Anthony N. Hollenberg
Thyrotropin (TSH) is the major regulator of normal thyroid hormone production, which, in turn, is a major regulator of TSH secretion, and measurement of serum TSH has become the principal screening test used by clinicians worldwide for the diagnosis of thyroid dysfunction. Thus, an understanding of the mechanisms by which TSH secretion is controlled is crucial for an understanding of normal thyroid physiology and interpreting the results of measurements of serum TSH. In addition, regulation of TSH secretion by the thyrotroph cells of the pituitary is an ideal model in which to study the regulation of gene expression by thyroid hormones, thyrotropin-releasing hormone (TRH), and other substances. These regulators of TSH secretion will be considered individually, although it is likely that there are extensive interactions among them.
TSH is synthesized and secreted from the thyrotroph cells of the anterior pituitary and released into the circulation to regulate thyroid hormone synthesis in the thyroid (Fig. 10D.1). The thyrotrophs are exposed to circulating levels of thyroxine (T4) and triiodothyronine (T3), and these hormones, principally T3, tightly regulate the synthesis and secretion of TSH, mainly at the level of gene expression. In addition, the thyrotrophs are exposed to other hormones or drugs, such as glucocorticoids or retinoic acid analogs, which also may influence the synthesis and secretion of TSH. Besides the systemic circulation, the anterior pituitary receives direct communication from the hypothalamus via the capillaries that comprise the hypothalamic-pituitary portal system that arises from the median eminence. The most important hypothalamic regulator of TSH secretion is TRH. However, other hypothalamic neuropeptides and transmitters such as somatostatin and dopamine may also regulate TSH secretion. TRH neurons project from the paraventricular nucleus (located on either side of the third ventricle) to the median eminence, where TRH is released and carried through the portal system to the pituitary (1).
FIGURE 10D.1. Schematic diagram of the hypothalamic–pituitary–thyroid axis. Thyrotropin-releasing hormone (TRH) is synthesized in neurons within the paraventricular nucleus of the hypothalamus that project to the median eminence. At the median eminence, TRH is released into the hypothalamic–pituitary portal circulation and carried to the pituitary, where it activates TRH-1 receptors on the thyrotroph cells of the pituitary to synthesize and release thyrotropin (TSH). TSH acts on the thyroid via TSH receptors to stimulate the synthesis and secretion of thyroxine (T4) and triiodothyronine (T3). T4 and T3 inhibit TRH and TSH secretion, closing the feedback loop.
The thyrotroph cells of the pituitary originate from a common pituitary stem cell and differentiate early in embryonic development (see Chapter 2). TRH is not required for the normal development of the thyrotrophs (2). The thyrotrophs share lineage with other pituitary cell types, including lactotrophs and somatotrophs, as demonstrated by mutations in the transcription factor Pit-1, which lead to defects in all three cell types (3,4,5). In addition, the thyrotrophs share lineage with the gonadotroph cells, based on upon their shared expression of the common α-subunit of TSH, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), and the key role of the transcription factor Prophet of Pit-1 (PROP-1) in both gonadotroph and thyrotroph development in humans (6). It is clear that development of the thyrotrophs requires the action of several transcription factors (7).
REGULATION OF THYROTROPIN SECRETION
Like other pituitary hormones, TSH secretion is pulsatile, with 6 to 18 pulses per 24 hours in adult humans (8,9). The frequency and amplitude of the pulses are reasonably constant during the day, but there is an increase in both frequency and amplitude in the late evening and early morning (10 PM to 2 to 4 AM) (10,11,12,13). As a result, serum TSH concentrations are about twofold higher during this period than during the rest of the day. This nocturnal surge of TSH secretion appears soon after birth and wanes with age. The mechanisms underlying TSH pulsatility and its nocturnal accentuation are not known, but likely include pulsatile TRH secretion and some input from the suprachiasmatic nucleus of the hypothalamus, which plays a role in many circadian rhythms in mammals (14). The pulsatility, especially at night, of TSH secretion decreases during fasting and nonthyroidal illness (15,16).
Production of TRH in the paraventricular nucleus of the hypothalamus is essential for normal TSH secretion. TRH-knockout mice have central hypothyroidism, with slightly high serum TSH concentrations, but the biological activity of the TSH is decreased (17). In addition, mice with TRH deficiency and resistance to thyroid hormone have lower serum TSH concentrations than do mice with only resistance to thyroid hormone (18). Similarly, in humans TRH deficiency results in a decrease in both the amount of TSH that is secreted and its biological activity (19). While isolated TRH deficiency has not been found in humans, a patient with central hypothyroidism was found to be a compound heterozygote for mutations of the TRH receptor, with both mutant receptors being nonfunctional, confirming the essential role of TRH in humans (20).
Structure and Synthesis of Thyrotropin-Releasing Hormone
TRH is a tripeptide (pyroGlu-His-ProNH2) that is produced by the processing of a larger prohormone in the hypothalamus and elsewhere in the central nervous system (Fig. 10D.2) (1,21).
It was the first hypothalamic hormone to be identified (22,23). The complementary DNA (cDNA) sequence of murine and rat TRH encodes for a preprohormone (preproTRH) of approximately 29 kilodaltons (kDa), which includes five copies of the sequence of the TRH prohormone, whereas in humans the cDNA contains six copies (24,25,26). The hormone cDNA sequence is flanked by codons that encode pairs of basic amino acids that allow for processing of the progenitor TRH sequence (Gln-His-Pro-Gly) by two convertases, PC-1 and PC-2, and carboxypeptidase E (27,28). The progenitor TRH sequence is subsequently modified via cyclization at the N-terminus and amidation at the C-terminus to yield mature TRH (29,30).
FIGURE 10D.2. Structures of the thyrotropin-releasing hormone (TRH) gene, preproTRH, and TRH. The gene encoding human preproTRH is present on chromosome (CHR) 3 and is encoded by three exons. The first exon codes for the 5′-untranslated region, and exons 2 and 3 code for the full 29 kDa preproTRH. The 3′-untranslated region is also coded for in exon 3. Human preproTRH contains six copies of the TRH progenitor sequence (black boxes), which are cleaved and further modified (see text) to yield mature TRH. PC, prohormone convertase; CPE, carboxypeptidase E.
TRH mRNA also encodes several other peptides between the progenitor TRH sequences that can be isolated from the mammalian hypothalamus. TRH peptide 160–169, which lies between the third and fourth TRH progenitor sequences, has been isolated from rat hypothalamic tissue. This peptide can potentiate TRH-mediated TSH release from the anterior pituitary through a receptor separate from the TRH receptor that is probably located on nonendocrine cells of the pituitary (31,32,33). Rat TRH peptides 83–106 and 178–199 are both increased during suckling; they may contribute to prolactin release by blocking hypothalamic dopamine release (34). Several other pro–TRH-derived peptides have been isolated, including TRH-Gly, the direct precursor to TRH, but their role in vivo is not known (35). Related TRH peptides have also been identified in the human hypothalamus and placenta, but because the intervening sequences are different from those in rats, the sequences of these peptides are different (36,37), and their biological activity, if any, is not known.
Based on the role of processing in producing mature TRH, it is clear that TRH production can be controlled through the regulation of enzymes such as PC-1 and PC-2 and carboxypeptidase E. Indeed, both PC-1 and PC-2 are down-regulated by thyroid hormone, consistent with their key regulatory role in the production of TRH (38,39). Mice that are deficient in carboxypeptidase E have less mature TRH and more of its precursor forms than normal mice (28). A single human patient with defective PC-1 has been described; this patient had defective processing of several hormones, including insulin, and mild primary, not central, hypothyroidism (40,41).
TRH is rapidly deamidated after its release to TRH-free acid and histidyl-proline-diketopiperazine (His-Pro-DKP), a stable cyclized metabolite (30,42). The principal enzymes that degrade TRH are three forms of pyroglutamyl aminopeptidase (PAP I, PAP II, and thyroliberase), which yield His-Pro-DKP, and prolyl endopeptidase, which yields the TRH-free acid. PAP I is a cytosolic enzyme that has broad substrate specificity and has a role in the metabolism of other neuropeptides (42,43). In contrast, PAP II (also known as TRH-degrading enzyme) is present in synaptosomal fractions of neural tissue and in pituitary cell membranes (44,45,46,47). Furthermore, PAP II has a substrate specificity restricted to TRH and TRH-like peptides, which, together with its tissue distribution, suggests that it is the major peptidase that removes the amino-terminal pyroglutamate residue from TRH (42,48). His-Pro-DKP, which has a variety of endocrine and neural actions, was only reduced by 50% in the hypothalamus and cerebral cortex of TRH-knockout mice, suggesting that it has other sources (49). The third PAP enzyme, thyroliberase, is present in serum and appears to catabolize TRH there. It is similar to PAP II, but because it is not membrane bound it may regulate TRH concentrations during transport through the portal system. This enzyme may be a proteolytic product of the PAPII gene, which also is expressed in the liver (50). Thyroid hormone increases PAP II activity in the pituitary (44). Furthermore, TRH itself appears to down-regulate PAP II activity in the pituitary (51). Thus, the bioavailability of TRH can be modified at the protein level through changes in both its processing and its degradation.
The Thyrotropin-Releasing Hormone Gene
The structure of the murine, rat, and human TRH genes are identical. Each consists of three exons and two introns (Fig. 10D.2).
The sequences of the exons are well conserved among the species, whereas the sequences of the introns are less conserved. The first exon encodes the 5′-untranslated region, while the coding sequence for the preprohormone and the 3′-untranslated region are on exons 2 and 3. The promoter is immediately adjacent to exon 1, as demonstrated by the presence of a TATA box within 25 base pairs (bp) of the transcription start site. In the hypothalamus, the content of TRH mRNA is highest in the paraventriclar nucleus and the lateral hypothalamic area. It is also found in the preoptic region, the olfactory lobes, the periaqueductal gray region, and the medullary raphe neurons in the brainstem, which contain much of the TRH found in the spinal cord. In addition, TRH mRNA is present in peripheral tissues, including the heart and the pancreas, where TRH may play a role in glucose homeostasis (17). In areas of the brain outside the paraventricular nucleus, TRH mRNA and its derived peptides have several functions, including regulation of gastric motility and gastric acid secretion through activation of vagal outflow (52).
The cell bodies of the TRH neurons (hypophysiotropic neurons) that control TSH secretion are located in the paraventricular nucleus. This nucleus has two major groups of neurons: a magnocellular group that is located laterally and expresses oxytocin and vasopressin, and a parvocellular group that is located medially. TRH neurons are found throughout the parvocellular group, but only those TRH neurons in certain anatomic subdivisions project to the median eminence to regulate thyrotroph function (1). The hypophysiotropic group of TRH neurons is also defined by coexpression of the mRNA encoding the cocaine- and amphetamine-related neuropeptide (CART) (53,54) and by their ability to regulate preproTRH mRNA levels, as discussed later in the chapter. The role of TRH neurons in the paraventricular nucleus that do not express CART and do not project to the median eminence is not clear. A separate group of medial parvocellular neurons of the paraventricular nucleus contain corticotropin-releasing hormone, the key hypothalamic regulator of corticotropin (ACTH) secretion. Corticotropin-releasing hormone is also present in other areas of the paraventricular nucleus during stress (55).
The underlying mechanism allowing for cell-specific TRH gene expression is not known. However, the transcription factor simple-minded (Sim)-1 is required for the formation of all paraventricular-nucleus neurons in vivo, and mice that lack Sim-1 have little development of the paraventricular nucleus and also the supraoptic nucleus of the hypothalamus (56,57). It is likely that Sim-1 and its partner, the transcription factor arylhydrocarbon receptor nuclear translocator (Arnt)-2 act upstream of another transcription factor, Brn-2, to allow appropriate development of these two hypothalamic nuclei (58,59). In addition, the transcription factor orthopedia (Otp) is also critical for the development of the paraventricular nucleus; it acts before Sim-1 in the developing hypothalamus (60,61). The factors that cause the development of separate TRH neurons from other neuropeptide-expressing neurons in the paraventricular nucleus are not known, but it is likely that still-unidentified cell-specific factors are needed for the development of these neurons, perhaps by interacting directly with regulatory elements in the TRH promoter which are required for its hypothalamic expression (62).
Regulation of the Expression of the Thyrotropin-Releasing Hormone Gene
TRH gene expression is dynamically regulated in the medial and periventricular regions of the paraventricular nucleus in order to regulate TSH secretion and subsequently thyroid secretion in response to many conditions, including illness, starvation, cold, and thyroid disease. An understanding of the mechanisms involved can be discerned through an examination of both the milieu in which the hypophysiotropic TRH neurons lie and the regulatory elements present within the promoter region of the TRH gene.
The TRH neurons in the paraventricular nucleus receive input from other regions of the brain as well as from the circulation. The major afferent connections to these neurons include catecholamine neurons from the brain stem (63) and neurons from the arcuate nucleus (1,64). Catecholamine signaling likely plays an important role in the up-regulation of TRH gene expression during cold exposure (65), while input from the arcuate nucleus plays a critical role in the down-regulation of TRH gene expression during starvation and illness. Indeed, insight into the regulation of TRH neurons by inputs from the arcuate nucleus has shed considerable light on the importance of pathways via which energy expenditure regulates the axis.
Regulation by Pathways Important in Energy Expenditure
In rodents, fasting results in a rapid fall in serum TSH and thyroid hormone concentrations (66,67), caused, in part, by down-regulation of TRH gene expression in the paraventricular nucleus (68,69). Administration of the adipocyte hormone leptin, the production of which also falls during fasting, prevents the fasting-induced fall in thyroid secretion by preventing the fall in TRH mRNA levels (70,71). Thus, leptin is a key regulator of TRH gene expression (Fig. 10D.3). The actions of leptin in the hypothalamus are mediated by the long-form of the leptin receptor (ObRb), a member of the cytokine receptor family that is coupled to the janus kinase 2/signal transducer of activated transcription 3 (JAK-2/STAT-3) signaling cascade (72). The ObRb is expressed strongly in the arcuate nucleus and, in a more limited way, in the paraventricular nucleus (73,74,75), and is thus poised to regulate TRH expression both indirectly and directly (76,77). Lesions of the arcuate nucleus prevent the effects of leptin on TRH gene expression (78).
FIGURE 10D.3. Schematic diagram of the actions of leptin on thyrotropin-releasing hormone (TRH) gene expression. Leptin, produced in adipocytes, crosses the blood–brain barrier and regulates TRH gene expression via the melanocortin system in the arcuate nucleus, whose neurons project to TRH neurons in the paraventricular nucleus. In addition, leptin regulates TRH gene expression directly in TRH neurons. AgRP, agouti-related protein; CART, cocaine- and amphetamine-related neuropeptide; α-MSH, α-melanocyte-stimulating hormone; MC4-R, melanocortin-4 receptors; NPY, neuropeptide Y; NPY1,5-R, neuropeptide-1 and -5 receptors; ObRb, leptin receptor; POMC, proopiomelanocortin; PVH, paraventricular nucleus; 3V, third ventricle. (Adapted from Bjorbaek C, Hollenberg AN. Leptin and melanocortin signaling in the hypothalamus. Vitam Horm 2002;65:281, with permission.)
Two neuronal groups within the arcuate nucleus express leptin receptors and project to the TRH neurons in the paraventricular nucleus. The neurons in the first group contain both pro-opiomelanocortin (POMC) and CART (79,80,81). In the arcuate nucleus, unlike the pituitary, POMC is processed primarily to α-melanocyte-stimulating hormone (MSH), a potent anorexigenic peptide, which signals through both the melanocortin (MC)-3 and -4 receptors. TRH neurons in the paraventricular nucleus have MC-4 receptors and are directly innervated by α-MSH nerve terminals (82,83). In addition, central administration of α-MSH prevents the fasting-induced suppression of TSH and thyroid secretion (76,77). The second group of arcuate-nucleus neurons that have leptin receptors synthesize the neuropeptides agouti-related peptide (AgRP) and neuropeptide Y (NPY). These neurons also directly contact TRH neurons in the paraventricular nucleus. In contrast to POMC, both AgRP and NPY are orexigenic peptides and are down-regulated by leptin and up-regulated during fasting (84,85,86). Both AgRP, an MC-4–receptor antagonist (and possibly an inverse agonist), and NPY (through NPY receptor–isoforms expressed on TRH neurons), when administered centrally, can cause central hypothyroidism by down-regulating TRH mRNA expression in the paraventricular nucleus (87,88,89,90). In summary, the arcuate nucleus serves to integrate leptin signaling and assist in the regulation of TRH gene expression, as well as regulate food intake and energy expenditure (91).
The direct and indirect actions of leptin on TRH neurons to regulate TSH and thyroid secretion in situations of nutritional stress are probably mediated by changes in TRH gene transcription. The proximal regions of the promoter regions of the rodent and human TRH genes are structurally similar (Fig. 10D.4). Both of the genes have a TATAA box within 25 bp of the transcriptional start site and a region termed Site 4 between 60 and 52 bp upstream of the start site. First identified as a thyroid hormone receptor (TR)–binding site (see later in the chapter) (25,92), Site 4 is critical for basal activity of the TRH promoter in mammalian cell lines. In addition, Site 4 can bind the cyclic adenosine monophosphate (cyclic AMP) response element–binding protein (CREB), which is downstream in the signaling cascade induced by α-MSH via MC-4 receptors. Thus, modulation of CREB activity via α-MSH, AgRP, and NPY signaling should allow for varying degrees of TRH gene expression. In addition to Site 4, the TRH gene has a conserved STAT-binding site between 150 and 140 bp upstream of the start site. This site interacts with STAT3 and mediates transcriptional responses to leptin signaling via the ObRb (64,93). Thus, the promoter region of the TRH gene has sites whereby multiple inputs can be integrated to control TRH gene expression.
FIGURE 10D.4. Activation of the thyrotropin-releasing hormone (TRH) promoter by multiple signaling pathways. The TRH promoter responds to leptin, melanocortin, and T3 via a number of cis-acting elements. Binding of leptin to its receptor (ObRb) activates JAK2, leading to its phosphorylation and subsequent recruitment and phosphorylation (P) of STAT3. Phosphorylated STAT3 homodimers enter the nucleus and bind directly to the promotor region of the TRH gene to stimulate its expression. The proopiomelanocortin (POMC)-derived peptide, melanocyte-stimulating hormone (α-MSH), activates TRH transcription via protein kinase A (PKA) by binding to the melanocortin-4 receptor (MC4-R) on TRH neurons. PKA phophorylates cyclic AMP response element–binding protein (CREB) bound to Site 4 in the TRH promoter, which leads to TRH transcription. In contrast, triiodothyronine binds to the β2 isoform of TR (thyroid receptor), which also acts through Site 4 to down-regulate TRH gene expression. JAK2, janus-kinase 2; STAT3, signal transducer of activated transcription 3. Together they form the JAK2/STAT3 signaling cascade.
While most of the studies of nutritional regulation of TRH production, or at least gene expression, have been performed in rodents, there is increasing evidence that similar mechanisms affect TRH production and therefore TSH and thyroid secretion in humans during food deprivation and illness. Humans with mutations of the leptin receptor have central hypothyroidism, although humans who have a defective leptin gene do not (94). Furthermore, controlled caloric restriction in humans leading to weight loss results in a decline in serum thyroid hormone concentrations that can be reversed by leptin (95). Also, acute fasting in humans leads to a decrease in pulsatile TSH secretion, which also can be reversed by administration of leptin (15,96). However, humans with MC-4-receptor mutations, the most common genetic form of obesity, have normal serum TSH and thyroid hormone concentrations (97,98).
Regulation of Expression of the Thyrotropin-Releasing Hormone Gene by Nonthyroidal Illness
Many changes in pituitary-thyroid function occur in nonthyroidal illness, including a decrease in TSH secretion (see section on nonthyroidal illness in Chapter 11). One cause of the decrease in TSH secretion is a decrease in TRH secretion, as manifested by a decrease in TRH gene expression in the paraventricular nucleus (16,99). The frequent presence of anorexia in these patients suggests that the leptin and melanocortin signaling pathways may play a role in the decrease. However, it is also likely that cytokines such as interleukin-1, and -1β, tumor necrosis factor, and interferon contribute to the decrease. MC-4–receptor knockout mice do not develop cachexia in response to cytokine administration or an increased tumor burden, suggesting that the melanocortin signaling pathway plays an important role in the response. However, increased expression of arcuate-nucleus neuropeptides during cachexia does not by itself explain low TRH gene expression or, for that matter, decreased food intake. It is likely that interactions between the cytokine-signaling pathways and the melanocortin system contribute to the central suppression of TRH gene expression in the paraventricular nucleus during nonthyroidal illness, or that other cytokine-activated pathways are involved (100,101,102).
Regulation of Thyrotropin-Releasing Hormone Gene Expression by Thyroid Hormone
An important component of the control of TSH secretion is inhibition of TRH gene expression in the paraventricular nucleus by thyroid hormone, principally T3. Conversely, TRH gene expression increases in response to low serum T4 and T3 concentrations (103,104). TRH mRNA levels in other regions of the hypothalamus are not regulated by T3. In terms of serum hormone, T4 may be more important than T3 (105), with the T3 being produced locally from T4 by the action of type 2 iodothyronine deiodinase (D2), the principal deiodinase present in the central nervous system (see Chapter 7). This is supported by the finding that D2-knockout mice maintain TSH secretion in the presence of high serum T4 concentrations (106). D2 is not expressed in the TRH neurons in the paraventricular nucleus, but it is present in specialized cells termed tanycytes that line the third ventricle and whose processes extend to the TRH neurons. It is likely that T4 is converted to T3 in the tanycytes, and the T3 is then released to act on the neurons (107,108).
T3 regulates TRH gene expression at the level of transcription via the nuclear thyroid hormone receptor (TR) (Fig. 10D.4). There are three TR-isoforms, α1, β1, and β2, which bind T3 and mediate transcriptional regulation (see Chapter 8). The TRβ-isoforms are alternatively spliced products of a single gene and differ structurally only in the amino terminus, while TRα1 is the product of a separate gene but functions in the same way as the TRβ-isoforms (109,110). The greatest difference in each of the isoforms may be in their cell-specific expression patterns (111). Mice devoid of both TRβ-isoforms have normal or slightly high serum TSH and high T4 and T3 concentrations, findings that resemble those found in patients with resistance to thyroid hormone (see Chapter 81), consistent with the predominant expression of TRβ-isoforms in the pituitary and hypothalamus (112,113). The values are similar in mice with selective ablation of the β2-isoform, demonstrating the requirement for this isoform for negative regulation by T3 of TRH gene expression (114); this isoform is also required for the normal development of color vision due to its unique expression in the retina (115). In contrast to the TRH gene, T3 regulation of the genes for the α- and β-subunits of TSH is mediated not only via the β2 isoform of the TR, but also to some extent via the β1- and α1-isoforms (116,117).
On genes that are positively regulated by the TRs, the TR isoforms bind to thyroid hormone response elements present in the regulatory regions of target genes as either homo- or heterodimers with retinoid-X-receptors (RXR). In the absence of T3, or in the hypothyroid state, the TR isoforms repress gene transcription by recruiting nuclear corepressor molecules, which in turn recruit a multiprotein complex that modifies chromatin through histone deacetylation, leading to transcriptional repression (118,119,120,121,122). The presence of T3 leads to a conformational change in the TR, which results in release of the corepressor complex and the sequential recruitment of coactivators, which result in histone acetylation and transcriptional activation (123,124,125).
The molecular mechanism by which the TR isoforms inhibit gene transcription is less clear. In vitro, genes repressed by T3 are activated in the absence of T3and subsequently repressed by the addition of T3, the mirror image of the process in positively regulated genes. Proposed mechanisms for this effect include interference with other transcription factors (independent of DNA binding) by T3–TR complexes, and interaction of the T3–TR complexes with specific negative thyroid hormone response elements to cause down regulation. Recent studies have demonstrated that negative regulation of TRH and TSH α- and β-subunit gene expression requires binding of the β isoform of TR to DNA (126). Indeed, the promoter regions of both the TRH and TSHβ-subunit genes have TR-binding sites, which are required for negative regulation (25,92,127,128). The TR-binding site in the promoter region of the TRH gene includes Site 4, which is required for basal and cyclic AMP-stimulated activity of the promoter. Taken together, it is likely that negative regulation of the TRH gene by T3 requires binding of TR-β2 to Site 4 and the recruitment of coregulators such as members of the steroid receptor coactivator family (129).
REGULATION OF THYROTROPIN SECRETION FROM THE PITUITARY
TSH secretion is regulated not only by TRH, but also by thyroid hormone and other factors acting directly on the thyrotrophs (Fig. 10D.1). These inputs arise from either the peripheral circulation or other regions of the hypothalamus. Regulators from the periphery include thyroid hormones, glucocorticoids, and retinoids. Other hypothalamic peptides and neurotransmitters that regulate TSH secretion include somatostatin and dopamine. These signals are integrated in the thyrotrophs to determine TSH secretion.
Action of Thyrotropin-Releasing Hormone on the Pituitary
Once TRH reaches the pituitary it acts to increase the synthesis and secretion of TSH from the thyrotrophs (Fig. 10D.5). [TRH also stimulates prolactin secretion, and it stimulates growth hormone secretion in some patients with somatotroph adenomas (acromegaly) and FSH or LH secretion in some patients with gonadotroph adenomas (130,131,132).] In normal subjects, intravenous administration of TRH in doses of 50 to 500 µg results in a dose-dependent 4- to 20-fold rise in serum TSH concentrations within 15 to 30 minutes, followed by a small rises in serum T3 concentrations in 1.5 to 3 hours and small rises in serum T4 concentrations in 4 to 8 hours (133,134). Before the development of sensitive TSH assays and hypothalamic-pituitary imaging techniques, the TRH stimulation test was a useful test for the diagnosis of thyrotoxicosis (no rise in serum TSH) and differentiation of hypothalamic from pituitary hypothyroidism (delayed rise vs. no rise in serum TSH) (135). TRH stimulation of prolactin secretion is also dose-dependent, and the response is supranormal in patients with hypothyroidism (136,137).
FIGURE 10D.5. Regulation of thyrotropin (TSH) secretion in the thyrotrophs of the pituitary gland. TSH secretion is regulated via multiple cell-surface and nuclear receptors on the thyrotrophs. The receptors include thyrotropin-releasing hormone 1 (TRH-1) and sst-2 and sst-5 (somatostatin) receptors in the cell membrane, which act via cyclic AMP (cAMP) or protein kinase C (PKC) or calcium ions to alter the expression of the genes for the subunits of TSH. In addition, activation of TRH receptors stimulates release of stored TSH. Within the nucleus, the β2 isoform of thyroid receptor (TR), retinoid-X-receptor g (RXRγ), and glucocorticoid receptor (GR) regulate transcription of the genes for the α- and β-subunits of TSH (TSHα and TSHβ). All of these pathways probably interact with the transcription factors cAMP response element binding-protein (CREB) and pituitary transcription factor 1 (Pit-1).
The actions of TRH on the thyrotrophs are mediated by binding of TRH to cell-surface receptors, in particular type 1 TRH receptors (TRH-R1) (138). A second TRH receptor (TRH-R2) has been identified in rodents but is not known to have a role in humans (139,140). A third receptor was identified in Xenopus laevis, but its affinity for TRH is so low that it is probably not a TRH receptor (141). The two TRH receptors are, for the most part, expressed in separate regions of the central nervous system, with TRH-R1 being principally expressed in the anterior pituitary as well as in the hypothalamus, autonomic nervous system, and portions of the brainstem. In contrast, TRH-R2 is more extensively expressed in the thalamus, subthalamic area, and cortex (142,143).
Both TRH receptors are members of the G-protein–coupled receptor family, and both are coupled to guanine nucleotide-binding (G) proteins (Gq/11) to mediate signal transduction. TRH and its analogs bind to both receptors with similar affinity (143). TRH binding to the receptor activates phospholipase C in the cells, leading to the production of inositol 1,4,5-trisphosphate and an increase in intracellular calcium, which in turn leads to activation of protein kinase C. The TRH receptors may also couple to other G proteins and activate other intracellular signaling pathways (143,144). The activation of these signaling cascades leads to the transcriptional regulation of target genes; these include the genes for the α- and β-subunits of TSH in the thyrotrophs and the gene for prolactin in the lactotrophs (145). TRH-receptor signaling is further regulated by receptor internalization after TRH binding, which leads to either degradation or recycling of the receptor to the cell membrane (146,147,148).
Activation of TRH-R1 receptors in the pituitary appears to control transcription by regulating the interaction of several transcription factors and coactivators with the promoter regions of the target genes. The promoter of the gene for the α-subunit of TSH is regulated by both CREB and Lim-1 (149,150), the activity of which is increased by TRH through the recruitment of the cofactor CREB-binding protein (CBP) (151,152). In contrast, the gene for the β-subunit of TSH is regulated primarily through direct binding of the transcription factors Pit-1 and GATA-2. Pit-1 in turn recruits CBP in a TRH-dependent fashion (151,153). Pit-1 is also a key regulator of prolactin gene expression, and TRH signaling increases transcription of the prolactin gene through this pathway.
The paradoxical stimulatory effect of TRH on growth hormone secretion in patients with somatotroph adenomas is probably due to the expression of receptors of the TRH-R1 type in these adenomas (154,155,156); TRH-R1 mRNA can be detected in the adenomas of patients whose serum growth hormone concentrations increase in response to TRH (157). Some somatotroph adenomas also have an aberrantly spliced variant of TRH-R1 lacking the sixth and seventh transmembrane domains that would be predicted not to bind TRH, but this probably inactive isoform is not found in adenomas that respond to TRH (157).
TRH stimulates not only the expression of the genes for the subunits of TSH, but also the posttranslational glycosylation of TSH, which contributes significantly to its biological activity (18,158,159). Some patients with central hypothyroidism have slightly high serum TSH concentrations that increase in both amount and bioactivity after the administration of TRH (see Chapter 51). Similarly, TRH-knockout mice also have slightly high serum TSH concentrations that increase in response to TRH, but their TSH has decreased thyroid-stimulating activity (17).
The Effect of Thyroid Hormones in the Thyrotrophs
Since the advent of serum TSH assays in the late 1960s and the development of the TRH stimulation test it has been clear that TSH secretion is very tightly controlled by T4 and T3, and that very small changes in serum T4 and T3 concentrations have large effects on TSH secretion (Fig. 10D.6) (160,161,162). Indeed, there is a log/linear relationship between serum TSH and free T4 concentrations at all levels of thyroid secretion (163) (see Chapter 13). The sensitivity of the thyrotrophs to small changes in serum thyroid hormone concentrations underscores the importance of the latter as the most important regulator of TSH secretion in humans.
FIGURE 10D.6. Mean (±SE) serum thyrotropin (TSH) concentrations before and after intravenous administration of thyrotropin-releasing hormone (TRH) in eight normal subjects before and during administration of two combinations of triiodothyronine (T3) plus thyroxine (T4) (inset), each given for 3 to 4 weeks. The low T3 plus T4 combination reduced basal serum TSH concentrations and especially the serum TSH responses to 400 µg of TRH (all eight subjects) and 25 µg of TRH (six subjects). The higher T3 plus T4 combination abolished the serum TSH response to 400 µg of TRH (the response to 25 µg of TRH was not tested after this T3 plus T4 dose). At the time of the three studies, the subjects' mean serum T3 and T4 concentrations were: base line, serum T3 119 ng/dL (1.8 nmol/L) and T4 6.9 µg/dL (89 nmol/L); low T3 plus T4 combination, serum T3 110 ng/dL (1.7 nmol/L) and T4 7.0 µg/dL (90 nmol/L); and high T3 plus T4 combination, serum T3 141 ng/dL (2.2 nmol/L) and T4 7.8 µg/dL (101 nmol/L). (From Snyder PJ, Utiger RD. Inhibition of thyrotropin response to thyrotropin-releasing hormone by small quantities of thyroid hormones. J Clin Invest 1972;51:2077, with permission.)
As discussed previously, T3 plays a critical role in TRH gene expression. In addition, T3 acting on the thyrotrophs regulates the expression of the genes for both TSH subunits and the TRH receptor (TRH-R1). The actions of T3 to regulate both hypothalamic and pituitary gene expression allow for the very close control of TSH secretion, which is expressed clinically in the log/linear relationship described above. While the molecular mechanism underlying T3 regulation of TRH signaling in the pituitary is unclear, experimental data suggests that T3 may both inhibit TRH-R1 mRNA expression and decrease the level of TRH-R1 protein in the thyrotrophs (164,165,166).
T3, in conjunction with TR isoforms, inhibits the transcription of the genes for the α- and β-subunits of TSH in the thyrotrophs in vivo (167). At least in mice, all TR isoforms are present in the thyrotrophs and contribute to the regulation of TSH, though among them β-isoforms are probably the most important (Fig. 10D.7) (113,114,116,117,168). Indeed, mutations in the ligand-binding domain of the β-isoform have been found in TSH-secreting pituitary adenomas (169,170).
FIGURE 10D.7. Mechanisms of negative regulation of the genes for the α- and β-subunits of TSH by T3. A: The gene for the α-subunit appears to be regulated by the thyroid receptor (TR) via a mechanism that does not involve TR binding to DNA. In this model, the TR alone [absence of triiodothyronine (T3)] increases α-subunit gene expression by removing corepressors (COR) and histone deacetylase (HDAC) from the promoter, leading to activation of transcription by coactivators (COA) and increased histone acetylase (HAT) activity. In the presence of T3, the corepressors remain bound to the promoter, leading to histone deacetylation and repression. B: In contrast, inhibition of the gene for the β-subunit requires binding of complexes of T3 and TR to a site in exon 1. This binding recruits histone deacetylase activity either directly or via corepressors. Alternatively, the T3–TR complexes may recruit captivators that may paradoxically function to repress transcription. CREB, cyclic AMP response element-binding protein.
While the β-isoforms are required for T3 responsiveness, the molecular mechanism by which T3 inhibits the genes for the subunits of TSH appears to be different. The promoter region of the gene for the α-subunit has a TR-binding site just downstream from its TATA box (171), but this site is probably not used; instead T3 inhibition is achieved through protein–protein interactions between the TR and coregulators, which mediate the response of the promotor to cyclic AMP (172). In the absence of T3, TR binds to a repression complex containing corepressor molecules and histone deacetylases, leading to activation. The presence of T3 prevents the repression complexes from interacting with the TR, allowing the complex to remain on the promoter, which leads to repression (173).
In contrast, the promoter region of the gene for the β-subunit of TSH contains a negative thyroid hormone response element that binds TR isoforms. This element is located in exon 1, just downstream from the transcription start site; it appears to bind the TR as a monomer to mediate T3-dependent repression (128). RXR antagonizes negative regulation by T3 by competing with the TR for binding to the response element. The negative thyroid hormone response element and a region surrounding it have been termed the Z-region, and this region appears to bind β-isoforms of TR and histone deactylases only in the presence of T3. The Z-region is present in several other genes, including the gene for the α-subunit of TSH, but its role in these other genes is not known (174).
The molecular mechanisms governing negative regulation of the TSH subunit genes by T3 are becoming clearer; they are multiple, and they are not as straightforward as positive regulation. In vivo, T3-mediated negative regulation of TRH and TSH-subunit gene expression is prolonged, because TSH gene expression and indeed TSH secretion that has been inhibited by T3 can remain low for prolonged periods. The mechanism underlying this long-term effect is unknown.
Retinoids and Thyrotropin Secretion
The role of vitamin A analogues in the regulation of TSH secretion was noted in 1999 when patients with cutaneous T-cell lymphoma who were being treated with a novel RXR ligand, bexarotene, developed reversible central hypothyroidism (175). Heterodimeric complexes of RXR and one of the isoforms of TR mediate many actions of T3, although the complexed RXR usually binds little if any of its own ligand. In the case of bexarotene, the inhibition of TSH secretion is probably due solely to inhibition of TSH-β gene expression by RXRγ, the principal RXR isoform in the thyrotrophs (175,176). RXRγ knockout mice are resistant to thyroid hormone; they have high serum TSH and thyroid hormone concentrations (177). The interaction of RXRγ with the TSH-β promoter is probably through a separate response element that binds RXR homodimers; RXR ligands inhibit TSH secretion in mice in which the TRβ gene is knocked out (178). In rodents, RXR ligands inhibit TSH secretion within two hours after their administration, before there are any changes in TSH-subunit gene expression or protein levels (179). Thus, the effects of retinoids on the thyrotrophs are multifactorial. Whether endogenous RXR ligands regulate TSH secretion is not known.
Glucocorticoids and Thyrotropin Secretion
TSH secretion is inhibited by high doses of glucocorticoids and endogenous hypercortisolemia, and some patients have mild central hypothyroidism. In contrast, patients with Addison's disease tend to have slightly high serum TSH concentrations. Glucocorticoids act in the paraventricular nucleus to inhibit TRH gene expression in vivo (180), although the mechanism is unclear, and glucocorticoids stimulate TRH gene expression in primary cultures of hypothalamic tissue (181,182). In addition, glucocorticoids inhibit TSH secretion in cultured pituitary cells; this effect may be due to an increase in secretion of annexin-1 by the folliculostellate cells of the pituitary, which then inhibits TSH release (183).
Glucocorticoids may contribute to regulation of TSH secretion in normal subjects. In patients with Addison's disease, continuous administration of hydrocortisone in doses that mimicked the normal diurnal variation of serum cortisol concentrations resulted in normal serum TSH concentrations, including normal nocturnal pulses of TSH secretion (184). In normal subjects, administration of metyrapone, which blocks cortisol secretion, resulted in an increase in serum TSH concentrations and reduced nocturnal TSH secretion (185). Thus, small changes in endogenous cortisol secretion can alter TSH secretion, probably by perturbing TRH synthesis and release.
The doses of glucocorticoid needed to inhibit TSH secretion in humans have not been well defined, but it is clear that even prolonged administration of high doses does not cause hypothyroidism. The reason is that the potent effect of low serum T4 and T3 concentrations to raise TSH secretion overcomes the glucocorticoid-induced inhibition of TSH secretion. The same phenomenon probably explains why none of the other substances that inhibit TSH secretion (see following sections) cause hypothyroidism.
Somatostatin and Thyrotropin Secretion
Somatostatin is an inhibitor of growth hormone secretion that was identified initially as a 14-amino-acid–and subsequently also as a 28-amino-acid–secreted peptide (186,187,188). It is synthesized in neurons in the periventricular and arcuate nucleus as well as in nonneural tissue, most notably the pancreas (189). The actions of somatostatin are mediated through five receptors (sst-1 through -5), which are G-protein coupled and signal in part through inhibition of adenylyl cyclase and a reduction in intracellular levels of cyclic AMP (Fig 10D.5) (189). The five receptors bind the secreted forms of somatostatin equally well, but they are expressed differently and likely differ in their signaling capabilities in different tissues.
Early studies of the pituitary actions of somatostatin revealed that it inhibited not only growth hormone but also TSH release, and incubation of pituitary tissue in vitro with antisomatostatin antibodies and administration of the antibodies in vivo increased release of TSH (190,191,192). In humans, intravenous administration of high doses of somatostatin reduces serum TSH concentrations and blocks TRH-mediated stimulation of TSH release. This effect is of little if any importance in the overall regulation of TSH secretion. Growth hormone itself can reduce TSH secretion, probably by increasing endogenous somatostatin release.
Long-term administration of somatostatin analogues, for example in patients with acromegaly, does not cause hypothyroidism. However, these analogues (as well as somatostatin itself) do inhibit TSH secretion in patients with TSH-secreting pituitary adenomas, and long-term analogue therapy may result in a decrease in adenoma size. The efficacy of octreotide and other long-acting analogues of somatostatin in patients with TSH-secreting pituitary adenomas is based upon the compounds' high affinity for sst-2 and sst-5 receptors, which appear to be preferentially expressed in these tumors (193,194,195,196,197). These analogues are now the principal medical therapy for patients with TSH-secreting adenomas (see Chapter 24) (198,199).
Catecholamines and Thyrotropin Secretion
Networks of catecholamine-containing fibers make contact with TRH neurons in the paraventricular nucleus, and they may also make contact with the nerve terminals of TRH-secreting neurons in the median eminence. The majority of these neurons originate in discrete regions of the brain stem (55,200). Stimulation of the α-adrenergic signaling system increases TSH release via α2-adrenergic receptors, and inhibition of the same system blocks TSH release (134,201,202,203). The effect of cold exposure is the best example of the role of the catecholamine system in the regulation of TSH secretion. Both TRH release and TRH mRNA synthesis in the paraventricular nucleus increase within 1 hour after exposure to cold (65,204,205,206); this response can be blocked by α-adrenergic antagonist drugs (65). Activation of this pathway probably explains the rapid increase in TSH secretion that occurs soon after birth in humans (see Chapter 74) (207,208). Cold has little effect on TSH secretion in adults.
Dopamine-containing neurons of the arcuate nucleus that project to the median eminence are important regulators of prolactin secretion, and they may also play a role in TSH secretion (209). Dopamine blocks TSH release from pituitary cells, probably acting via the A2 isoform of the dopamine receptor to inhibit intracellular cyclic AMP accumulation. In addition, in cultured rat pituitary cells, dopamine inhibits the expression of the genes for the α- and β-subunits of TSH (210,211). In humans, intravenous administration of dopamine inhibits TSH secretion, and this is one cause of the low serum TSH concentrations often present in patients with severe nonthyroidal illness (see section on nonthyroidal illness in Chapter 11). The acute administration of metoclopramide or domperidone, both dopamine receptor antagonists, can lead to a rise in serum TSH concentrations, while the acute administration of bromocriptine, a dopamine receptor agonist, lowers serum TSH concentrations (209,212,213,214). The effects of these drugs are not clinically important, in that long-term therapy has no effect on TSH secretion.
CLINICAL ASSESSMENT OF THYROTROPIN SECRETION
The first assays for serum TSH were not sufficiently sensitive to detect TSH in the serum of all normal subjects. That is no longer the case, and assays are now available that detect far less TSH than is present in all normal subjects. As a result, measurement of serum TSH has become the standard test to screen for thyroid dysfunction. However, there are some clinical situations in which a patient's serum TSH concentration is not a reliable indicator of clinical status (see Chapter 13).
Measurement of serum TSH is an excellent screening test for thyrotoxicosis. However, because of the log/linear relationship between serum TSH and free T4concentrations, serum TSH concentrations may be low in patients with normal serum free T4 concentration (see Chapter 79). Furthermore, the serum free T4and TSH values are often dissociated during treatment, with the former falling to normal or even below normal, but the latter remaining low (see Chapter 45) (215,216).
Inappropriate Thyrotropin Secretion
Serum TSH concentrations may be inappropriately high or normal in patients with normal or high serum free T4 concentrations. This can occur when the patient has serum heterophile antibodies; these antibodies interfere in TSH assays to raise the value (217). It also occurs in patients with a TSH-secreting pituitary adenoma (see Chapter 24), in patients with resistance to thyroid hormone (see Chapter 81), and in patients who take a large dose of T4 just before testing (218). All these situations are rare; among 7908 consecutive serum samples submitted to the laboratory of a large tertiary care hospital, the free T4concentration was high and the TSH concentration was >1 mU/L in 18 samples (from 17 patients) (219).
A substantial proportion of patients with nonthyroidal illness have abnormal serum TSH concentrations. Some of them have low serum TSH and low free T4concentrations (220), or what might be called illness-related central hypothyroidism. During recovery, their serum TSH concentrations may be high transiently (221). The mechanisms underlying the suppression of TSH secretion in these patients are probably multiple, and include cytokines, glucocorticoids, and possibly the leptin-signaling pathway. A major question is whether this central hypothyroidism is a beneficial or harmful adaptation to illness. This problem is discussed in detail in the section on nonthyroidal illness in Chapter 11.
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