In Physiology

The two main factors that control the physiology of the thyroid after embryogenesis are the requirement for thyroid hormones and the supply of its main and specialized substrate iodide (Table 3-1). Thyroid hormone plasma levels and action are monitored by the hypothalamic supraoptic nuclei and by the thyrotrophs of the anterior lobe of the pituitary, where they exert a negative feedback through T₃ receptor β. In normal rats, serum thyroid-stimulating hormone (TSH) levels are inversely related to thyrocyte sensitivity to TSH.¹ The corresponding homeostatic control is expressed by TSH (thyrotropin). The TSH receptor is also stimulated by a new, different natural hormone cloned by homology, thyrostimuline. The physiologic role of this protein is unknown, but its level is not controlled by a thyroid hormone feedback, and it does not participate in the homeostatic control of the thyroid.² Iodide supply is monitored in part indirectly as a substrate for the synthesis of thyroid hormones and therefore through its effects on the plasma level of thyroid hormones, but mainly in the thyroid itself, where it depresses various aspects of thyroid function and the response of the thyrocyte to TSH. These two major physiologic regulators control the function and size of the thyroid: TSH positively, iodide negatively.^3–6 In mice embryo, other unknown factors control differentiation and organ growth which takes place normally until birth in the absence of TSH receptor.^7,8 In humans, in whom birth occurs later in development, homozygous inactivating mutations of the TSH receptor in familial congenital hypothyroidism were found to be associated with a very hypoplastic thyroid gland.⁹ Although the thyroid contains receptors for thyroid hormones, and a direct effect of these hormones on thyrocytes would make sense,¹⁰ as yet little evidence has indicated that such control plays a role in physiology.¹¹ However, expression of dominant-negative thyroid hormone receptors in mice represses PPARγ expression and induces thyroid tumors in thyroid.¹² The role of increased TSH plasma level in this induction is not defined. Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) at high levels activate the TSH receptor and thus directly stimulate the thyroid. This effect accounts for the depression of TSH levels and sometimes elevated thyroid activity at the beginning of pregnancy.^13–15

The thyroid gland is also influenced by various other nonspecific hormones.¹⁶ Hydrocortisone exerts a differentiating action in vitro.¹⁷ Estrogens affect the thyroid by unknown mechanisms, directly or indirectly, as exemplified in the menstrual cycle, in pregnancy, and in the higher prevalence of thyroid tumors and other pathologies in women. Growth hormone (GH) induces thyroid growth, but its effects are thought to be mediated by locally produced somatomedins (insulin-like growth factor 1 [IGF-1]). Nevertheless the presence of basal TSH levels might be a prerequisite for the growth-promoting action of IGF-1, because a GH replacement therapy did not increase thyroid size in patients deficient for both GH and TSH.¹⁸ Conversely, TSH does not induce the proliferation of human thyrocytes in the absence of IGF-1 or high levels of insulin. The anomalously low endemic goiter prevalence among pygmies living in iodine-deficient areas,¹⁹ a population genetically resistant to IGF-1, is also compatible with an in vivo permissive effect of IGF-1 and IGF-1 receptor on TSH mitogenic action. In dog and human thyroid primary cultures, the presence of insulin receptors strictly depends on TSH, suggesting that thyroid might be a more specific target of insulin than generally considered.^20,21 Effects of locally secreted neurotransmitters and growth factors on thyrocytes have been demonstrated in vitro and sometimes in vivo, and the presence of some of these agents in the thyroid has been ascertained. The set of neurotransmitters acting on the thyrocyte and their effects vary from species to species.^3,22 In human cells, well-defined, direct but short-lived responses to norepinephrine, ATP, adenosine, bradykinin, and thyrotropin-releasing hormone (TRH) have been observed.^3,23,24 Growth-factor signaling cascades demonstrated in vitro can exert similar effects in vivo. In nude mice, the injection of epidermal growth factor (EGF) promotes DNA synthesis in thyroid and inhibits iodide uptake in xenotransplanted rat²⁵ and human thyroid tissues.²⁶ By contrast, the injection of fibroblast growth factor (FGF) induces a colloid goiter in mice, with no inhibition of iodide metabolism or thyroglobulin and thyroperoxidase mRNA accumulation.²⁷ These effects are exact replicas of initial observations from the dog thyroid primary culture system²⁸ and other thyroid primary culture systems.^29–32 EGF and FGF have since been reported to be locally synthesized in the thyroid gland, as a possible response to thyroxine and TSH,³³ respectively. Their exact role as autocrine and/or paracrine agents in the development, function, and pathology of the thyroid gland of different species has yet to be clarified.^34,35 Transforming growth factor beta (TGF-β) constitutes another category of cytokines that are growth factors produced locally by thyrocytes and influence their proliferation and the action of mitogenic factors.^34,36 TGF-β inhibits proliferation and prevents most of the effects of TSH and cAMP in human thyrocytes in vitro.^37,38 TGF-β is synthesized as an inactive precursor which can be activated by different proteases produced by thyrocytes. TGF-β expression is up-regulated during TSH-induced thyroid hyperplasia in rats, suggesting an autocrine mechanism limiting goiter size.³⁹ Also present in the thyroid are activin A and bone morphogenic peptide (BMP), which are related to TGF-β and inhibit thyrocyte proliferation in vitro.⁴⁰ Unlike TGF-β, they are directly synthesized in an active form. Elements of a Wnt/β catenin signaling pathway (Wnt factors, Frizzled receptors, and disheveled isoforms) have been identified in human thyroid and thyroid cancer cell lines.⁴¹ The eventual role in vivo in humans of most of these factors remains to be proved and clarified.

In Pathology

Mutated, constitutively active TSH receptors and G_s proteins cause thyroid autonomous adenomas.42,43 Mutations which confer to the TSH receptor a higher sensitivity to LH/hCG cause hyperthyroidism in pregnancy.^44,45 Pathologic extracellular signals play an important role in autoimmune thyroid disease. Thyroid-stimulating antibodies (TSAbs), which bind to the TSH receptor and activate it, reproduce the stimulatory effects of TSH on the function and growth of the tissue. Their effects on cAMP accumulation are slower and much more persistent than those of TSH.⁴⁶ Their abnormal generation is responsible for the hyperthyroidism and goiter of Graves’ disease. Thyroid-blocking antibodies (TBAbs) also bind to the TSH receptor but do not activate it and hence behave as competitive inhibitors of the hormone. Such antibodies are responsible for some cases of hypothyroidism in thyroiditis. Both stimulating and inhibitory antibodies from mothers with positive sera induce transient hyperthyroidism or hypothyroidism in newborns.⁴ The existence of thyroid growth immunoglobulins has been hypothesized to explain the existence of Graves’ disease with weak hyperthyroidism and prominent goiter. The thyroid specificity of such immunoglobulins would imply that they recognize thyroid-specific targets. This hypothesis is now abandoned.^47–49 Discrepancies between growth and functional stimulation may instead reflect kinetics or cell intrinsic factors. Local cytokines have been shown to influence, mostly negatively, the function, growth, and differentiation of thyrocytes in vitro and thyroid function in vivo. Because they are presumably secreted in loco in autoimmune thyroid diseases, these effects might play a role in the pathology of these diseases, but this notion has not yet been proved.^3,16 Moreover, in selenium deficiency, secretion of TGF-β by macrophages has been implicated in the generation of thyroid fibrosis⁵⁰ and the pathogenesis of thyroid failure in endemic cretinism. The overexpression of both FGF and FGF receptor 1 in thyrocytes from human multinodular goiter might explain their relative TSH independence.⁵¹ On the other hand, the subversion of tyrosine kinase pathways similar to those normally operated by local growth factors (i.e., the activation and thyrocyte expression of Ret⁵² and TRK,⁵³ the overexpression of Met/HGF receptor, sometimes in association with hepatocyte growth factor (HGF),⁵⁴ or erbB/EGF receptor in association with its ligand, TGF-α⁵⁵) can be causally associated with TSH-independent thyroid papillary carcinomas. An autocrine loop involving IGF-2 and the insulin receptor isoform-A is also proposed to stimulate growth of some thyroid cancers.⁵⁶ Thyroid cancer cells often escape growth inhibition by TGF-β.⁵⁷

The Cyclic Adenosine Monophosphate Cascade

The cyclic adenosine monophosphate (cAMP) cascade in the thyroid corresponds, as far as it has been studied, to the canonic model of the β-adrenergic receptor cascade⁴ (Fig. 3-1). It is activated in the human thyrocyte by the TSH and the β-adrenergic and prostaglandin E receptors. These receptors are classical seven-transmembrane receptors controlling transducing guanosine triphosphate (GTP)-binding proteins. Activated G proteins belong to the G_s class and activate adenylyl cyclase; they are composed of a distinct α_s subunit and nonspecific β and γ monomers. Activation of a G protein corresponds to its release of guanosine diphosphate (GDP) and binding of GTP and to its dissociation into α_GTP and βγ dimers; α_sGTP directly binds to and activates adenylyl cyclase. Inactivation of the G protein follows the spontaneous, more or less rapid hydrolysis of GTP to GDP by α_s GTPase activity and the reassociation of α_GDP with βγ. The effect of stimulation of the receptor by agonist binding is to increase the rate of GDP release and GTP binding, thus shifting the equilibrium of the cycle toward the α_GTP active form. Unless constrained in a multiprotein complex, one receptor can consecutively activate several G proteins (hit-and-run model). A similar system negatively controls adenylyl cyclase through G_i. It is stimulated in the human thyroid by norepinephrine through α₂-receptors and moderately by the TSH receptor. Adenosine at high concentrations directly inhibits adenylyl cyclase. The thyroid contains mainly three isoforms of adenylyl cyclase: III, VI, and IX.⁵⁸ The cAMP generated by adenylyl cyclase binds to the regulatory subunit of protein kinase A (PKA) that is blocking the catalytic subunit and releases this now-active unit. The activated, released catalytic unit of protein kinase phosphorylates serines and threonines in the set of proteins containing accessible specific peptides that it recognizes. These phosphorylations, through more or less complex cascades, lead to the observed effects of the cascade. Two isoenzymes (I, II) of cAMP-dependent kinases have been found, the first of which is more sensitive to cAMP; as yet no clear specificity of action of these kinases has been demonstrated. In the case of the thyroid, this cascade is activated through specific receptors, by TSH in all species, and by norepinephrine receptors and prostaglandin E in humans, with widely different kinetics: prolonged for TSH and short lived (minutes) for norepinephrine and prostaglandins.⁵⁹ Other neurotransmitters have been reported to activate the cascade in thyroid tissue, but not necessarily in the thyrocytes of the tissue.²⁴ Besides PKA, cAMP activates EPAC (exchange protein directly activated by cAMP) or Rap guanine nucleotide exchange factor 1 (GEF1) and the less abundant GEF2, which activates the small G protein Rap.⁶⁰ It does so in the thyroid.⁶¹ However, despite high expression of EPAC1 in thyrocytes and its further increase in response to TSH, all the physiologically relevant cAMP-dependent functions of TSH studied in dog thyroid cells, including acute regulation of cell functions (including thyroid hormone secretion) and delayed stimulation of differentiation expression and mitogenesis, are mediated only by PKA activation.⁶¹ The role of the cAMP/EPAC/Rap cascade in thyroid thus remains largely unknown. Activation of PKA inactivates small G proteins of the Rho family (RhoA, Rac1, and Cdc42), which reorganizes the actin cytoskeleton and could play an important role in stimulation of thyroid hormone secretion and induction of thyroid differentiation genes.⁶² Of the other known possible effectors of cAMP, cyclic guanosine monophosphate (cGMP)-dependent protein kinases are present in the thyroid but cyclic nucleotide–activated channels have not been looked for.

In the thyrocyte, the cAMP cascade is controlled by several negative feedbacks, including the direct activation by phosphorylation of phosphodiesterases and the induction of several proteins inhibiting the cascade.⁶³

The thyrocyte is very sensitive to small changes in cAMP concentrations; a mere doubling of this concentration is sufficient to cause maximal thyroglobulin endocytosis.⁶⁴

The Calcium–Inositol 1,4,5-Triphosphate Cascade

The calcium (Ca²⁺)–inositol 1,4,5-triphosphate (IP₃) cascade in the thyroid also corresponds, as far as has been studied, to the canonic model of the muscarinic or α₁-adrenergic receptor–activated cascades. It is activated in the human thyrocyte by TSH—through the same receptors that stimulate adenylyl cyclase—and by ATP, bradykinin, and TRH through specific receptors. In this cascade, as in the cAMP pathway, the activated receptor causes the release of GDP and the binding of GTP by the GTP-binding transducing protein (G_q, G_11, G₁₂), and their dissociation into α_q and βγ. In turn, α_GTP then stimulates phospholipase C. The TSH receptor activates G_s and G_q, with a higher affinity for G_s.65,66 Phospholipase C hydrolyzes membrane phosphatidylinositol 4,5-bisphosphate (PIP₂) into diacylglycerol and IP₃. IP₃ enhances calcium release from its intracellular stores, followed by an influx from the extracellular medium. The rise in free ionized intracellular Ca²⁺ leads to the activation of several proteins, including calmodulin. The latter protein in turn binds to target proteins and thus stimulates them (e.g., cyclic nucleotide phosphodiesterase and, most importantly, calmodulin-dependent protein kinases). These kinases phosphorylate a whole set of proteins exhibiting serines and threonines on their specific peptides and thus modulate them and cause many observable effects of this arm of the cascade. Calmodulin also activates constitutive nitric oxide (NO) synthase in thyrocytes. The generated NO itself enhances soluble guanylyl cyclase activity in thyrocytes and perhaps in other thyroid cells and thus increases cGMP accumulation.⁶⁷ Nothing is yet known about the role of cGMP in the thyroid cell.

Diacylglycerol released from PIP₂ activates protein kinase C, or rather the family of protein kinases C, which by phosphorylating serines or threonines in specific accessible peptides in target proteins causes the effects of the second arm of the cascade.⁶⁸ It inhibits phospholipase C or its G_q, thus creating a negative feedback loop. In the human thyroid, the PIP₂ cascade is stimulated through specific receptors by ATP, bradykinin, TRH, and TSH.^24,66,69,70 The effects of bradykinin and TRH are very short lived. Acetylcholine, which is the main activator of this cascade in the dog thyrocyte, is inactive on the human cell, although it activates nonfollicular (presumably endothelial) cells in this tissue.²⁴

Other Phospholipid-Linked Cascades

In dog thyroid cells and in a functional rat thyroid cell line (FRTL5), TSH activates PIP₂ hydrolysis weakly and at concentrations several orders of magnitude higher than those required to enhance cAMP accumulation. Of course, these effects have little biological significance. However, at lower concentrations in dog cells, TSH increases the incorporation of labeled inositol and phosphate into phosphatidylinositol. Similar effects may exist in human cells, but they would be masked by the stimulation of the PIP₂ cascade. They may reflect increased synthesis, perhaps coupled to and necessary for cell growth.⁷¹

Diacylglycerol can be generated by other cascades than the classic Ca²⁺-IP₃ pathway. Activation of phosphatidylcholine phospholipase D takes place in dog thyroid cells stimulated by carbamylcholine. Because it is reproduced by phorbol esters (i.e., by stable analogs of diacylglycerol), it has been ascribed to phosphorylation of the enzyme by protein kinase C, which would represent a positive feedback loop.⁷² Although such mechanisms operate in many types of cells, their existence in human thyroid cells has not been demonstrated.⁷³

Release of arachidonate from phosphatidylinositol by phospholipase A₂ and the consequent generation of prostaglandins by a substrate-driven process are enhanced in various cell types through G protein–coupled receptors, by intracellular calcium, or by phosphorylation by protein kinase C. In dog thyroid cells, all agents enhancing intracellular calcium concentration, including acetylcholine, also enhance the release of arachidonate and the generation of prostaglandins. In this species, stimulation of the cAMP cascade by TSH inhibits this pathway. In pig thyrocytes, TSH has been reported to enhance arachidonate release. In human thyroid, TSH, by stimulating PIP₂ hydrolysis and intracellular calcium accumulation, might be expected to enhance arachidonate release and prostaglandin generation, but such effects have not yet been proved.

Regulatory Cascades Controlled by Receptor Tyrosine Kinases

Many growth factors and hormones act on their target cells by receptors that contain one transmembrane segment. They interact with the extracellular domain and activate the intracellular domain, which phosphorylates proteins on their tyrosines. Receptor activation involves in some cases a dimerization and in others a conformational change of a preexisting dimer. The first step in activation is protein-tyrosine phosphorylation, followed by binding of various protein substrates on tyrosine phosphates containing segments of the receptor. Such binding through src homology domains (SH2) leads to phosphorylation of these proteins on their tyrosines. In turn, this phosphorylation causes sequential activation of the ras and raf proto-oncogenes, mitogen-activated protein (MAP) kinase kinase, MAP kinase, and so on, on the one hand, and phosphatidylinositol-3-kinase (PI-3-kinase), protein kinase B (PKB), and TOR (target of rapamycin) on the other hand. The set of proteins phosphorylated by a receptor defines the pattern of action of this receptor. In thyroids of various species, insulin, IGF-1, EGF, FGF, HGF, but not platelet-derived growth factor activate such cascades.74,75 In the human thyroid, effects of insulin, IGF-1, EGF, FGF, but not HGF have been demonstrated.^{3,21,51,76–78} TGF-β, acting through the serine threonine kinase activity of its receptors and its phosphorylated protein targets (Smad), inhibits proliferation and specific gene expression in human thyroid cells.^37,38,79 TSH and cAMP do not activate either the MAPK-ERK nor the JUNK and p38 phosphorylation pathways in dog and human thyroids.⁷⁵

Cross-Signaling Between the Cascades

Calcium, the intracellular signal generated by the PIP₂ cascade, activates calmodulin-dependent cyclic nucleotide phosphodiesterases and thus inhibits cAMP accumulation and its cascade.⁸⁰ This activity represents a negative cross-control between the PIP₂ and the cAMP cascades. Activation of protein kinase C enhances the cAMP response to TSH and inhibits the prostaglandin E response, which suggests opposite effects on the TSH and prostaglandin receptors.⁸¹ No important effect of cAMP on the PIP₂ cascade has been detected. On the other hand, stimulation of protein kinase C by phorbol esters inhibits EGF action.

Cross-signaling between the cyclic AMP pathway and growth factor–activated cascades have been observed in various cell types.82,83 In ovarian granulosa cells, FSH (through cAMP) activates MAP kinases and the PI3 kinase pathway.⁸⁴ In FRTL5, but not in WRT cell lines, TSH (through cAMP) activates MAP kinase; in WRT cells but not in PCCl3 cells, TSH and cAMP activate PKB.^85,86 Such cross signalings have not been observed in human or dog thyroid cells. Ras, MAPK, p38, Jun kinase and ERK5, as well as PI3 kinase and PKB, are not modulated by cAMP.^74,75,87,88

Other growth-activating cascades have been little investigated in the thyroid. In dog and human cells, TSH or cAMP have no effect on STAT phosphorylations (i.e., on the Jak-STAT pathways). The nuclear factor κB (NF-κB) pathway has not yet been investigated in thyroid cells.

Structure of the Protein

The β subunits of glycoprotein hormones, to which TSH belongs, are encoded by paralogous genes displaying substantial sequence similarity (Fig. 3-3). The corresponding receptors, FSHR, LH/CGR and TSHR, are members of the rhodopsin-like G protein–coupled receptor family. As such, TSHR contains a “serpentine” portion with seven transmembrane helices with many (but not all) of the sequence signatures typical of this receptor family. In addition, and a hallmark of the subfamily of glycoprotein hormone receptors, it contains a large (about 400 residues) aminoterminal ectodomain responsible for the high affinity and selective binding of TSH.^102–104 The higher-sequence identity of the serpentine portions of glycoprotein hormone receptors (about 70%), when compared with the ectodomains (about 40%, see Fig. 3-3), suggested early that the former are interchangeable modules capable of activating the G proteins (mainly G_as) after specific binding of the individual hormones to the latter.⁴ Contrary to other rhodopsin-like GPCRs, binding of the hormones to their ectodomains can be observed with high affinity in the absence of the serpentine.¹⁰⁵ The intramolecular transduction of the signal between these two portions of the receptors involves mechanisms specific to the glycoprotein hormone receptor family (see later). The relatively high sequence identity between the hormone-binding domains of the TSH and LH/CG receptors opens the possibility of spillover phenomena during normal or, even more so, molar or twin pregnancies, when hCG concentrations are several orders of magnitude higher than TSH. This provides an explanation to cases of pregnancy hyperthyroidism.^15,15

The TSHR contains six sites for N-glycosylation, of which four have been shown to be effectively glycosylated.¹⁰⁵ The functional role of the individual carbohydrate chains is still debated. It is likely that they contribute to the routing and stabilization of the receptor through the membrane system of the cell.

Alone among the glycoprotein hormone receptors, the TSHR undergoes cleavage of its ectodomain, severing it from the serpentine domain.¹⁰⁶ This phenomenon has been related to the presence in the TSHR of a 50-amino-acid “insertion” for which there is no counterpart in the FSHR or LH/CGR. The initial cleavage step, by a metalloprotease, takes place around position 314 (within the 50-amino-acid insertion), followed by nibbling of the aminoterminal extremity of the serpentine-containing portion.^107,108 The aminoterminal and serpentine portions of the resulting dimer remain bound to each other by disulphide bonds. The functional meaning of this TSHR-specific posttranslational modification remains unclear. Whereas all wild-type TSHR at the surface of thyrocytes seem to be in cleaved form, it has been shown that noncleavable mutant constructs are functionally undistinguishable from cleaved receptors when expressed in transfected cells.¹⁰⁶ Noteworthy, when transiently or permanently transfected in nonthyroid cells, the wild-type human TSHR is present at the cell surface as a mixture of monomer and cleaved dimer. The cleavage and shedding of the ectodomain from a portion of the mature receptors has been related to the triggering or maintenance of autoimmunity in Graves’ disease.^109,110

The TSH receptor is specifically inserted into the basolateral membrane of thyrocytes. This phenomenon involves signals encoded in the primary structure of the protein, as it is conserved when the receptor is expressed in the MDCK cell, a polarized cell of nonthyroid origin.¹¹¹

The possibility that TSHR are present at the cell surface as “dimers of cleaved dimers” has recently been raised following demonstration that most rhodopsin-like GPCRs do dimerize¹¹² (see following).

The TSH Receptor Gene

The gene coding for the human TSH receptor has been localized on the long arm of chromosome 14 (14q31).113,114 It spreads over more than 60 kb and is organized into 10 exons displaying an interesting correlation with the protein structure.¹⁰⁴ The extracellular domain is encoded by a series of 9 exons, each of which corresponds to one or an integer number of leucine-rich repeat (LRR) motifs. The C-terminal half of the receptor containing the C-terminal part of the ectodomain and the serpentine domain is encoded by a single large exon. This finding is reminiscent of the fact that many G protein–coupled receptor genes are intronless. A likely evolutionary scenario derives from this gene organization¹¹⁵: the glycoprotein hormone receptor genes would have evolved from the condensation of an intronless classic G protein–coupled receptor with a mosaic gene encoding a protein with LRRs. Triplication of this ancestral gene and subsequent divergence led to the receptors for LH/CG, FSH, and TSH. The existence of 10 exons in both the TSH and FSH receptor genes (as opposed to the 11-exon LH/CG receptor), suggests the following phylogeny: one ancestral glycoprotein hormone receptor duplicating in the LH receptor and in the ancestor of TSH and FSH receptor, which lost one intron. The latter would later duplicate yielding the TSH and FSH receptors.

The gene promoter has been cloned and sequenced in humans and rats.115,116 It has characteristics of “housekeeping” genes in that it is GC-rich and devoid of TATA boxes; in the rat it was shown to drive transcription from multiple start sites.¹¹⁶

Expression of the TSH receptor gene is largely thyroid specific. Constructs made of a chloramphenicol acetyltransferase reporter gene under control of the 5′ flanking region of the rat gene show expression when transfected into FRTL5 cells and FRT cells but not into nonthyroid HeLa or a rat liver cell line (BRL) cells.¹¹⁶ However, TSH receptor mRNA has been clearly demonstrated in fat tissue of the guinea pig¹¹⁷ and following differentiation of preadipocytes into adipocytes.^118,119 Functional significance in man of reports showing its presence in lymphocytes, extraocular tissue, and recently in cartilage and bone requires additional studies.^109,120

Recently, functional expression of the TSH receptor has been demonstrated in pars tuberalis of the pituitary in quails and ependymal cells in mice, in relation with the control of photoperiodic behavior.118,119,121,122 No extrapolation of these studies to man has been attempted yet. Expression of the TSH receptor in thyroid cells is extremely robust. It is moderately up-regulated and down-regulated by TSH in vitro¹²³ and down-regulated by iodide in vivo.¹⁰¹

Functional Aspects

Thyroid Hormone Synthesis

Thyroid hormone synthesis requires the uptake of iodide by active transport, thyroglobulin biosynthesis, oxidation and binding of iodide to thyroglobulin, and within the matrix of this protein, oxidative coupling of two iodotyrosines into iodothyronines. All these steps are regulated by the cascades just described.

Thyroid Hormone Secretion

Secretion of thyroid hormone requires endocytosis of human thyroglobulin, its hydrolysis, and the release of thyroid hormones from the cell. Thyroglobulin can be ingested by the thyrocyte by three mechanisms.6,183–185

In macropinocytosis, which is the first, pseudopods engulf clumps of thyroglobulin. In all species this process is triggered by acute activation of the camp/PKA cascade and therefore by TSH.¹⁸⁶ Inactivation of the Rho small G proteins, resulting in microfilament depolarization and stress-fiber disruption, is probably involved in the process.⁶² Stimulation of macropinocytosis is preceded and accompanied by an enhancement of thyroglobulin exocytosis and thus of membrane to the apical surface.^182,187,188 By micropinocytosis, the second process, small amounts of colloid fluid are ingested. This process does not appear to be greatly influenced by acute modulation of the regulatory cascades. It is enhanced in chronically stimulated thyroids and thyroid cells with induction of vesicle transport proteins Rab 5 and 7.^189,190 It probably accounts for most of basal secretion. A third (hypothesized) process is receptor-mediated endocytosis; it would be enhanced in chronically stimulated thyroid cells.^191–193 The protein involved could be megalin¹⁹⁴ and/or asialoglycoprotein. This process probably accounts for transcytosis of thyroglobulin.¹⁹⁵

Contrary to the last named, the first two processes are not specific for the protein. They can be distinguished by the fact that macropinocytosis is inhibited by microfilament and microtubule poisons and by lowering of the temperature (below 23° C). Whatever its mechanism, endocytosis is followed by lysosomal digestion, with complete hydrolysis of thyroglobulin. The main iodothyronine in thyroglobulin is thyroxine. However, during its secretion, a small fraction is deiodinated by type I 5-deiodinases (DIO1 and DIO2) to triiodothyronine (T₃), thus increasing relative T₃ (the active hormone) secretion.¹⁹⁶

The free thyroid hormones are released by an unknown mechanism, which may be transport or exocytosis. The iodotyrosines are deiodinated by specific deiodinases and their iodide recirculated in the thyroid iodide compartments. Under acute stimulation, a release (spillover) of amino acids and iodide from the thyroid is observed. A mechanism for lysosome uptake of poorly iodinated thyroglobulin on N-acetylglucosamine receptors and recirculation to the lumen has been proposed. Under normal physiologic conditions, endocytosis is the limiting step of secretion, but after acute stimulation, hydrolysis might become limiting with the accumulation of colloid droplets. Secretion by macropinocytosis is triggered by activation of the cAMP cascade and inhibited by Ca²⁺ at two levels: cAMP accumulation and cAMP action. It is also inhibited in some thyroids by protein kinase C downstream from cAMP. Thus the PIP₂ cascade negatively controls macropinocytosis.⁸¹

The thyroid also releases thyroglobulin. Inasmuch as this thyroglobulin was first demonstrated by its iodine, at least part of this thyroglobulin is iodinated; thus it must originate from the colloid lumen. Release is inhibited in vitro by various metabolic inhibitors and therefore corresponds to active secretion.187,197 The most plausible mechanism is transcytosis from the lumen to the thyrocyte lateral membranes.¹⁸⁸ As for thyroid hormone, this secretion is enhanced by activation of the cAMP cascade and TSH and inhibited by Ca²⁺ and protein kinase C activation. Because thyroglobulin secretion does not require its iodination, it reflects the activation state of the gland regardless of the efficiency of thyroid hormone synthesis. Thyroglobulin serum levels and their increase after TSH stimulation constitute a very useful index of the functional state of the gland when thyroid hormone synthesis is impaired, as in iodine deficiency, congenital defects in iodine metabolism, treatment with antithyroid drugs, and the like.¹⁹⁸ Regulated thyroglobulin secretion should not be confused with the release of this protein from thyroid tumors, which corresponds in large part to exocytosis of newly synthesized thyroglobulin in the extracellular space rather than in the nonexistent or disrupted follicular lumen. In inflammation or after even mild trauma, opening of the follicles can cause unregulated leakage of lumen thyroglobulin. Transcytosis or leakage from the lumen yields iodinated thyroglobulin, whereas newly synthesized exocytotic thyroglobulin is not iodinated.

Thyroglobulin

The regulatory DNA elements of the thyroglobulin gene have been characterized in several species.199,205,225 The proximal promoter, as defined in transfection experiments, extends over 200 base pairs and contains binding sites for transcription factors TTF-1, TTF-2, and Pax 8 (Fig. 3-7). An upstream enhancer element containing binding sites for TTF-1 has been identified in beef and man.²²⁶ In the latter, the enhancer region is longer and harbors additional binding sites for TTF-1 and cAMP responsive element–binding (CREB) protein.²²⁷ Both TTF-1 and Pax 8 proteins were individually shown to exert a major control on thyroglobulin gene transcription.^228,229 By contrast, TTF-2 activity appears to be dispensable, because the thyroglobulin gene is expressed in cells devoid of TTF-2 protein.²²⁸ Synergism in the transcriptional activation of the gene by TTF-1 and Pax 8 appears to rely on a direct interaction between these two factors²³⁰ and on their coordinated action involving both the enhancer and proximal promoter sequences.²³¹ It has also been proposed recently that the transcriptional coactivators p300²³² and (or ?) TAZ, the transcriptional coactivator with PDZ-binding motif,²³³ are involved in this activation. The known thyroglobulin gene regulatory elements were shown to be sufficient to drive the thyroid-restricted expression of a linked gene in living mice.²³⁴ This thyroid-restricted expression likely results from the requirement for the simultaneous presence of both TTF-1 and Pax 8, which occurs in thyroid only. It is associated with the tissue-specific demethylation of thyroglobulin gene sequences.²³⁵ Demethylation of the DNA is supposed to relieve the constitutive silencing of the gene.²³⁶

Thyroglobulin gene transcription has been shown to require the presence of circulating TSH in the adult rat²³⁷ and to be highly dependent on an elevated cAMP level in dog thyroid tissue slices,²³⁸ in primary cultured cells,²²² and to a much lower extent in immortalized thyroid cell lines like FRTL-5.²³⁹ Although they are devoid of classical cAMP-responsive element (CRE), the proximal promoter sequences are essentially involved in this control, as indicated by the observation of TSH/cAMP-induced changes in their chromatin structure²⁴⁰ and their TSH/cAMP-dependent activity in transfection experiments.²⁴¹ It has, however, been demonstrated recently that the onset of thyroglobulin gene expression during thyroid development takes place normally in mouse strains deprived of either circulating TSH or functional TSH receptors.^7,8 This may be consistent with the observation that the thyroglobulin gene displays a low level of cAMP-independent transcription in primary cultured thyrocytes,²²² which might depend on insulin, as observed in different culture models.^242–244 In primary cultures of dog thyrocytes, the transcriptional activation of the thyroglobulin gene by cAMP after transient TSH withdrawal is also delayed as compared to that of the thyroperoxidase gene,²²² and unlike thyroperoxidase transcription, it requires an active protein synthesis for sustained transcription.²²² The increase in Pax 8 concentration consecutive to TSH/cAMP stimulation of the thyrocyte is not sufficient to account for the observed control on thyroglobulin gene transcription; TSH is still required for transcriptional activation even in cells expressing high levels of Pax 8 protein.²⁰⁷ Thus, besides TTF-1 and Pax 8, at least one additional, still unidentified, factor is likely to play a key role in the control of thyroglobulin gene expression, as also suggested by the observation that in the course of thyroid development, both TTF-1 and Pax 8 are present well before a thyroglobulin gene is expressed.

In addition to the full-length thyroglobulin mRNA, a shorter transcript accumulates in the rat thyroid in response to TSH stimulation.²⁴⁵ This transcript results from differential splicing and polyadenylation of the primary transcript and encodes a protein limited to the very N-terminal part of thyroglobulin. Because this truncated protein still contains a major hormonogenic site,²⁴⁶ it could suggest that in conditions in which the balance of thyroid metabolism would favor hormone synthesis over iodine storage (e.g., shortage of iodine), the rat thyrocyte would manufacture a shorter thyroglobulin with a preserved hormonogenic ability but lacking many of the nonhormonogenic tyrosines.

Thyroperoxidase

In the species studied so far, the architecture of the proximal promoter region of the thyroperoxidase gene strikingly resembles that of the corresponding region of the thyroglobulin gene247,248 (see Fig. 3-7). The upstream enhancer element also encompasses a pair of TTF-1 binding sites and contains an additional binding site for Pax 8, as compared to its counterpart in the thyroglobulin gene.^249,250 Here again, the combination of the upstream enhancer and proximal promoter supports the synergistic action of TTF-1 and Pax 8 on gene transcription.²³¹ The transcriptional coactivator p300 has also been reported to be involved in the activation of this promoter.²⁵¹

Despite the existence of this high similarity, thyroperoxidase gene transcription is more tightly and more rapidly controlled by TSH and cAMP than that of the thyroglobulin gene in primary cultured thyrocytes and does not require a concomitant protein synthesis.222,252 Contrary to the thyroglobulin gene, the thyroperoxidase gene is not expressed in the absence of circulating TSH or functional TSH receptors in intact animals.⁸ On the other hand, the constitutive hyperactivation of the cAMP cascade leads to an increased expression of the gene as compared to the normal situation.²⁵³ In spite of their lack of a classical CRE, the proximal promoter sequences have been shown to mediate this TSH/cAMP control on transcription in transfection experiments.²⁴⁷ Exposure to a high dose of iodide reduces thyroperoxidase gene expression, as well as that of thyroglobulin, sodium/iodide symporter, and thyrotropin receptor genes in PCCl3 thyroid cells.²²⁴ Low doses of iodide also decrease thyroperoxidase gene expression in vivo, while the expression of thyroglobulin remains unaffected.¹⁰¹ Thus, apart from their basic dependence on the presence of the transcription factors TTF-1 and Pax 8, which insures their shared thyroid-restricted expression, the thyroperoxidase and thyroglobulin genes distinguish themselves significantly regarding the control of their transcription. It is worth mentioning in this context that a synergistic action of Pax8 and pRb, the retinoblastoma protein, appears to be required for thyroperoxidase promoter activation, whereas this is not the case for thyroglobulin promoter activation.²⁵⁴ It has also been postulated recently that the hormone-induced developmental activation of the thyroperoxidase gene involves the concerted action of TTF-2 and NF-1, both of which bind neighboring sequences in the gene promoter (see Fig. 3-7), resulting in the initial opening of the chromatin structure of the promoter.²⁵⁵

The existence of a major thyroperoxidase mRNA isoform has been detected in man.²⁵⁶ It appears to encode a protein devoid of its normal enzymatic activity.

Sodium/Iodide Symporter

Although the sodium/iodide symporter plays a key role in thyroid hormonogenesis, the expression of the corresponding gene is not restricted to the thyroid. Accordingly, the proximal promoter sequences identified so far do not exhibit a thyroid-specific activity in vitro,257,258 even if this activity may be marginally increased in the presence of TTF-1.²⁵⁹ The robust and appropriately controlled expression of this gene in the thyroid seems to be mediated essentially by the upstream enhancer element, which contains binding sites for both TTF-1 and Pax 8, and a cAMP-responsive element (CRE)-like DNA motif, which is involved in the control by TSH/cAMP^173,260 (see Fig. 3-7). The cAMP-response element modulator (CREM) has recently been proposed to be involved in this control.²⁶¹ As for the thyroperoxidase gene, TSH signaling is indispensable for sodium/iodide symporter gene transcriptional activation in vivo,^7,8 and iodide down-regulates the expression of the gene.^101,224 In addition, synergy between Pax8 and pRb appears to be required for the activation of both thyroperoxidase and sodium/iodide symporter promoters.²⁵⁴ A very similar control is thus exerted on the expression of both of these genes in the thyroid, despite the fact that their known regulatory regions bear only limited resemblances.

Thyrotropin Receptor

The TSH receptor gene is also expressed in tissues other than the thyroid. Again, the promoter elements identified presently, which include binding sites for thyroid hormone receptor (TR)-α1/retinoid-X receptor (RXR) heterodimer,²⁶² GA-binding protein (GABP),²⁶³ CREB protein,²⁶⁴ and TTF-1²⁶⁵ (see Fig. 3-7), do not display a clear thyroid-specific activity in transfection experiments, as could be expected. Contrary to the promoters described so far, the promoter of the TSH receptor gene does not contain a TATA-box motif but encompasses a GC-rich region preceding the multiple neighboring transcription start sites. Consistent with the presence of TTF-1 binding sites in the promoter region, the TSH receptor gene exhibits decreased activity in animals expressing reduced levels of TTF-1.²⁶⁶ No other regulatory DNA element specifically involved in the thyroid-specific expression of this gene has been identified as yet. On the other hand, DNA demethylation events in the promoter region have been observed in thyroid cells expressing the TSH receptor gene, as compared to nonexpressing cells.²⁶³

Control of TSH receptor gene expression has been studied in the FRTL5 cell line,267–269 the canine thyrocyte in primary culture,¹²³ cultured human thyrocytes,^270,271 and human thyroid cancer.^272,273 The general conclusion emerging from these studies is the extreme robustness of TSH receptor gene expression as compared with the other markers of thyroid cell differentiation (thyroglobulin and thyroperoxidase). In the dog, levels of TSH receptor mRNA remain virtually unchanged in animals subjected for 28 days to hyperstimulation by TSH secondary to treatment with methimazole or to TSH withdrawal achieved by administration of thyroxine.¹²³ In the same study, the effect of TSH or forskolin has been investigated in dog thyrocytes in primary culture. This experimental system has the advantage that the differentiation state of the cells can be manipulated at will: cAMP agonists maintain expression of the differentiated phenotype, whereas agents such as EGF, tetradecanoyl phorbol acetate (TPA), and serum lead to “dedifferentiation.”²⁷⁴ The results demonstrate that the dedifferentiating agents reduce accumulation of the receptor mRNA. However, contrary to what is observed with thyroglobulin and thyroperoxidase mRNA, the inhibition is never complete. TSH or forskolin are capable of promoting reaccumulation of the receptor mRNA, a maximum being reached after 20 hours. As with thyroglobulin but at variance with the thyroperoxidase gene, this stimulation requires ongoing protein synthesis.¹²³ Chronic stimulation of cultured dog thyrocytes by TSH for several days does not lead to any important down-regulation in mRNA. Similar data have been obtained with human thyrocytes in primary culture.^123,271 By contrast, negative regulation of receptor mRNA accumulation has been observed in immortal FRTL5 cells after treatment with TSH or TSAB.^267,269 This difference from human and canine cells must probably be interpreted in the general framework of the other known differences in phenotype and regulatory behavior of this cell line as compared with primary cultured thyrocytes (see later).⁸⁵ The presence in the promoter region of a CRE-like DNA motif which appears to be able to bind the CREB protein,²⁶⁴ a transcriptional activator directly activated by cAMP, as well as the CREM isoform ICER,²¹² a transcriptional repressor induced by cAMP, could explain both reported increase and decrease in gene expression following TSH stimulation, depending on the relative amounts of these factors (and likely of other CRE-binding proteins also) preexisting in the studied cells and the kinetics of the individual observations. Moreover, the binding site of the TRα1/RXR heterodimer identified in this promoter encompasses the CRE-like motif (see Fig. 3-7), which may add a further level of complexity depending on the availability of thyroid hormone in the experimental system. Recently, thyrotropin receptor mRNA was shown to be decreased in PCCl3 cells exposed to a high iodide concentration.²²⁴

The effect of malignant transformation on the amounts of TSH receptor mRNA has been studied in spontaneous tumors in humans,272,273 in a murine transgenic model of thyroid tumor promoted by expression of the simian virus-40 large T oncogene,²⁷⁵ and in FRTL5 cells transformed with v-ras.²⁶⁸ In the last two models, expression of the TSH receptor gene was suppressed: the tumor or cell growth became TSH independent. In the transgenic animal model, loss of TSH receptor mRNA seemed to take place gradually, with early tumors still displaying some TSH dependence for growth. In the human tumors, a spectrum of phenotypes was observed. As expected, anaplastic tumors had completely lost the receptor mRNA, as well as other markers of thyrocyte differentiation (thyroglobulin and thyroperoxidase). In papillary carcinoma, variable amounts of TSH receptor mRNA were invariably found,²⁷² even in the tumors that had lost the capacity to express the thyroglobulin or thyroperoxidase genes.²⁷² These data agree well with the observations of thyrocytes in primary culture: expression of the TSH receptor gene is robust, and it persists in the presence of agents (or after several steps in tumor progression) that promote extinction of the other markers of thyroid cell differentiation. This evidence leads to the conclusion that the basic marker of the thyroid phenotype is probably the TSH receptor itself, which makes sense: the gene encoding the sensor of TSH—the major regulator of thyroid function, growth, and differentiated phenotype—is virtually constitutive in thyrocytes. From a pragmatic viewpoint, these data provide a rationale for the common therapeutic practice of suppressing TSH secretion in patients with a differentiated thyroid tumor.²⁷⁶

Thyroid Oxidases

Two distinct genes, ThOX1 and ThOX2 (also known as DUOX-1 and DUOX-2), both significantly related to the gene encoding the phagocyte NADPH oxidase gp91^Phox, are expressed essentially in the thyroid, but not exclusively, at least for ThOX2.179,277 In the dog, ThOX mRNAs accumulate in response to TSH/cAMP stimulation.¹⁷⁹ This effect is much less apparent in man,¹⁷⁹ and in the rat, conflicting results were obtained in vivo and in the established FRTL-5 cell line, respectively.²⁷⁷ The proximal promoter sequences of both human THOX1 and THOX2 genes have been delineated using a functional assay.²⁷⁸ These promoters do not exhibit a thyroid cell-restricted activity in vitro and are not controlled positively by TSH/cAMP, as could be expected. No known regulatory DNA motif could be identified within these promoter sequences. Notably, like the promoter of the TSH receptor gene, both of these promoters are devoid of a TATA-box motif. A recent report identified the ThOX-2 promoter as a target for either Pax8 or TTF-1,²⁷⁹ but another study indicated that this control is likely to involve regulatory sequences other than the ones identified and cloned so far.²⁸⁰

Recently, genes encoding proteins required for the functional maturation of the thyroid oxidases were identified in the close vicinity of both ThOX genes, and named DUOXA1 and DUOXA2, respectively.²⁸¹ Within each ThOX-DUOXA pair, the two genes are arranged in a head-to-head configuration, the distance separating the putative transcription starts being in the range of only 100 bp. This raises the question as to whether the ThOX promoter sequences identified previously²⁷⁸ may also control transcription in the other direction.

Thyroid Cell Turnover

The thyroid is composed of thyrocytes (70%), endothelial cells (20%), and fibroblasts (10%) (proportions measured in dog thyroid). In a normal adult, the weight and composition of the tissue remain relatively constant. Because a low but significant proliferation is demonstrated in all types of cells, it must be assumed that the generation of new cells is balanced by a corresponding rate of cell death.3,282,283 The resulting turnover is on the order of 1 per 5 to 10 years for human thyrocytes (i.e., 6 to 8 renewals in adult life, as in other species).²⁸³ Normal cell population can therefore be modulated mainly at the level of proliferation and only secondarily by cell death. In growth situations—that is, either in normal development or after stimulation—the different cell types grow more or less in parallel, which implies a coordination between them.^{16,34,284,285} Because TSH receptors and iodine metabolism and signaling coexist only in the thyrocyte in thyroid, this cell, sole receiver of the physiologic information, must presumably control the other types of cells by paracrine factors such as VEGF, FGF, IGF-1, NO, and the like.¹⁶ The successful isolation of human thyroid endothelial cells will allow a more detailed study of these interactions.²⁸⁶ TSH has been demonstrated to up-regulate the production of vascular endothelial cell growth factor (VEGF) by human thyrocytes.²⁸⁷ It is interesting in this regard that the vascular support of the follicles reflects their activity, suggesting the concept of angiofollicular units.^288,289

The Mitogenic Cascades

In the thyroid, at least three families of distinct mitogenic pathways have been well defined (Fig. 3-8): (1) the hormone receptor–adenylyl cyclase–cAMP-dependent protein kinase system, (2) the hormone receptor–tyrosine protein kinase pathways, and (3) the hormone receptor–phospholipase C cascade.^3,290 The receptor–tyrosine kinase pathway may be subdivided into two branches; some growth factors, such as EGF, induce proliferation and repress differentiation expression, whereas others, such as FGF in dog cells or IGF-1 and insulin, are either mitogenic or are necessary for the proliferation effect of other factors without being mitogenic by themselves, but they do not inhibit differentiation expression.^28,242 In human thyroid cells, IGF-1 is required for the mitogenic action of TSH or EGF, but by itself it only weakly stimulates proliferation.⁷⁸ In dog and human thyrocytes in primary cultures, after induction of insulin receptors by TSH, physiologic concentrations of insulin permit the proliferative action of TSH.^20,21 In FRTL5 and rat cells, and in mouse thyroid in vivo,²⁹¹ IGF-1 is weakly mitogenic per se,²⁹² whereas in pig thyroid cells, it produces a strong effect.²⁹³ Expression of both human IGF-1 and IGF-1R in mouse thyroid induces a mild goiter and a decrease in serum TSH level—that is, an increase in non-TSH-activated thyroid function.²⁹¹ Similarly, relatively high serum IGF-1 levels in a human population are associated with higher goiter frequency.²⁹⁴

It should be noted that TSH directly stimulates proliferation while maintaining the expression of differentiation. Differentiation expression, as evaluated by NIS or by thyroperoxidase and thyroglobulin mRNA content or nuclear transcription, is induced by TSH, forskolin, cholera toxin, and cAMP analogs.³ These effects are obtained in all the cells of a culture, as shown by in situ hybridization experiments.²⁴² They are reversible; they can be obtained either after the arrest of proliferation or during the cell-division cycle.^223,242 Moreover, the expression of differentiation, as measured by iodide transport, is stimulated by concentrations of TSH lower than those required for proliferation.⁷⁸

All the proliferation effects of TSH are mimicked by nonspecific modulators of the cAMP cascade (e.g., cholera toxin and forskolin, which stimulate adenylate cyclase), cAMP analogs (which activate the cAMP-dependent protein kinases), and even synergistic pairs of cAMP analogs acting on the different sites of these two kinases.78,295,296 They are reproduced in vitro and in vivo by expression of the adenosine A₂ receptor, which is constitutively activated by endogenous adenosine,²⁵³ and by constitutively active G_sα²⁹⁷ and cholera toxin.²⁹⁸ They are inhibited by antibodies blocking G_s.²⁹⁹ Inhibition of cAMP-dependent protein kinases (PKA) inhibits the proliferation and differentiation effects of cAMP.^300,301 Stimulation of PKA by selective cAMP analogs that do not activate EPAC proteins is sufficient to fully mimic mitogenic effects of TSH and forskolin in dog thyrocytes.⁶¹ There is, therefore, no doubt that the mitogenic and differentiating effects of TSH are mainly and probably entirely mediated by cAMP-dependent protein kinases. A complementary role of the Rap guanyl nucleotide exchange factor EPAC and of Rap1 has been proposed in cell lines and in mice,^302–304 but it is not observed in canine thyroid primary cultures.⁶¹ Permanent abolition of G_q expression in mouse thyroid leads as expected to an inhibition of iodide oxidation and thyroid hormone synthesis but also to hypotrophy of the gland.³⁰⁵ This suggests at least a necessary chronic permissive role of the G_q phospholipase C cascade. In dog thyroid cells, carbamylcholine acting through the G_q cascade has the same permissive effects on TSH mitogenic action as IGF-1, perhaps through activation of PKB.^88,306

EGF also induces proliferation of thyroid cells from various species.3,16,78 However, the action of EGF is accompanied by a general and reversible loss of differentiation expression, assessed as described earlier.²⁷⁴ The effects of EGF on differentiation can be dissociated from its proliferative action. Indeed, they are obtained in cells that do not proliferate in the absence of insulin and in human cells, in which the proliferative effects are weaker, or in pig cells at concentrations lower than the mitogenic concentrations.³

Finally, the tumor-promoting phorbol esters, the pharmacologic probes of the protein kinase C system, and analogs of diacylglycerol also enhance the proliferation and inhibit the differentiation of thyroid cells. These effects are transient because of desensitization of the system by protein kinase C inactivation.3,78,307,308 Activation of the G_q/phospholipase C (PLC)/PKC cascade by a more physiologic agent, such as carbamylcholine, in dog thyroid cells does not reproduce all the effects of phorbol esters. In particular, prolonged stimulation of this cascade by carbamyl choline permits the cAMP-dependent mitogenesis of dog thyrocytes,³⁰⁶ but unlike phorbol esters, it does not induce proliferation in the presence of insulin.³⁰⁹ The Ras protooncogene is strongly activated by phorbol esters but very weakly by carbachol.⁸⁸ Thus we cannot necessarily equate the effects of phorbol esters and prolonged stimulation of the PLC cascade. The dedifferentiating effects of phorbol esters do not require their mitogenic action either. Thus the effects of TSH, EGF, and phorbol esters on differentiation expression are largely independent of their mitogenic action.³

In several thyroid cell models, very high insulin concentrations are necessary for growth even in the presence of EGF. We now know that this prerequisite mainly reflects a requirement for IGF-1 receptor.3,76,292,310 It is interesting that in FRTL5 cells, as in cells from thyroid nodules, this requirement may disappear, perhaps because the cells secrete their own somatomedins and thus become autonomous with regard to these growth factors.^76,311 By contrast, in primary cultures of normal dog and human thyrocytes, very low concentrations of insulin, acting on insulin receptors, are sufficient to support the mitogenic effects of TSH and cAMP when insulin receptors have been induced to high levels by TSH.^20,21 This puzzling regulation, which is reminiscent of the induction of insulin receptors during the differentiation of adipocytes, suggests that thyroid might well be revealed as a more specific target of circulating insulin than hitherto recognized.

In the action of growth factors on receptor protein tyrosine kinase pathways, the effects on differentiation expression vary with the species and the factor involved: from stimulation (e.g., insulin, as well as IGF-2 in dog and FRTL5 cells),²⁴³ to an absence of effect,³¹² to transitory inhibition of differentiation during growth (FGF and HGF in dog cells),^28,313 to full but reversible dedifferentiation effects (EGF in dog and human cells).^77,274 Ret/PTC rearrangements, activating mutations of Ras, as well as oncogenic mutation of B-Raf, which are responsible for most differentiated carcinoma, constitutively activate the signaling cascades of growth factors.^314–316

The kinetics of the induction of thymidine incorporation into nuclear DNA of dog thyroid cells is similar for TSH, forskolin, EGF, and TPA. Whatever the stimulant, a minimal delay of about 16 to 20 hours takes place before the beginning of labeling—that is, the beginning of DNA synthesis.³¹⁷ This is the minimal amount of time required to prepare the necessary machinery. For the cAMP and EGF pathways, the stimulatory agent has to be present during this whole prereplicative period; any interruption in activation (e.g., by washing out the stimulatory forskolin) greatly delays the start of DNA synthesis.³¹⁸ This limitation explains why norepinephrine and prostaglandin E, which also activate the cAMP cascade, do not induce growth and differentiation: the rapid desensitization of their receptors does not allow a sustained rise in cAMP levels.

The three main types of mitogenic cascade, specifically the growth factor–protein tyrosine kinase, phorbol ester–protein kinase C, and TSH-cAMP cascades, are fully distinct at the level of their primary intracellular signal and/or the first signal-activated protein kinase.³

Iodide actually inhibits the cAMP and the Ca²⁺-phosphatidylinositol cascades and in a more delayed and chronic effect decreases the sensitivity of the thyroid to the TSH growth response. These effects are relieved, according to the general paradigm of Van Sande, by perchlorate and methimazole95,319 in that they are mediated by a postulated organified derivative of iodide, iodohexadecanal.⁹⁷

Proliferation an Differentiation (Fig. 3-10)

The incompatibility at the cell level of a proliferation and differentiation program is commonly accepted in biology. In general, cells with a high proliferative capacity are poorly differentiated, and during development, such cells lose this capacity as they progressively differentiate. Some cells even lose all potential to divide when reaching their full differentiation, a phenomenon called terminal differentiation. Conversely, in tumor cells, proliferation and differentiation expression are inversely related. Activation of Ras and p42/p44 MAP kinases, induction of c-Jun, sustained expression of c-myc, induction of cyclin D1 and down-regulation of p27^kip1 all have been shown to be causatively associated not only with proliferation but also with loss of differentiation in a large variety of systems, sometimes independently of proliferation effects. It is therefore not surprising that in thyroid cells, the general mitogenic agents and pathways, phorbol esters and the protein kinase C pathway, EGF, and in calf and porcine cells, FGF and the protein tyrosine kinase pathway, induce both proliferation and the loss of differentiation expression. The effects of the cAMP cascade are in striking contrast with this general concept. Indeed, TSH and cAMP induce proliferation of dog thyrocytes while maintaining differentiation expression; both proliferation and differentiation programs can be triggered by TSH in the same cells at the same time.²⁴² This situation is by no means unique, because neuroblasts in the cell cycle may simultaneously differentiate. It is tempting to relate this apparent paradox to the unique characteristics of the cAMP-dependent mitogenic pathway, such as the lack of activation (or even the inhibition) of the Ras/MAP kinase/c-Jun/cyclin D1 cascade, as demonstrated in dog and human thyrocytes. For instance, if one generalization could be made about proto-oncogenes, it is the dedifferentiating role of c-myc. A rapid and dramatic decrease in c-myc mRNA by antisense myc sequences induces differentiation of a variety of cell types. It is therefore striking that in the case of the thyrocyte, in which activation of the cAMP cascade leads to both proliferation and differentiation, the kinetics of the c-myc gene appears to be tightly controlled. After a first phase of 1 hour of higher levels of c-myc mRNA, c-myc expression is decreased below control levels. In this second phase, cAMP decreases c-myc mRNA levels, as it does in proliferation-inhibited fibroblasts. It even depresses EGF-induced expression. The first phase could be necessary for proliferation, whereas the second phase could reflect stimulation of differentiation by TSH.^210,347 The specific involvement of cyclin D3 in the cAMP-dependent mitogenic stimulation of dog and human thyrocytes is also interesting in this context.³⁴³ We have recently shown that the differential utilization of cyclin D1 or cyclin D3 affects the site specificity of the pRb-kinase of CDK4, including in dog and human thyrocytes.^343,348 In addition to inhibiting E2F-dependent gene transcription related to cell-cycle progression, pRb plays positive roles in the induction of tissue-specific gene expression by directly interacting with a variety of transcription factors, including Pax8 in thyroid cells.²⁵⁴ Whether the selective utilization of cyclin D3 in the TSH cascade, associated with a more restricted pRb-kinase activity, could allow the preservation of some differentiation-related functions of pRb thus remains to be examined. Indeed, unlike cyclins D1 and D2, cyclin D3 is highly expressed in several quiescent tissues in vivo, and its expression is not only stimulated by mitogenic factors but also induced during several differentiation processes associated with a repression of cyclin D1.³⁴⁹

We now consider the distinct cAMP-dependent mitogenic pathway, which appears to be adjuncted to the more general mechanisms used by growth factors, as pertaining to the specialized differentiation program of thyroid cells.⁸² In dog thyrocytes, the proliferation in response to serum or growth factors specifically extinguishes their capacity to respond to TSH/cAMP as a mitogenic stimulus.³⁵⁰ Similarly, in less differentiated thyroid cancers generated by the subversion of growth factor mechanisms, the TSH dependence of growth is generally found to be lost, and in various cell lines derived from thyroid carcinomas, cAMP and PKA activation even inhibit CDK4 activity and cell-cycle progression.³⁵¹

Because the cell renewal rate is very low in the thyroid (once every 8 years in adults), the role of apoptosis is also very low. However, under different circumstances, the apoptotic role can greatly increase, such as after the arrest of an important stimulation in vitro³⁵² and in vivo.^353–355

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