**3.1. Calcitonin and calcitonin family of peptides**

In 1961, Copp et al. were the first in postulating the existence of a hypocalcemic hormone secreted by the thyro-parathyroid complex which was named calcitonin (CT) [67]. Subsequently, in 1963, Kumar et al. [68] confirmed the secretion of this hypocalcemic hormone by the thyroid gland. The evidence that parafollicular cells contained calcitonin was provided by immunofluorescent techniques in dog and pig thyroids [39], and these findings led to the name of 'C' cells for parafollicular cells. In more recent years, however, accumulating evidence supports the existence of nonthyroidal calcitonin or CT-like peptides in brain, prostate and uterus, although their biological role is unknown [69].

Calcitonin is expressed in many species including fish, amphibians, reptiles, birds and mammals, and it is normally a 32 amino acid peptide with a carboxy terminal proline amide and a disulfide bridge between cysteine residues at positions 1 and 7 [70, 71]. The CT amino acid sequence in the amino terminal loop region is highly conserved within species, but displays less homologies in the rest of the sequence [69].

The hypocalcemic action of calcitonin is due to inhibition of bone resorption and to effects on other tissues indirectly related to bone remodeling such as kidneys. Additional cellular actions of calcitonin include its effect on the growth of breast cancer cells [72].

Calcitonin is synthesized as a 136 amino acid precursor which is processed by proteolytic cleavage and by amidation of the carboxy terminal proline residue before being secreted. The gene transcript encoding CT also encodes calcitonin gene related peptide (CGRP), another peptide in the CT family of peptides. The CT/CGRP gene was one of the first described examples of tissue-specific alternative RNA processing, it has six exons, of which the first four are spliced together to generate calcitonin mRNA in C cells, which encodes calcitonin and katacalcin (CCP), while the transcripts from CT/CGRP gene in neurons are processed into CGRP mRNA containing exons 1-3, 5 and 6 [30, 73] (see figure 5). In thyroid C cells 95% of the transcripts encode calcitonin, while more than 98% of the transcripts encode CGRP in the nervous system [69].

58 Thyroid Hormone

demonstrated the presence of TSH receptors on C cells [64], we can infer that those hyperplastic changes may be due to the corresponding increased TSH-serum levels. Nevertheless, the influence of local secretion of growth factors by follicular cells themselves,

The main secretagogue of C cells is calcium, which is concordant with the classical role of calcitonin as a plasma calcium- and bone metabolism-related hormone. Nevertheless, C-cell activity appears to be subjected to a more complex regulation, for which different regulatory peptides, either with inhibitory (somatostatin) or stimulatory effect (glucagon, pentagastrin), have being reported [65]. Furthermore, Suzuki et al. in 1998 [66] reported that C cells expressed the thyroid transcription factor 1 (TTF-1), which is typically expressed by follicular cells and known for its critical role in thyroid-specific gene expression. According to these authors, TTF-1 is calcium-modulated and coordinately regulates genes involved in calcium homeostasis in C cells. All these data constitute new evidence of interdependence

**3. Description of the different regulatory factors synthesized by C cells** 

The most important objective of the current review is to present new evidence of the paracrine regulation exerted by C cells upon follicular cells, through the secretion of numerous factors synthesized by C cells in the immediate vecinity of the follicular epithelium, which could act in a synergistic or antagonist manner. To accomplish that objective, we will expose the functional role played by the most studied C-cell secreted

In 1961, Copp et al. were the first in postulating the existence of a hypocalcemic hormone secreted by the thyro-parathyroid complex which was named calcitonin (CT) [67]. Subsequently, in 1963, Kumar et al. [68] confirmed the secretion of this hypocalcemic hormone by the thyroid gland. The evidence that parafollicular cells contained calcitonin was provided by immunofluorescent techniques in dog and pig thyroids [39], and these findings led to the name of 'C' cells for parafollicular cells. In more recent years, however, accumulating evidence supports the existence of nonthyroidal calcitonin or CT-like peptides

Calcitonin is expressed in many species including fish, amphibians, reptiles, birds and mammals, and it is normally a 32 amino acid peptide with a carboxy terminal proline amide and a disulfide bridge between cysteine residues at positions 1 and 7 [70, 71]. The CT amino acid sequence in the amino terminal loop region is highly conserved within species, but

The hypocalcemic action of calcitonin is due to inhibition of bone resorption and to effects on other tissues indirectly related to bone remodeling such as kidneys. Additional cellular

factors on the activity of follicular cells mediated through their specific receptors.

in brain, prostate and uterus, although their biological role is unknown [69].

actions of calcitonin include its effect on the growth of breast cancer cells [72].

or by the surrounding tissue could not be discarded.

between the two endocrine cell populations of the thyroid gland.

**3.1. Calcitonin and calcitonin family of peptides** 

displays less homologies in the rest of the sequence [69].

**and their potential effects on thyroid hormone synthesis** 

**Figure 5.** Schematic representation of alternative RNA processing of CT/CGRP gene. Transcription of the CT/CGRP gene produces a pre-mRNA, which has two polyadenylation sites. From the primary transcript two different mRNAs are produced, one coding for the calcitonin (CT)/katacalcin (CCP) precursor and the other coding for the CGRP precursor. The blue boxes indicate sequences complementary to exons, the lines between them are the sequences complementary to introns and the black boxes indicate the noncoding sequences of RNA.

The alternative product of the CT/CGRP gene, a neuropeptide referred to as CGRP, is a 37 amino acid peptide widely expressed in nerves in both the central and peripheral nervous systems and in several neuroendocrine tissues [74]. CT and CGRP have some homologies in the amino terminal region, but are almost entirely different in the rest of the molecule [74]. CGRP is a potent vasodilator, relaxant of mesangial cells and has functional roles in response to painful stimuli [75].

CT interacts with a member of seven-transmembrane-domain G-protein-coupled receptor superfamily cloned in 1991 [76]. High-affinity CT receptors (CTRs) were characterized on isolated osteoclasts, the physiological CT target cells. Moreover, calcitonin receptors have been identified in a variety of tissues, such as brain, testis, spermatozoa, kidney, skeletal

muscle, breast cancer cell lines and human primary breast cancer cells [77]. Numerous studies have demonstrated that multiple CTR mRNA isoforms are present in a number of species. In humans, two isoforms (h-CTR1 and h-CTR2) differing by a 16 amino-acid insert in the first intracellular loop have been reported [78]. Finally, studies have revealed h-CTR2 expression in normal thyroid glands, in medullary thyroid carcinoma (MTC), and in a model of human MTC cell line (TT), suggesting an autocrine regulation of CTR in C cells [79]. Nevertheless, there is still no evidence of the CTR expression in follicular cells.

Paracrine Regulation of Thyroid-Hormone Synthesis by C Cells 61

inhibitory effects on the endocrine system, gut exocrine secretion, adrenal glands, kidneys, and on proliferation of lymphocytes, cartilage and bone precursor cells [86, 87, 89, 90]. Other effects consist on the inhibition of the release of growth factors (IGF1, EGF, PDGF) and cytokines (IL6, IFN-g) [91]. All of these diverse effects of SS can be explained by the inhibitory actions of the

The above-listed functions of SS are mediated by a family of receptors that recognizes these ligands with different affinities [92]. Originally, five different somatostatin receptor types (SSR1-5) were identified, cloned and found able to bind not only native SS forms (SS-14 and SS-28), but also their synthetic analogues [93]. SS receptors belong to the family of G protein-coupled receptors, typically consisting of extracellular domains forming the ligand binding site, a single polypeptide chain with seven transmembrane domains and intracellular domains mediating signal transduction. Each SS receptor subtype mediates different biological actions of SS, via the activation of different intracellular systems such as inhibition of adenylate cyclase (with decrease of intracellular cyclic AMP levels), reduction of intracellular calcium levels, and stimulation of phosphotyrosine phosphatase or MAP

Within the thyroid, SS coexists with CT in a subpopulation of C cells, with a variable extension. Thus, while the frequency for somatostatin-immunoreactive (SS-IR) CC varies in rats throughout their lives, as they are scarce in the foetus, abundant at the time of birth and scarce again in adults (see Figure 6) [33, 95-97], the majority of calcitonin immunoreactive (CT-IR) CC in guinea pigs and rabbits are also SS-IR, besides positivity is seen in the parathyroid gland [33]. On the contrary, SS-IR CC are observed very occasionally in normal

**Figure 6.** Immunohistochemical demonstration of calcitonin (A), somatostatin (B) and serotonin (C) in consecutive sections of the rat thyroid gland. Only a subpopulation of calcitonin-positive C cells is also immunopositive for somatostatin. Serotonin is exclusively located in mast cells, being C cells negative in

The effects of SS on the thyroid include the inhibition of the TSH-stimulated release of T4 and T3 [99], as well as the inhibition of TSH-stimulated radioiodine turnover accumulation in thyroid glands of euthyroid volunteers [100]. SS inhibition of basal as well as TSHstimulated adenyl cyclase activity has been demonstrated in normal and neoplastic human

peptide on two key cellular processes: secretion and cell proliferation.

kinase activity [94].

the rat. (x400).

pig and human thyroid glands [98].

A family of receptors for CGRP and CT has been demonstrated throughout the nervous systems of both man and rat. CGRP-specific binding sites have been found in the cerebellum, spinal cord and other brain regions including vessels and pia mater. In contrast to calcitonin receptors found in bone, these receptors are not linked to adenylate cyclase, however, CGRP stimulates adenylate cyclase activity by acting at the calcitonin receptor in more than one tissue. Furthermore, specific cAMP linked CGRP receptors have been identified both in the intima and media of vessels as well as the atria and ventricles of the heart [80]. Seifert et al. [81] demonstrated the presence of a novel specific receptor for CGRP in the acinar cells of the pancreas. Finally, specific CGRP binding has also been demonstrated on cloned lung and bone cancer cells [82].

CGRP has a double location in the thyroid, one in nerve fibers around blood vessels and follicles and the other in C cells. This may suggest a functional role on the follicular cells; however, it seems not to affect basal or TSH-stimulated thyroid hormone secretion [83]. In contrast, CT, CGRP and katacalcin, which have been demonstrated to have no effect on basal and TSH-stimulated thyroid hormone secretion when separately administered alone, inhibited TSH-stimulated thyroid hormone secretion when the three peptides were applied together [83]. Despite this, further studies about the CTRs or calcitonin like receptors (CLRs) expression in follicular cells are necessary to dilucidate the role of CT/CGRP family peptides in the thyroid gland function.
