression. Serum calcium is the major secretagogue for CT, a hormone product whose biosynthesis is the main biological activity of thyroid C-cells. Taking advantage of this regulatory mechanism, long-term vitamin D3-induced hypercalcemia has been extensively used as a model to produce hyperactivation, hyperplasia and even proliferative lesions of C-cells, supposedly to reduce the sustained high calcium serum concentrations. We have recently demonstrated that CT serum levels did not rise after long-term hypervitaminosis D3. Moreover, C-cells did not have a proliferative response, rather a decrease in CT-producing C-cell number was observed. In order to confirm the inhibitory effect of vitamin D3 on C-cells, Wistar rats were administered vitamin D3 chronically (25,000 IU/d) with or without calcium chloride (CaCl2). Under these long-term vitamin D3-hypercalcemic conditions, calcium, active metabolites of vitamin D3, CT and PTH serum concentrations were determined by RIA; CT and PTH mRNA levels were analysed by Northern blot and in situ hybridization; and, finally, the ultrastructure of calciotrophic hormone-producing cells was analysed by electron microscopy. Our results show, that, in rats, long term administration of vitamin D3 results in a decrease in hormone biosynthetic activities of both PTH and CT-producing cells, albeit at different magnitudes. Based upon these results, we conclude that hypervitaminosis D3-based methods do not stimulate C-cell activity and can not be used to induce proliferative lesions of calcitonin-producing cells.

Expression of a neu/c-erbB-2-like product in neuroendocrine cells of mammals.

The neu/c-erbB-2 oncogene encodes a 185 kDa protein closely homologous to the epidermal growth factor receptor. The protein product (p185) is a glycoprotein with an external domain and an internal domain with tyrosine kinase activity. Amplification and/or overexpression of p185 is related to several human adenocarcinomas. Subsequent studies demonstrated its presence in certain neuroendocrine (NE) neoplasms, including phaeochromocytomas, insulinomas and medullary thyroid carcinomas. However, relatively little is known about its role in normal cell growth regulation and development. Therefore, our objective was to determine whether neu/c-erbB-2 was expressed in normal NE tissues of different mammals, specially in humans, as it was in their neoplasms. We have examined by immunohistochemistry different endocrine glands (thyroid, pancreas, suprarrenal and hypophysis) and the small intestine of human beings, rats and guinea pigs, using two polyclonal antibodies raised against the intracytoplasmic part of the protein, and specific antigen absorption controls. We have found that a neu/c-erbB-2-like product occurs in all normal NE tissues examined: C cells of the thyroid gland, chromaffin cells of the adrenal medulla, pancreatic islets, enteroendocrine cells of the small intestine and, finally, scattered cells of the adenohypophysis, according to a typical granular immunohistochemical pattern. Our results indicate that normal NE cells share a new common antigen in their cytoplasms, a neu/c-erbB-2-like product, with a similar immunostaining pattern to that presented by the neoplasms derived from them.

Expression of c-erbB-2 oncoprotein in human thyroid tumours.

AIMS: c-erbB-2 expression has been found to be a potential marker of aggressive biological behaviour in some tumours, but the role played by this oncoprotein in the development and maintenance of thyroid tumours is still controversial. Therefore our objective was to determine whether c-erbB-2 was overexpressed in a large retrospective series of human thyroid tumours, including both from follicular and C-cell differentiation. METHODS AND RESULTS: We have studied 67 thyroid tumours (10 follicular adenomas, 11 follicular carcinomas, three anaplastic carcinomas, 25 papillary carcinomas and 18 medullary carcinomas and 16 metastases) by immunohistochemistry using an antigen retrieval method for paraffin-embedded material and a specific polyclonal antibody against the intracytoplasmic part of c-erbB-2 oncoprotein. There are marked differences in the pattern of c-erbB-2 immunoreactivity depending on the type of thyroid tumour. Thus, no expression of the oncoprotein has been found in follicular adenomas, follicular carcinomas and anaplastic carcinomas, but 52% of papillary carcinomas (membranous and diffuse cytoplasmic patterns) and all medullary carcinomas (granular cytoplasmic pattern) are immunopositive. CONCLUSIONS: Our results indicate that overexpression of c-erbB-2 oncoprotein is easily identifiable by immunohistochemistry in paraffin sections of certain thyroid tumours after applying an antigen retrieval method. This study suggests that c-erbB-2 oncoprotein may play some role in disease progression in papillary and medullary thyroid carcinomas, but the significance of the different immunohistochemical patterns merits further investigations.

Chronic hypervitaminosis D3 determines a decrease in C-cell numbers and calcitonin levels in rats.

Many papers have reported that chronic hypercalcemia induced either by large doses of vitamin D or by the administration of calcium or parathormone, produces hypertrophy and hyperplasia of C cells. However, more recent studies suggest that the effect of elevated calcium or 1.25(OH)2D3 concentration on the production of calcitonin may be more complex than previously suspected. To assess the validity of such a response an experimental model, where hypercalcemia was induced with vitamin D3 overdose, was designed. Male Wistar rats were administered vitamin D3 chronically (50,000 IU per 100 ml of drinking water with or without CaCl2). Serum calcium and calcitonin levels were determined. C cells were stained by immunohistochemistry using calcitonin and neuronal specific enolase (NSE) antibodies and their percentage was calculated by a morphometric analysis. We also investigated the ultrastructural characteristic of the C cells under experimental conditions. C cells did not have a proliferative response rather a decrease in their number was observed after 1 month of treatment with 25,000 IU of vitamin D3 (1.55 vs 2.43% in control animals) and 3 months with vitamin plus CaCl2 (2.27% vs 3.62% in control animals). In addition, no significant changes in serum calcitonin levels were observed during the experimental period. We conclude that rat C cells do not respond with hypertrophic and hyperplastic changes in a hypercalcemic state due to an intoxication with vitamin D3.

Expression and prognostic value of c-erbB-2 oncogene product in human phaeochromocytomas.

AIMS: Amplification of c-erbB-2 proto-oncogene has been reported in endocrine tumours, but the results were unclear and no predictive prognostic value has been established in the case of phaeochromocytoma. We investigated the immunohistochemical expression of c-erbB-2 oncogene in 34 cases of human phaeochromocytoma (27 sporadic, seven familial type MEN (multiple endocrine neoplasm)) in order to find out if it could be used to differentiate sporadic and familial forms and whether c-erbB-2 expression is related to tumour biological behaviour. METHODS AND RESULTS: All the cases showed diffuse, generally heterogeneous, intracytoplasmic granular c-erbB-2 staining. The percentage of tumour cells expressing c-erbB-2 was used as the comparative variable. The percentage of c-erbB-2 positive cells had a statistically significant (P < 0.001) relationship with tumour aggressiveness, as manifested by the presence of distant metastasis or association with other malignant neoplasms. We also found significantly higher levels (P = 0.007) of c-erbB-2 overexpression in MEN phaeochromocytoma than in sporadic cases. CONCLUSIONS: These results clarify the important role of c-erbB-2 proto-oncogene in the pathogenesis of human phaeochromocytoma and confirm the unfavourable prognostic significance of c-erbB-2 expression.

Postnatal variations in the number and size of C-cells in the rat thyroid gland.

The heterogeneous distribution of thyroid C-cells has until now hindered an objective evaluation of changes caused by age or experimental stimuli. To overcome this, a rigorous methodology has been designed to detect variations in shape, size, and number of C-cells throughout development. Using this methodology, we have demonstrated that C-cells do not significantly alter their shape with age. However, their volume increases gradually from 472 microns3 in newborn rats to 1653 microns3 in 120-day-old animals. Over the same time period, the mean number of C-cells within the thyroid gland increased 9-fold (from 1.6 x 10(4) to 1.5 x 10(5), and the number of C-cells per unit area decreased (from 6.15 x 10(4)/mm3 to 2.6 x 10(4)/mm3). We conclude that there are marked variations in size, total number, and number of C-cells per unit area in the rat thyroid gland after birth.

Mitotic activity of the endocrine cells in rat thyroid glands during postnatal life.

This paper presents the results of investigations into the mitotic rates of thyroid endocrine cells in normal postnatal rats, aged 1-120 days. Our study revealed considerable age-dependent shifts in the mean mitotic activity of follicular cells and C-cells. The maximum indices of endocrine cell renewal were reached during the first 10 days of life, decreasing gradually and significantly until 25 days. At 1 month, there was a significant recuperation of the division rate for both cell types, which declined in the adult rat. These results mean that the proliferation of C-cells and follicular cells is inversely proportional to age. The preferential zone of localization of mitoses of both cell types is the central region of the thyroid lobe. The present paper provides new evidence for the postnatal origin of C-cells and follicular cells from the preexisting endocrine cells.

Evidence of the occurrence of calcitonin cells in the ultimobranchial follicle of the rat postnatal thyroid.

A study on thyroid glands of Wistar rats of ages ranging from 1 to 120 days was carried out. The glands were serially sectioned and stained for calcitonin using the peroxidase antiperoxidase method. All the thyroids contained ultimobranchial follicles (UBF) located partially embedded among the usual follicles but in a 5-day-old rat this structure showed an unusual position in the interstitium of connective tissue between the cartilage of the trachea and the thyroid gland. We have observed in the wall of that UBF the presence not only of resting C cells but also mitotic figures of C cells. Furthermore, on the opposite side of the same UBF an active area of formation of thyroid follicles was found. These observations provided the first evidence of the contribution of the UBF in the formation of C cells during the postnatal life of the rat. Furthermore, it is suggested that some C cells may share a common origin with ultimobranchially derived follicular cells.