TSHβ Subunit Gene Structure

The human TSHβ subunit gene has been isolated and characterized.¹⁵ This single-copy gene is 4527 base pairs (bp) in size, and is located on the short arm of chromosome 1.¹⁶ The gene structure consists of three exons and two introns (Fig. 2-1, top panel). The first exon has 37 bp and contains the 5′-untranslated region of the gene. It is separated from the second exon by a large first intron of 3.9 kb. The coding region of the gene is contained within the second (163 bp) and third (326 bp) exons, which are separated by a 0.45 kb intron, while the 3′-untranslated region is contained within the third exon.

DNA sequences close to the transcriptional start site in the promoter of the TSHβ gene contain elements responsible for initiating transcription and regulating expression. A consensus TATA box is located 28 bp upstream of the transcriptional start site and is important for positioning RNA synthesis. Progressive 5′ deletions of the mouse TSHβ promoter linked to a luciferase reporter following expression in thyrotrope cells defined the cis-acting sequences required for expression to the first 270 bp of the promoter.17,18 Although these sequences defined the minimal promoter, other studies have shown that enhancer sequences located more than 6 kb upstream are also required for the promoter to express in pituitary thyrotropes in transgenic mice.¹⁹

Promoter deletion studies have demonstrated that the mouse TSHβ promoter from −271 to −80 is sufficient to confer thyrotrope-specific activity,¹⁷ and thyrotrope transcription factors can bind to the proximal promoter.²⁰ Within this broad area, four regions of protein interaction have been identified using nuclear extracts from thyrotrope cells.¹⁸ Two factors, Pit-1, a homeodomain factor, and Gata2, a zinc finger transcription factor, can bind to TSHβ promoter sequences from −135 to −88²¹ (Fig. 2-2). Both factors can bind independently to the promoter, form a heteromeric complex with DNA, physically interact with each other, and functionally synergize to activate TSHβ promoter activity. Recently, an additional transcription factor, TRAP220 (Med1, PBP), was shown to be recruited to the TSHβ proximal promoter, where it was shown to play a role in transcriptional activation.²²

TRAP220 was originally defined as part of a transcriptional mediator complex that interacts with hormone-occupied thyroid/steroid hormone receptors.²³ Mice with a single copy of this gene were hypothyroid with reduced levels of pituitary TSHβ transcripts.²⁴ TRAP220 is recruited to the TSHβ gene by virtue of its physical interaction with both Pit-1 and Gata2, since the protein itself does not bind DNA. Co-transfections in nonpituitary cells showed that Pit-1, Gata2, or TRAP220 alone could not stimulate the TSHβ promoter. However, maximal activity resulted when all three factors were expressed. Interaction studies showed that all three factors interact with each other in vivo and in vitro. The regions of interaction were important for maximal function. Chromatin immunoprecipitation demonstrated in vivo occupancy on the proximal TSHβ promoter.²² Thus, the TSHβ gene is activated by a unique combination of transcription factors present in pituitary thyrotropes, including those that act via binding to the proximal promoter, as well as others that are recruited to the promoter via protein-protein interactions.

α Subunit Gene Structure

The human glycoprotein hormone α subunit gene is located on chromosome 6 at position 6q12-q21.²⁵ It is present as a single copy gene that is 9635 kb in size and contains four exons and three introns and contains a consensus TATA box located 26 bp upstream of the transcriptional start site.²⁶ The first exon (94 bp) contains 5′-untranslated sequences and is separated from the second exon by a 6.4 kilobase (kb) intron. The second exon contains 7 bp of 5′-untranslated sequence and 88 bp of the coding region. The coding sequence continues in the third (185 bp) and fourth (75 bp) exons, and the 3′-untranslated region (220 bp) is contained within the fourth exon (Fig. 2-1, bottom panel).

The α subunit gene is expressed in thyrotropes, gonadotropes, and placental cells but is differentially regulated. Cell-specific expression in each cell type is dependent on different regions of the promoter. Whereas the region downstream of −200 is sufficient for placental expression,²⁷ gonadotropes require sequences between −225 and −200,²⁸ and regions farther upstream appear to be critical for thyrotrope expression.²⁹ Transgenic mouse studies have shown 480 bp of the mouse α subunit 5′-flanking DNA could target transgenic expression to both gonadotropes and thyrotropes.³⁰ A region from −225 to −200 that binds steroidogenic factor 1 appears to be critical for gonadotrope expression,³¹ but not for thyrotrope expression.³² Another important sequence involves the pituitary glycoprotein hormone basal element, extending from −342 to −329, that is critical for both thyrotrope and gonadotrope expression.³³ The element interacts with P-LIM, a pituitary-specific LIM-homeodomain transcription factor that is important for other pituitary cells.³⁴ Several sequences within the region from −480 to −300 appear to be important for mouse α subunit expression in thyrotropes but not gonadotropes.²⁹ Among these is the sequence from −434 to −421, which interacts with the developmental homeodomain transcription factor Msx1.³⁵ Other sequences within the 480 bp promoter have been found to interact with the pituitary-specific homeodomain factor Ptx-1, and a synergism between Ptx-1 and P-LIM, mediated by the co-activator C-LIM, has been described.³⁶ Recent studies with the mouse promoter in transgenic mice showed that an upstream DNA element located between −4.6 and −3.7 kb further enhanced expression in both thyrotropes and gonadotropes, and contained consensus binding sites for Gata, SF1, Sp1, ETS, and bHLH factors, which suggests cooperativity between factors binding both to proximal cis-acting elements and to the distal enhancer.³⁷

Transcription of TSH Subunit Genes

The DNA information contained in the TSHβ and α subunit genes is transcribed into a precursor RNA by a complex of enzymes, as directed by their respective promoters in the presence of both ubiquitous and specific transcription factors. The transcribed RNAs undergo a precise series of splicing events at the exon-intron junctions that lead to the production of the mature messenger RNA (mRNA) that then exits the nucleus and is translated to protein in the cytoplasm prior to posttranslational modification, subunit association, storage, and finally secretion. Transcription of the TSHβ and α subunit genes is coordinated under the influence of physiologic regulators, the most important of which are T₃ and TRH.

Translation of TSH Subunits

The next steps in TSH biosynthesis are summarized in Fig. 2-3.³⁸ The mRNAs for the TSHβ and α subunits are independently translated by ribosomes in the cytoplasm. The first peptide sequences consist of “signal” peptides of 20 amino acids for TSHβ and 24 amino acids for α.³⁹ These signal peptides are hydrophobic, allowing insertion through the lipid bilayer of the membrane of the rough endoplasmic reticulum. Translation into TSHβ and α pre-subunits continues into the lumen of the rough endoplasmic reticulum, and cleavage of the signal peptide occurs before translation is completed. This results in the formation of a 118 amino acid TSHβ subunit⁴⁰ and a 92 amino acid α subunit. Cleavage of TSHβ to a protein of 112 amino acids appears to be an artifact of purification. Synthesis of recombinant TSHβ subunit has resulted in two products of 112 and 118 amino acids, both of which are similarly active in vitro.⁴¹

Glycosylation of TSH

Glycosylation of TSH has a significant impact on its biological activity.⁴² The TSHβ subunit has a single glycosylation site, the asparagine residue at position 23, whereas the α subunit is glycosylated in two sites, the asparagine residues at positions 52 and 78⁴³ (see Fig. 2-3). Excess free α subunit is glycosylated at an additional site, the threonine residue at position 39.⁴⁴ This residue is located in a region believed to be important for combination with the TSHβ subunit. It is not known whether glycosylation at this residue is a regulated step that inhibits combination with the TSHβ subunit, or whether it occurs in excess free α subunits because this site is exposed.

Extensive studies on the processes of TSH subunit glycosylation have been carried out. Glycosylation of the TSHβ and α subunits begins before translation is completed (co-translational glycosylation), and addition of the second oligosaccharide in the α subunit occurs after translation is completed (posttranslational glycosylation). The first step in this process involves the assembly of a 14-residue oligosaccharide, (glucose)3-(mannose)9-(N-acetylglucosamine)2 on a dolichol-phosphate carrier. This oligosaccharide then is transferred to asparagine residues by the enzyme olygosaccharyl transferase, which recognizes the tripeptide sequence (asparagine)-(X)-(serine or threonine).⁴⁵ This mannose-rich oligosaccharide is progressively cleaved in the rough endoplasmic reticulum and Golgi apparatus. An intermediate with only six residues is produced and then other residues are added, resulting in complex oligosaccharides.⁴⁶ The residues added include N-acetylglucosamine, fucose, galactose, and N-acetylgalactosamine. Oligosaccharides before the six-residue intermediate are termed high mannose and are sensitive to endoglycosidase H, which releases the oligosaccharide from the protein, whereas the intermediate and complex oligosaccharides are endoglycosidase H resistant. Complex oligosaccharides usually consist of two branches (biantennary), but sometimes three or four branches are seen, as are hybrid oligosaccharides consisting of one complex and another high-mannose branch. Sulfation and sialation occur late in the pathway, within the distal Golgi apparatus. Sulfate is bound to N-acetylgalactosamine residues, and sialic acid, or its precursor N-acetylneuraminic acid, is bound to galactoside residues.⁴⁷ Thus, activation of the enzymes sulfotransferase and N-acetylgalactosamine transferase may involve important regulatory steps that affect the ratio of sulfate to sialic acid. As demonstrated with LH, it appears that sulfation increases and sialylation decreases the bioactivity of TSH,⁴⁷ because the exclusively sialylated recombinant glycoprotein produced in Chinese hamster ovary cells has been found to have attenuated activity in vitro.⁴⁸

Processing of complex oligosaccharides appears to occur at a slower rate for secreted glycoproteins, such as TSH, when compared with that for nonsecreted glycoproteins. For example, after an 11 minute pulse labeling with [³⁵S]methionine and a 30 minute chase, only a few α subunits were endoglycosidase H resistant, and only 76% reached this stage after an 18 hour chase.⁴⁹ Secretion was observed after a 60 minute chase, and the secreted products—TSH, free α subunit but no free β subunit—had mostly complex oligosaccharides associated with them.⁴³ It may be important to note that many of the studies described were carried out in thyrotropic tumor tissue obtained from hypothyroid mice, and glycosylation may differ in the euthyroid as compared with the hypothyroid state. In addition, differences between species have been noted, such as the human TSH may contain more sialic acid than the bovine TSH.⁴⁰

Folding, Combination, and Storage of TSH

Elucidation of the crystal structure of human CG (hCG)⁵⁰ allowed the construction of a model of human TSH, as supported by other evidence.^51,52 This model has greatly facilitated the interpretation of structure-function studies of the protein backbone. However, crystallization was achieved only with partly deglycosylated hCG, so it is likely that the conformation of the glycosylated protein may differ to some extent, although nuclear magnetic resonance studies suggest that the α subunit carbohydrate moieties project outward and may be freely mobile.⁵³ Nevertheless, this model predicts that the tertiary structure of each TSH subunit consists of two hairpin loops on one side of a central knot formed by three disulfide bonds and a long loop on the opposite side. In this tertiary structure, the glycoprotein hormones share features in common with transforming growth factor β, nerve growth factor, platelet-derived growth factor, vascular endothelial growth factor, inhibin, and activin, all of which are now grouped in the family of “cystine knot” growth factors.⁵⁴

Folding of nascent peptides begins before translation is completed. It has been shown that proper folding is dependent on glycosylation, because the drug tunicamycin, which prevents the initial oligosaccharide transfer to the asparagine residue, results in a peptide that does not fold properly and is degraded intracellularly.⁵⁵ Site-directed mutagenesis of a single glycosylation site also disrupted processing and decreased TSH secretion in transfected Chinese hamster ovary cells.⁵⁶ Folding is a critical step that allows correct internal disulfide bonding, which stabilizes the tertiary structure of the protein, allowing subunit combination.

Combination of TSH β and α subunits begins soon after translation is completed in the rough endoplasmic reticulum and continues in the Golgi apparatus.⁴³ Subunit combination then accelerates and modifies oligosaccharide processing of the α subunit.⁵⁷ In fact, studies have suggested that the conformation of the α subunit differs after combination with each type of β subunit,⁵⁸ and this may affect subsequent processing. The rate of combination of TSHβ and α subunits has been examined in mouse thyrotropic tumors. After a 20 minute pulse labeling with [³⁵S]methionine, 19% of TSHβ subunits were combined with α subunits, and this percentage increased to 61% after a additional 60 minute chase incubation.⁴³ Recent studies have shown that the combination of the TSHβ and α subunits, as is also the case with other glycoprotein hormones, occurs after “latching” of the disulfide “seatbelt” of the β subunit, with subsequent “threading” of loop 2 and the attached oligosaccharide of the α subunit beneath that “seatbelt.”⁵⁹

The sequence of the TSHβ subunit from amino acid 27 to 31 (CAGYC) is highly conserved among species and is thought to be important for combination with the α subunit. In a case of congenital hypothyroidism, a point mutation in the CAGYC region (see “Disorders of TSH Production”) results in the synthesis of altered TSHβ subunits that are unable to associate with α subunits, with consequent lack of intact TSH production.⁶⁰ A lack of free circulating TSHβ subunit was also observed, suggesting that combination with α subunit is necessary for TSHβ subunit secretion. This phenomenon was also demonstrated in studies where synthesis of wild-type recombinant TSHα subunit was carried out in the presence or absence of recombinant β subunit.⁶¹ Using site-directed mutagenesis, another study showed that a mutation at residue 25 in the glycosylation recognition site, which substitutes a serine for a threonine, does not alter glycosylation but decreases TSH production by 70%, possibly because of disruption of the nearby CAGYC region.⁶²

After TSH and free α subunit are processed in the distal Golgi apparatus, they are transported into secretory granules or vesicles.⁶³ The secretory granules constitute a regulated secretory pathway, mainly influenced by TRH and other hypothalamic factors. These granules contain mostly TSH, whereas free α subunit is contained in the secretory vesicles that constitute a nonregulated secretory pathway.

Ontogeny of TSH Levels

At 8 to 10 weeks of gestation in the human, TRH is measurable in the hypothalamus, with progressive increases in TRH levels until term. By 12 weeks of gestation, immunoreactive TSH cells are present in the human pituitary gland,⁶⁶ and TSH is detectable in the pituitary and the serum.^67,68 Serum and pituitary TSH levels remain low until week 18, when TSH levels increase rapidly, followed by increases in serum T₄ and T₃ concentrations. Fetal serum TSH and T₄ concentrations continue to increase between 20 and 40 weeks of gestation. Pituitary TSH begins to respond to exogenous TRH early in the third trimester, and negative feedback control of TSH secretion develops during the last half of gestation and the first 1 to 2 months of life.⁶⁹

An abrupt rise in serum TSH levels occurs within 30 minutes of birth in term infants. This is followed by an increase in serum T₃ concentrations within 4 hours and a lesser increase in T₄ levels within the first 24 to 36 hours. The initial increase in serum TSH levels appears to be stimulated by cooling in the extrauterine environment. Serum TSH levels fall to the adult range by 3 to 5 days after birth, and serum thyroid hormone levels stabilize by 1 to 2 months. Serum TSH levels in healthy premature infants (less than 37 weeks gestational age) are highly variable but tend to be lower at birth compared with those of term infants. TSH levels decrease slightly during the first week of life, followed by a gradual increase to normal term levels. Serum TSH levels are even lower in ill premature infants but rise toward normal levels during recovery.70,71

Patterns of TSH Secretion

TSH is secreted from the pituitary gland in a dual fashion, with secretory bursts (pulses) superimposed upon basal (apulsatile) secretion (Fig. 2-4, upper panel). Basal TSH secretion accounts for 30% to 40% of the total amount released into the circulation, and secretory bursts account for the remaining 60% to 70%. TSH pulses occur approximately every 2 to 3 hours, although considerable variability is noted among individuals.⁷² TSH pulses appear to directly stimulate T₃ secretion from the thyroid gland, as cross-correlation analysis has shown that a free T₃ peak occurs between 0.5 and 2.5 hours after a TSH peak. However, changes in free T₃ levels from nadir to peak are only 11% of mean free T₃ levels, probably because T₃ has a long serum half-life, and most T₃ does not arise from the thyroid gland.⁷³

In healthy euthyroid subjects, TSH is secreted in a circadian pattern, with nocturnal levels increasing to up to twice daytime levels⁷² (Fig. 2-4, upper panel). Peak TSH levels occur at between 2300 and 0500 hours in subjects with normal sleep-wake cycles, and nadir levels occur at about 1100 hours. The TSH circadian rhythm is not present in infants younger than 4 weeks old but emerges between 1 and 2 months of life and is well established in healthy children.⁷⁴ The circadian variation in TSH levels is due to increased mass of TSH secreted per burst at night, as well as slightly increased frequency of bursts and more rapid increase to maximal TSH secretion within a burst.⁷² The nocturnal increase in TSH levels can precede the onset of sleep, and sleep deprivation enhances TSH secretion. Therefore, in contrast to other pituitary hormones with a circadian variation, the nocturnal rise in TSH levels is not sleep entrained. Instead, a sleep-related inhibition of TSH release is of insufficient magnitude to counteract the nocturnal TSH surge.

Subjects with mild primary hypothyroidism retain the nocturnal rise in TSH levels, and patients with severe primary hypothyroidism have markedly increased TSH pulse amplitude throughout the day with loss of circadian variation in TSH levels (Fig. 2-4, middle panel).⁷² l-Thyroxine therapy reestablishes the normal TSH circadian variation. In contrast, patients with hypothalamic-pituitary causes of hypothyroidism secrete less TSH over a 24 hour period, with loss of the nocturnal TSH surge in pulse amplitude⁷⁵ (Fig. 2-4, lower panel). A similar pattern of reduced 24 hour TSH secretion occurs in critical illness.⁷⁶

The origin of pulsatile and circadian TSH secretion is not known. Thyroid hormones alter TSH pulse amplitude but have little effect on pulse frequency, and therefore are unlikely to participate in TSH pulse generation. The TSH pulse generator may reside in the hypothalamus, with TRH neurons acting in concert to stimulate a burst of TSH secretion from the pituitary gland. However, constant TRH infusions do not change TSH pulse frequency in humans, which casts doubt on this theory.⁷⁷ Dopamine suppresses TSH pulse amplitude but does not alter TSH pulse frequency, and therefore dopamine does not appear to control pulsatile TSH secretion. A diurnal variation in the activity of anterior pituitary 5′-monodeiodinase in the rat may control circadian TSH secretion.⁷⁸ However, this has not been confirmed in the human.

Physiologic serum cortisol levels may control circadian TSH secretion, although cortisol does not appear to affect TSH pulse frequency. When subjects with adrenal insufficiency were studied under conditions of glucocorticoid withdrawal, daytime TSH levels were increased, and the usual TSH circadian rhythm was abolished. When these subjects were given physiologic doses of hydrocortisone in a pattern that mimicked normal pulsatile and circadian cortisol secretion, daytime TSH levels were decreased, and the normal TSH circadian rhythm was reestablished. Hydrocortisone infusions at the same dose given as pulses of constant amplitude throughout the 24 hour period also decreased 24 hour TSH levels, but no circadian variation was noted.⁷⁹ Similarly, when healthy subjects were given metyrapone (an inhibitor of endogenous cortisol synthesis), TSH levels increased during the day, leading to abolition of the usual TSH circadian variation.⁸⁰ These data suggest that the normal early morning increase in endogenous serum cortisol levels decreases serum TSH levels and leads to the observed normal circadian variation in TSH.

Hypothalamic Regulation of TSHβ Subunit Biosynthesis

Thyrotropin-releasing hormone (TRH) is a tripeptide that is secreted from the hypothalamus and transported to the pituitary via the hypothalamic-hypophyseal portal system; it is a major activator of TSH production with a significant three- to fivefold increase in the transcription of both TSHβ and α subunit mRNAs.⁸¹ TRH from maternal or fetal sources is not required for normal thyrotrope development during ontogeny, and TRH-deficient mice are not hypothyroid at birth. However, TRH is required for the postnatal maintenance of TSH activation.⁸²

TRH binding to its receptor initiates a cascade of intracellular events. In GH₃ cells, the TRH-receptor complex interacts with a guanine nucleotide binding regulatory protein (G) that then binds and activates GTP(G′). G′ binds to phospholipase C (C) and activates it (C′). C′ catalyzes the hydrolysis of phosphatidylinositol 4,5 bisphosphate, which results in the formation of two intracellular “second messengers,” inositol triphosphate (InsP₃) and 1,2-diacylglycerol (1,2-DG). InsP₃ diffuses from the cell-surface membrane to the endoplasmic reticulum, where it causes the release of sequestered Ca²⁺. This activates the movement of secretory granules to the cell surface and their exocytosis. Simultaneous with these events is a parallel activation of protein kinase C by 1,2-DG, which also leads to phosphorylation of proteins involved in exocytosis. TRH has been shown to stimulate a nuclear protein, Islet-brain-1 (IB1)/JIP-1, in the anterior pituitary gland and in cultured rat GH₃ cells⁸³ and has been implicated in the action of TRH in stimulating the TSHβ gene in thyrotropes. Studies in somatomammotrope cells, where TRH stimulates prolactin production, have suggested that phosphatidylinositol, protein kinase C, and calcium-dependent pathways may be involved,⁸⁴ although TRH stimulation of the TSHβ subunit promoter may be mediated by AP1.⁸⁵

Two TRH-response regions are located from −128 to −61 and from −28 to +8 of the human TSHβ promoter.⁸⁶ The upstream region contains binding sites for the pituitary-specific transcription factor, Pit-1, suggesting a role for this or a similar factor in the regulation of the TSHβ subunit gene by TRH. In the rat TSHβ subunit gene, responsiveness to TRH has been localized to regions upstream of −204, where Pit-1 binding sites are also found.⁸⁷ Furthermore, it has been shown that both protein kinase C and protein kinase A pathways can phosphorylate Pit-1 at two sites in response to phorbol esters and cyclic adenosine monophosphate (cAMP),⁸⁸ thus altering the binding to Pit-1 transactivation elements on the human TSHβ gene.⁸⁹

Dopamine acting via DA2 dopamine receptors inhibits TSHα and β subunit gene transcription by decreasing the intracellular levels of cAMP.⁸¹ Studies of the TSHβ subunit gene have localized two regions of the promoter necessary for cAMP stimulation, from −128 to −61 bp and from +3 to +8 bp. The upstream region coincides with the TRH-responsive region and contains Pit-1 binding sites. The downstream region resides within the regions responsive to T₃ (+3 to +37) and TRH (−28 to +8). The downstream region also overlaps with an AP1 binding site (−1 to +6). The sequence from −1 to +6 appears to cooperate with Pit-1 in mediating responses to cAMP and TRH.⁸⁵ Thus, multiple interactions between transcription factors and hormonal regulators appear to converge on sequences close to the transcriptional start site.

Peripheral Regulation of TSHβ Subunit Biosynthesis

Thyroid hormone is thought to act predominantly through a classical TR-mediated genomic model. T₄ serves as a minimally active prohormone that is converted into a metabolically active T₃ via a family of tissue deiodinases, termed D1, D2, and D3. These selenoprotein enzymes are membrane bound and can activate or inactivate substrate in a time- and tissue-specific manner.⁹⁰ D2 is the major T₄-activating deiodinase. It is present on the endoplasmic reticulum close to the nucleus and produces 3,5,3′-triiodothyronine (T₃) through the removal of an iodine residue from the outer ring of thyroxine. D2 activity is rapidly lost in the presence of its substrate T₄ by a ubiquitin proteasomal mechanism.⁹¹ Rat pituitary thyrotropes coexpress D2 RNA and protein, and both are increased in hypothyroidism. Murine thyrotropes in TtT-97 tumors or the TαT1 cell line have extremely high levels of D2, which accounts for the sustained production of T₃ by thyrotropes even in the presence of supraphysiologic T₄ levels. Serum TSH levels in normal mice are suppressed by administration of T₄ or T₃, although only T₃ was effective in the mouse with targeted disruption of the D2 gene. The observed phenotype of pituitary resistance to T₄ demonstrated the critical importance of D2 in controlling negative thyroid hormone regulation of TSH in thyrotropes.

T₄ can also act, in some cases, via nongenomic mechanisms that do not involve classical nuclear TR mechanisms. T₄ can bind to a cell-surface integrin αVβ3 receptor followed by activation of a mitogen-activated protein kinase cascade, which transduces the signal into a complex series of cellular and nuclear actions. These nongenomic hormone actions are likely to be contributors to basal rate setting of transcription of some genes, and they may control complex cellular events.⁹²

TSHβ and α subunit gene transcription rates are markedly inhibited by treatment with triiodothyronine (T₃). Studies using mouse TtT-97 thyrotropic tumors have demonstrated that suppression of TSHβ and α subunit mRNA transcription rates measured by nuclear run-on assays is evident by 30 minutes after treatment and is maximal by 4 hours.⁹³ This effect was seen in the presence of the protein synthesis inhibitor cycloheximide, indicating that it did not require an intermediary protein. Other studies using mouse and rat pituitaries along with mouse thyrotropic tumors have demonstrated that steady-state mRNA levels of TSHβ and α subunits are dramatically decreased by T₃⁹⁴ (Fig. 2-5). The mechanism of action of T₃ involves interaction with nuclear receptors that act mainly at the transcriptional level. The transcriptional response to T₃ is proportional to the nuclear receptor occupancy,⁹⁵ and the time course of T₃ nuclear binding and that of transcriptional inhibition are also in agreement⁹⁶ (see Fig. 2-5).

The T₃ inhibitory effect on the TSHβ gene requires ligand-occupied T₃ receptor (TR), specifically the TRβ1 or TRβ2 isoform, because patients with thyroid hormone resistance and inappropriate secretion of TSH have abnormalities only in the TRβ, not the TRα, gene.⁹⁷ TRβ interacts with specific cis-acting DNA sequences close to the transcriptional start. T₃ response elements have been reported to be located between +3 and +37 of the human TSHβ gene.⁹⁸ Two T₃ receptor binding sites, from +3 to +13 and from +28 to +37, may mediate T₃ inhibition. T₃ responses can be mediated through receptor monomers, homodimers, or heterodimers involving retinoid X receptors (RXR).⁹⁹ An RXR selective ligand was shown in vitro to inhibit TSHβ expression in TtT-97 thyrotropic tumor cells¹⁰⁰ and in cultured TαT1 thyrotropes.¹⁰¹ This finding has been confirmed in vivo and resulted in central hypothyroidism (low T₄ and low TSH) in cancer patients treated with the retinoid bexarotene.¹⁰² The RXR-selective retinoid (LG 268) decreased circulating TSH and T₄ levels in mice with marked lowering of pituitary TSHβ mRNA without decreasing TRH, suggesting a direct effect on thyrotropes.¹⁰¹

Other, more recent studies have disputed the requirement of the negative response element located in exon 1 of the human gene, because its deletion did not eliminate T₃ suppression of TSHβ promoter activity in a reconstitution system.¹⁰³ These studies showed that liganded TRβ can associate with Gata2 in vitro and in vivo via direct interaction between the zinc fingers of Gata2 and the DNA binding domain of TRβ. In addition, T₃-occupied TR can physically interact with TRAP220.²⁴ Thus, it is likely that interference with the transactivation function of the Pit-1/Gata2/TRAP220 complex on the proximal TSHβ promoter plays an important role in T₃ negative regulation.

Abundant information exists as to the mechanisms involved in gene stimulation by T₃. Generally, TRs bind to cis-acting DNA response elements (TRE) in the absence of ligand, interact with a family of nuclear receptor corepressor molecules that recruit histone deacetylases, and locally modify chromatin structure to result in repression of the target gene.¹⁰⁴ In the presence of T₃, the corepressor complexes rapidly dissociate and are replaced by coactivator complexes that bind to TRs and increase histone methylation and acetylation locally on the chromosomal DNA, which unwinds the chromatin into an open configuration.¹⁰⁵ Other activating transcription factors such as TRAP220 are then recruited to the TR via protein-protein interactions, which then activate RNA polymerase II–mediated transcription.

In contrast, the molecular mechanisms involved in negative T₃ regulation, such as the TSH subunit genes, have not been well characterized. Liganded TRβ has been reported to recruit histone deacetylase 3 and reduce histone H4 acetylation that modifies histones, resulting in a fully repressed chromosomal state of the TSHβ gene.¹⁰³ Several recent studies have demonstrated the requirement of an intact DNA binding domain of TRβ in the negative regulation of the TSHβ gene in vitro¹⁰⁶ and in vivo.¹⁰⁷ In one study, a combination of Pit-1 and Gata2 activated a human TSHβ (−128/+37) reporter construct along with vectors containing TRβ1 constructs in the absence or presence of T₃. These investigators found that unliganded TRβ1 did not stimulate promoter activity, whereas a mutation lacking the N-terminus and DNA binding domain of TRβ1 lost the ability of T₃-treated cells to negatively regulate TSHβ promoter activity. This demonstrated the importance of various modular domains that constitute the molecular structure of TRs. Moreover, using a gene targeting approach in transgenic mice and replacing the wild-type TRβ gene with a mutant that abolished DNA binding in vitro did not alter ligand and cofactor interactions.¹⁰⁷ Homozygous mutant mice demonstrated central thyroid hormone resistance with 20-fold higher serum TSH in the face of two- to threefold higher T₃ and T₄ levels, which were similar to those of TRβ homozygous null mice.

Although thyrotrope cells contain all TRs—TRα1, TRβ1, and TRβ2, as well as non-T₃ binding variant α2¹⁰⁸—it is TRβ2 that is expressed predominantly in the pituitary and in T₃-responsive TRH neurons¹⁰⁹ and is most critical for the regulation of TSH.¹¹⁰ Moreover, TRβ2-deficient mice had a phenotype consistent with pituitary resistance to thyroid hormone, with increased TSH and thyroid hormone levels, even in the presence of TRβ1 and TRα1, showing the lack of compensation between TR isoforms.¹¹¹ However, TRβ1 and TRα1 still may play a role, in that they are able to form heterodimers with TRβ2. Heterodimers of a TR and a TR accessory protein, such as RXR, may also bind to DNA,¹¹² constituting heterodimeric complexes that may have different affinities for specific DNA sequences and different functional activities. A particular RXR isoform, RXRγ1, is uniquely expressed in thyrotropes and appears to mediate the inhibition by 9-cis-retinoic acid through a region extending from −200 to −149 of the mouse TSHβ subunit promoter, an area upstream and distinct from that mediating negative regulation by thyroid hormone.¹⁰⁰ Other proteins that interact with TR include the coactivators, such as the glucocorticoid receptor interacting protein-1 (GRIP-1) and the steroid receptor coactivator-1 (SRC-1),¹¹³ and corepressors, such as the silencing mediator for retinoid receptors and thyroid hormone receptors (SMRT) and the nuclear receptor corepressor (NCoR).¹¹⁴ These coactivators and corepressors modulate the effects of many members of the steroid-thyroid hormone receptor superfamily. Their role in the regulation of TSH subunit promoters by thyroid hormone remains to be elucidated in detail.

Studies with genetic knockout mouse models in which both TRH and TRβ genes were removed have recently showed an unexpected dominant role for TRH in vivo in regulating the hypothalamic-pituitary-thyroid axis. It appears that the presence of both TRβ and TRH is necessary for a normal thyrotroph response during hypothyroidism, suggesting that unliganded TRβ stimulates TSH subunit gene expression.¹¹⁵

Posttranscriptional effects of T₃ have also been described. T₃ decreases the half-life of TSHβ subunit mRNA and decreases the size of the poly(A) tail.¹¹⁶ The shortening of the poly(A) tail is thought to cause mRNA instability. Leedman et al. showed that T₃ increased the binding of an RNA-binding protein present in rat pituitary to the 3′-untranslated region of the rat TSHβ mRNA¹¹⁷ and also induced a shortening of the poly(A) tail of the mouse TSHβ mRNA from 160 to 30 nucleotides.¹¹⁸

Steroid hormones, specifically glucocorticoids, inhibit TSH production, but TSH subunit mRNA levels do not change significantly.¹¹⁹ Their major effect may be seen at the secretory level. Estrogens mildly reduce both α and TSHβ subunit mRNA in hypothyroid rats compared with euthyroid controls.¹²⁰ In this study, estrogen also abolished the early rise in subunit mRNA levels seen following T₃ replacement. Testosterone has been shown to increase TSHβ subunit mRNA in castrate rat pituitary and mouse thyrotropic tumor.¹²¹

Leptin and neuropeptide Y have opposite effects on TSH biosynthesis. Leptin is the product of the ob gene, which is found mainly in adipose tissue and regulates body weight and energy expenditure.¹²² Neuropeptide Y (NPY) is a neuropeptide that is synthesized in the arcuate nucleus of the hypothalamus and plays many roles in neuroendocrine function.¹²³ In dispersed rat pituitary cells, leptin stimulated and NPY inhibited TSHβ mRNA levels in a dose-related manner.¹²⁴ In contrast, both agents increased α subunit steady state mRNA levels.

Hypothalamic Regulation of α Subunit Biosynthesis

TRH stimulates α subunit biosynthesis through a novel mechanism. A CRE-binding protein that binds to the region from −151 to −135 of the human α subunit promoter appears to be important for TRH regulation, as well as a Pit-1–like protein that binds to a more distal region from −223 to −190.¹²⁵ The CRE of the human glycoprotein hormone α subunit gene promoter consists of an 18 bp repeat and extends from −146 to −111.¹²⁶ The mechanisms involved in TRH stimulation of the α subunit gene appear to involve two transcription factors, P-Lim and CREB binding protein (CBP). When stimulated with TRH, both of these factors transcriptionally cooperate to activate α subunit promoter activity caused by direct protein-protein interactions.¹²⁷ Both of these factors synergistically activated the α subunit gene promoter during TRH stimulation and interact in a TRH-dependent manner. P-Lim binds to the α subunit promoter directly, but CBP does not possess a DNA binding domain, so it must be recruited to the promoter via interaction with another factor. The P-Lim/CBP binding is formed in a TRH signaling-specific manner, in contrast to forskolin, which mimics protein kinase A signaling and dissociates both the binding and the transcriptional synergy. α subunit gene expression in thyrotropes is inhibited by dopamine in coordination with expression of the TSHβ subunit gene. Its action is mediated by decreases in intracellular cAMP levels.

Peripheral Regulation of α Subunit Biosynthesis

Thyroid hormone inhibition of α subunit gene transcription is observed in thyotropes in coordination with that of the TSH β subunit. The T₃ response element of the human α subunit gene promoter has been reported to be located from −22 to −7.¹²⁸ Similar to the TSHβ subunit gene, the T₃ response elements of human and of mouse¹²⁹ and rat¹³⁰ genes are located close to the transcriptional start. T₃ inhibition may be mediated by different isoforms of the T₃ receptor¹³¹ in combination with the corepressors SMRT and NCoR.¹³² Studies have suggested that mutations of the T₃ response element of the human α subunit promoter that eliminate TR binding do not abrogate the inhibitory effect of T₃, suggesting that protein-protein interactions may be more important than protein-DNA binding.¹³³

Steroid hormone regulation of α subunit gene transcription is probably of limited importance. Androgen inhibition and androgen receptor (AR) binding have been localized to a region from −120 to −100. Negative regulation by estrogen was described in the gonadotropes of transgenic mice expressing a reporter gene under the control of both human and bovine promoters, but no binding of these regions to the estrogen receptor (ER) was detected, suggesting an indirect effect.¹³⁴ However, other studies using rat somatomammotropes have found positive regulation by estrogen localized to the proximal 98 bp of 5′-flanking DNA of the human α subunit gene and binding of the ER to the T₃ response element from −22 to −7.¹³⁵ Transcriptional inhibition by glucocorticoids may be mediated by binding of the glucocorticoid receptor to sequences between −122 and −93 of the human α subunit gene.¹³⁶ However, no direct binding was detected in other studies, suggesting that the GR inhibits transcription by interfering with other transactivating proteins.¹³⁷

Regulation of TSH Glycosylation

Glycosylation is a regulated process that is primarily modulated by TRH and thyroid hormone.¹³⁸ Primary hypothyroidism¹³⁹ and TRH administration¹⁴⁰ have been found to increase oligosaccharide addition, which results in an increased bioactivity of TSH.¹⁴¹ The same was noted in patients with resistance to thyroid hormone. TSH glycosylation patterns were found to differ in several pathologic states such as central hypothyroidism, TSH-producing pituitary adenomas, and euthyroid sick syndrome.¹⁴² Also observed were changes in the sulfation and sialylation of the oligosaccharide residues, which modulate bioactivity.^139,143,144 Recently, thyroid hormone was shown to increase TSH bioactivity, and this was correlated with decreased sialylation.¹⁴⁵

Hypothalamic Regulation of TSH Secretion

TRH directly affects TSH secretion in vivo and in vitro at concentrations that exist in the pituitary portal blood.¹⁴⁶ Immunoneutralization of TRH in animals leads to a decline in thyroid function,¹⁴⁷ and TRH knockout mice have a reduced postnatal TSH surge, followed by impaired baseline thyroid function with a poor TSH response to hypothyroidism. Lesions of the paraventricular nucleus (PVN) decrease circulating TRH and TSH levels in normal or hypothyroid animals and cause hypothyroidism¹⁴⁸ and electrical stimulation of this area causes TSH release. Although baseline levels of TSH are reduced in animals with lesions of the PVN, TSH levels still show appropriate responses to changes in circulating thyroid hormone levels. Thus, TRH likely determines the set point of feedback control by thyroid hormones.

Acute intravenous administration of TRH to human subjects causes a dose-related release of TSH from the pituitary. This occurs within 5 minutes and is maximal at 20 to 30 minutes. Serum TSH levels return to basal levels by 2 hours. More prolonged (2 to 4 hour) infusions of TRH lead to biphasic increases in serum TSH levels in humans and animals.¹⁴⁹ The early phase may reflect release of stored TSH, and the later phase may reflect release of newly synthesized TSH. Interpretation of TSH responses to even more prolonged TRH infusions is complicated by the increase in serum T₃ levels, with feedback to suppress further TSH release.⁷⁷ Continuous TRH administration in vitro also causes desensitization of TSH responses, which may further explain decreased TSH levels with long-term TRH exposure.¹⁵⁰

Somatostatin (SS) in humans and animals inhibits basal and TRH-stimulated TSH secretion in vivo and in vitro at concentrations that exist in the pituitary portal blood.¹⁵¹ In the hypothalamus, the highest concentrations of SS occur in the anterior paraventricular region.¹⁵² From this region, axonal processes of SS-containing neurons project to the median eminence. Animals that have undergone sectioning of these fibers have depletion of SS content of the median eminence and increased serum TSH levels.¹⁵³ Similarly, immunoneutralization of SS in animals increases basal TSH levels and TSH responses to TRH.¹⁵⁴ In humans, SS infusions suppress TSH pulse amplitude, slightly decrease TSH pulse frequency, and abolish the nocturnal TSH surge.¹⁵⁵ Thus, TSH secretion probably is regulated through a simultaneous dual-control system of TRH stimulation and SS inhibition from the hypothalamus.

SS binds to specific, high-affinity receptors in the anterior pituitary gland. Five subtypes of the SS receptor (SST1 through SST5) have been identified,¹⁵⁶ and SST1 and SST5 have been localized to thyrotropes.¹⁵⁷ Binding of SS to its receptor inhibits adenylate cyclase via the inhibitory subunit of the guanine nucleotide regulatory protein, which lowers protein kinase A activity¹⁵⁶ and decreases TSH secretion. SS may also exert some effects by cAMP-independent actions on intracellular calcium levels. Hypothyroidism reduces the efficacy of SS in decreasing TSH secretion in vitro, which is reversed by thyroid hormone administration.¹⁵⁸ Additional studies in mouse thyrotropic tumors indicate that both SST1 and SST5 are markedly downregulated in hypothyroidism and are induced by thyroid hormone.¹⁵⁷ Although short-term infusions of SS lead to pronounced suppression of TSH secretion in humans, long-term treatment with SS or its analogues does not cause hypothyroidism.¹⁵⁹ This probably reflects compensatory mechanisms in the thyroid hormone feedback loop. GH deficiency is associated with increased TSH responses to TRH, and GH administration decreases basal and TRH-stimulated TSH secretion,¹⁶⁰ possibly owing to GH stimulation of hypothalamic SS release.

Dopamine also inhibits basal and TRH-stimulated TSH secretion in vivo and in vitro at concentrations that exist in the pituitary portal blood.161,162 Exogenous dopamine antagonists, including those that do not penetrate the blood-brain barrier, increase TSH levels.¹⁶³ In humans, dopamine infusions rapidly suppress TSH pulse amplitude, do not affect TSH pulse frequency, and abolish the nocturnal TSH surge¹⁵⁵; administration of a dopamine antagonist has the opposite effect.¹⁶⁴ Dopamine also has direct effects on hypothalamic hormone secretion that may indirectly affect TSH secretion. For example, dopamine and dopamine-agonist drugs stimulate both TRH and SS release from rat hypothalami.¹⁶⁵

In the hypothalamus, dopamine is secreted by neurons in the arcuate nucleus.¹⁶⁶ From the arcuate nucleus, neuronal processes project to the median eminence. Dopamine acts by binding to type 2 dopamine receptors (DA₂) on thyrotrope cells.¹⁶⁷ This leads to inhibition of adenylate cyclase, which decreases the synthesis and secretion of TSH. In addition, TSH may downregulate its own release through the induction of DA₂ receptors on thyrotrope cells.¹⁶⁸ The inhibitory effects of dopamine on TSH secretion vary according to sex steroids, body mass, and thyroid status. Dopamine antagonist drugs cause greater increases in serum TSH levels in women than in men. Recent studies show that obesity is associated with enhanced TSH secretion, which may be mediated via blunted central dopaminergic tone.¹⁶⁹ Dopamine inhibition of TSH release is greater in patients with mild hypothyroidism than in normal subjects, although subjects with severe hypothyroidism may be less responsive.¹⁷⁰ Although short-term infusions of dopamine lead to pronounced suppression of TSH secretion, long-term treatment with dopamine agonists does not cause hypothyroidism. This probably reflects compensatory mechanisms in the thyroid hormone feedback loop.

Adrenergic effects have also been reported in vivo and in vitro. α-Adrenergic activation stimulates TSH release directly from the rat pituitary gland at physiologic concentrations of catecholamines.¹⁷¹ α-Adrenergic agonists stimulate TSH release in rats, and blockade of norepinephrine synthesis or treatment with adrenergic receptor blockers decreases TSH levels.^166,172 It is unclear whether these effects are mediated via changes in TRH and/or SS levels. In humans, data regarding adrenergic effects on TSH secretion are limited. α-Adrenergic blockade diminishes serum TSH responses to TRH.¹⁷³ However, administration of epinephrine does not alter TRH-stimulated TSH secretion.¹⁷⁴ These data suggest that endogenous adrenergic pathways do not have a major role in TSH secretion. Noradrenergic stimulation of TSH secretion is mediated by high-affinity α₁-adrenoreceptors linked to adenylate cyclase.¹⁷³ Therefore, dopamine and epinephrine appear to exert opposing actions on thyrotropes through opposite effects on cAMP generation.

Opioid administration to rats suppresses basal or stimulated TSH levels, and the opioid receptor antagonist naloxone reverses these effects.¹⁷⁵ Acute opiate administration in humans may slightly stimulate TSH levels, and acute naloxone administration has little effect.¹⁷⁶ In contrast to these acute studies, when naloxone is given over 24 hours, 24 hour TSH secretion decreases, primarily owing to a decrease in nocturnal TSH pulse amplitude.¹⁷⁷ TSH responses to TRH are also decreased. Serum T₃ levels are decreased as well, suggesting that the magnitude of TSH suppression is sufficient to affect thyroid gland function. These findings suggest that endogenous opioids may play a role in tonic stimulation of TSH secretion.

Peripheral Regulation of TSH Secretion

Thyroid hormone directly blocks pituitary secretion of TSH.¹⁷⁸ Acute administration of T₃ suppresses TSH levels within hours, and chronic administration leads to further suppression. Slight changes in serum thyroid hormone levels within the normal range alter basal and TRH-stimulated TSH levels, confirming the sensitivity of the pituitary gland to thyroid hormone feedback. Thyroid hormones alter tonic TSH secretion and TSH pulse amplitude without affecting pulse frequency, because subjects with primary hypothyroidism have a near-normal number of TSH pulses, and T₄ replacement leads to a decrease in TSH pulse amplitude without much change in pulse frequency.⁷²

In addition to direct effects on TSH secretion, thyroid hormones have other actions that affect TSH secretion. At the pituitary level, thyroid hormones decrease the number of TRH receptors¹⁷⁹ and stimulate the activity of the pituitary TRH-degrading enzymes,¹⁸⁰ which act in concert to decrease TRH stimulation of TSH secretion. At the hypothalamic level, TRH mRNA levels in the PVN are increased in hypothyroidism and are reduced by T₃ and T₄.¹⁸¹ Hypothalamic SS content is decreased in hypothyroid rats and is restored by T₃ treatment. Finally, T₃ directly stimulates SS release from hypothalamic tissue.¹⁸² These combined effects of thyroid hormones on TRH and SS decrease TRH release from the hypothalamus and indirectly decrease TSH secretion.

Glucocorticoids at pharmacologic doses or high endogenous cortisol levels (Cushing’s syndrome) suppress basal TSH levels, blunt TSH responses to TRH, and diminish the nocturnal TSH surge in humans and animals.183,184 In healthy subjects, infusions of hydrocortisone that increase serum cortisol to levels seen in mild to moderate stress suppress 24 hour TSH secretion.^185,186 Glucocorticoid-induced changes in TSH levels are due to decreased TSH pulse amplitude without alteration in TSH pulse frequency, with more profound suppression of nocturnal TSH secretion and abolition of the TSH surge.

Physiologic glucocorticoid levels also affect TSH secretion.79,80 Untreated patients with adrenocortical insufficiency can have elevated serum TSH levels that resolve with steroid replacement. Complimentary studies of metyrapone (an inhibitor of cortisol synthesis) administration to healthy subjects confirm that endogenous cortisol levels suppress TSH secretion, and physiologic hydrocortisone replacement in patients with adrenal insufficiency decreases daytime TSH levels back to those seen in healthy subjects.

Glucocorticoid suppression of TSH levels may occur directly at the pituitary gland. Animal studies suggest that glucocorticoids exert direct effects on thyrotropes to impair TSH secretion, although these appear to be highly dependent on dose and time course of administration.187,188 It does not appear that glucocorticoids directly affect TSH gene transcription. In humans, TSH pulse frequency is maintained during glucorticoid administration, TSH pulse amplitude is reduced, and TSH responses to exogenous TRH are attenuated, suggesting a direct effect on TSH secretion. In addition to direct pituitary effects, it appears that glucocorticoids may have hypothalamic actions that affect TSH levels. Dexamethasone increases hypothalamic TRH levels, and circulating TRH levels are decreased.¹⁸⁹ Reports that the proposed consensus sequence for glucocorticoid receptor binding is present in the 5′-flanking region of the TRH gene also support these data.¹⁹⁰

Patients with Cushing’s syndrome or subjects receiving prolonged courses of glucocorticoids may have low serum T₄ and TSH levels. Whether such patients have true hypothyroidism, or whether they should be treated with thyroid hormone, is unclear; however, patients with acute or chronic illnesses and similar abnormalities in thyroid hormone levels do not appear to benefit from thyroid hormone therapy.

Leptin is primarily a product of adipocytes, although it is also located in thyrotrophs. It regulates food intake and energy expenditure, decreasing acutely with fasting in animals and humans.191–193 Exogenous leptin administration to fed rats raises serum TSH levels, probably by increasing TRH gene expression and TRH release. Similarly, leptin administration to fasted rats or humans reverses fasting-induced decrements in TSH levels, also by increasing TRH gene expression and release. This suggests that fasting-related reductions in leptin levels play a role in suppressing TSH secretion. However, immunoneutralization of leptin increases TSH levels; therefore endogenous leptin may inhibit TSH release, at least in rats. In healthy subjects, leptin and TSH have coordinated pulsatility and circadian rhythms in plasma, with a nadir in the late morning and a peak in the early morning; however, leptin-deficient subjects have disorganized pulsatile and circadian rhythms.

Sex steroids may account for higher serum and pituitary TSH concentrations in male compared with female rats. In addition, TSH content is reduced by castration and is restored by androgen administration.¹²¹ Testosterone administration to castrated male or female rats increases basal and TRH-stimulated serum TSH levels.¹⁹⁴ In contrast, androgen administration to intact female rats does not alter serum or pituitary levels of TSH.¹⁹⁵ Estrogen administration to euthyroid rats does not alter serum TSH levels. In euthyroid humans, most studies suggest that changes in endogenous or exogenous sex steroid levels do not affect basal or TRH-stimulated TSH levels.¹⁹⁶ No significant gender difference in the basal mean and pulsatile secretion of TSH or in the TSH response to TRH has been noted.¹⁹⁷ Therefore, sex steroids do not appear to play a major regulatory role in TSH secretion in humans.

Cytokines are circulating mediators of the inflammatory response that are produced by many cells and have systemic effects on the hypothalamic-pituitary-thyroid axis. Administration of tumor necrosis factor (TNF) or interleukin-6 (IL-6) decreases serum TSH levels in healthy human subjects, and TNF and interleukin-1 (IL-1) decrease TSH levels in animals.198–200 Administration of some of these cytokines may mimic the alterations in thyroid hormone and TSH levels seen in acute nonthyroidal illness. In rats, TNF reduces hypothalamic TRH content and pituitary TSH gene transcription. IL-1 stimulates type II 5′-deiodinase activity in rat brain, which may decrease TSH secretion by increasing intrapituitary T₃ levels.

Autocrine and paracrine peptides may alter regulatory pathways within the pituitary gland for TSH secretion, acting in concert with the central and peripheral factors described above.²⁰¹ Peptides that have been implicated in this role include neurotensin, opioid-related peptides, galanin, substance P, epidermal growth factor (EGF), fibroblast growth factor (FGF), IL-1, and IL-6. Of particular interest is neuromedin B, a mammalian peptide that is structurally and functionally related to the amphibian peptide bombesin.²⁰² Neuromedin B is present in high concentrations in thyrotrope cells, with levels that change according to thyroid status. Administration of neuromedin B to rodents decreases TSH levels, and intrathecal administration of neuromedin B antiserum increases TSH levels. Therefore, neuromedin B appears to act as an autocrine factor that exerts a tonic inhibitory effect on TSH secretion. Additional data suggest that neuromedin B may modulate the action of other TSH secretagogues and release inhibitors, including TRH and thyroid hormones.

Determinants of TSH Binding

Specificity of TSH binding is conferred by the TSH β subunit. It appears that amino acid residues from 58 to 69, within the βL3 loop, and from 88 to 105, in the “seatbelt” region of the TSH β subunit,²⁰⁴ play an important role in binding to and activating the TSH receptor. The carboxylterminal end of the TSH β subunit contains multiple lysine residues (positions 101, 107, and 110) and a cysteine at position 105, all of which are critical for the ability to bind to the receptor.²⁰⁵ Congenital hypothyroidism due to biologically inactive TSH was found to result from a frameshift mutation with loss of β-cysteine105²⁰⁶ (see “Disorders of TSH Production”). Several regions of the α subunit are also important for TSH activity, particularly the residues α11-20 and α88-92.^51,56,207 In addition, the oligosaccharide chain at position α-asparagine52 plays an important role in both binding affinity and receptor activation. A mutant TSH lacking the α-asparagine52 oligosaccharide showed increased in vitro activity, although this same mutation had the opposite effect on CG binding to its native receptor.⁵⁶ However, such a mutation also increased TSH clearance and this decreased in vivo activity. In addition, the oligosaccharide chains on the TSH subunits are critically important for signal transduction.^42,208 In this regard, the α subunit oligosaccharides are important for all the pathways activated by the receptor, whereas the TSHβ subunit oligosaccharide influences only the adenylate cyclase pathway.²⁰⁹ The mechanism by which the oligosaccharides influence signal transduction is not known. A model for the action of the glycoprotein hormones has been proposed that suggests a role for the oligosaccharides in directly modulating the influx of calcium into the target cell.²¹⁰

Effects of TSH on the TSH Receptor

TSH has been shown to inhibit the expression of the rat TSH receptor by stimulating cAMP production.²¹¹ TSH receptor desensitization also has been described both in vivo and in vitro, whereby prior TSH stimulation leads to a 30% to 70% decrease in the subsequent cAMP response to TSH stimulation.²¹² Recent studies using recombinant TSH receptor have shown that desensitization does not occur when the receptor is expressed in nonthyroidal cells, suggesting that this phenomenon requires a cell-specific factor.²¹³

Actions of TSH on the Thyroid Gland

The effects of TSH on the thyroid gland include changes in thyroid gland growth, cell morphology, iodine metabolism, and synthesis of thyroid hormone. The TSH receptor is coupled to the G_s protein cascade, and binding of TSH activates adenylate cyclase to produce cAMP.214,215 The G_q/phospholipase C/inositol phosphates/Ca²⁺ pathway is also activated²¹⁶ and appears to play a role, particularly in regulating iodination.²¹⁷ TSH is also able to signal through the JAK/STAT²¹⁸ and mTOR/S6K1²¹⁹ pathways, with important roles in thyroid cell growth. The end point of TSH action is the production of thyroid hormone by the thyroid gland. The process begins with thyroglobulin gene transcription, which in itself is able to occur independently of TSH.²²⁰ However, the transcriptional rate and possibly the mRNA stability are increased by TSH.²²¹ TSH regulates the expression and activation of Rab5a and Rab7, which are rate-limiting catalysts of thyroglobulin internalization and transfer to lysosomes.²²² TSH stimulates iodide uptake and organification, then acts on the iodinated thyroglogulin stored in the luminal colloid and stimulates its hydrolysis, resulting in release of the constituent amino acids, including the iodotyronines T₃ and T₄.

Extrathyroidal Actions of TSH

The ocurrence of precocious puberty in cases of severe juvenile primary hypothyroidism²²³ has suggested that high levels of TSH are able to cross-activate the gonadotropin receptors. This interaction has now been demonstrated using recombinant human TSH, which has been found to be capable of activating the FSH²²⁴ but not the CG/LH receptor.²²⁵

Expression of thyrotropin receptor has been reported in the brain²²⁶ and pituitary gland.²²⁷ In the brain, both astrocytes and neuronal cells were found to express TSH receptor mRNA and protein²²⁶ and to stimulate arachidonic acid release and type II 5′-iodothyronine-deiodinase activity.²²⁸ In the pituitary gland, the TSH receptor was localized to folliculo-stellate cells and may be involved in paracrine feedback inhibition of TSH secretion, which also may occur in response to TSH receptor autoantibodies.²²⁷

Expression of both TSH and its receptor has been reported in lymphocytes²²⁹ and erythrocytes,²³⁰ and this has led to speculation that TSH may have other nonclassic functions. The TSH receptor also was found to be expressed in adipocytes²³¹ and to stimulate proliferation and inhibit differentiation of preadipocytes in vitro.²³² TSH activated the nuclear factor-κB (NF-κB) pathway to induce the release of interleukin-6 (IL-6).²³³ Direct effects of TSH were described in bone,²³⁴ where it modulates tumor necrosis factor α (TNFα)²³⁵ and receptor activator of NF-κB ligand (RANKL)²³⁶ production to decrease osteoclast differentiation. More studies are needed to determine the physiologic significance of the extrathyroidal effects of TSH, as this may affect the safety of future treatment modalities for thyroid cancer that may attempt to target radioisotopes to the TSH receptor.²³⁷

Free TSH β and α Subunit Measurements

TSHβ and α subunits were purified in 1974 from human TSH, and specific antibodies to them developed.²⁵² Radioimmunoassays were developed first, and then immunometric assays for the free α subunit. In general, free TSHβ levels are detectable only in primary hypothyroidism and therefore are of limited utility. Free α subunit levels have been useful in the evaluation of pituitary and placental disease. Free α subunit is detectable and measurable in both euthyroid and eugonadal human subjects.²⁵³ Elevated values of free α subunit are found in the sera of patients with TSH-secreting or gonadotropin-secreting pituitary tumors^254–256 or choriocarcinoma,²⁵⁷ and with a variety of nonpituitary and nonplacental malignancies, including cancers of the lung, pancreas, stomach, prostate, and ovary.^258–260

Provocative Testing of TSH

TRH directly stimulates TSH biosynthesis and secretion. Given intravenously, intramuscularly, or orally, TRH causes a reproducible rise in serum TSH levels in euthyroid subjects.²⁶¹ The test is performed by giving 200 to 500 µg of TRH intravenously and measuring TSH at 0, 20 to 30, 60, 120, and 180 minutes after injection. In euthyroid subjects, an immediate release of TSH rises to peak levels approximately 20 to 30 minutes after TRH injection, usually reaching values fivefold to 10-fold higher than basal. With the use of third- and fourth-generation assays across a wide spectrum of normal and abnormal basal TSH levels, the increase in TSH after TRH stimulation has been between 2.8- and 22.9-fold²⁴⁶ (Fig. 2-8). In hyperthyroid subjects, the undetectable basal serum TSH levels correlate with absent TSH responses to TRH. Patients with low basal serum TSH levels secondary to pituitary insufficiency (secondary hypothyroidism) or hypothalamic disease (tertiary hypothyroidism) have absent or attenuated TSH responses to TRH.²⁶² In contrast, patients with elevated TSH levels due to primary hypothyroidism have exuberant responses to TRH stimulation. However, elevated TSH levels in patients with pituitary TSH-secreting tumors respond less than twofold to TRH stimulation.²⁵⁴

Drugs and TSH Levels

Among the most common causes of abnormal TSH levels are pharmacologic interventions that alter TSH production.²⁶³ These can be divided into those that directly affect hypothalamic-pituitary function, those that affect thyroid gland function, and those that alter the distribution of thyroid hormones between free and protein-bound thyroid hormones in plasma.

Hypothalamic Disorders

The hypothalamus is the source of three important molecules that regulate TSH secretion: TRH, dopamine, and somatostatin. TRH is the only positive regulator of TSH production from the hypothalamus. Structural abnormalities in the hypothalamus have resulted in clinical hypothyroidism, which presumably is due to decreased or defective TRH production (Table 2-1). Exogenous administration of TRH to patients with hypothalamic hypothyroidism can restore serum thyroid hormone levels toward normal, resulting in clinical improvement.²⁶⁵ Hypothalamic disease has not been reported to elevate endogenous SS or dopamine, resulting in decreased TSH and hypothalamic hypothyroidism. However, administration of GH has been associated with this syndrome,¹⁶⁰ presumably by stimulating hypothalamic SS production or enhancing the conversion of T₄ to T₃, both of which may result in decreased TRH production. In contrast, acute exogenous administration of both dopamine and somatostatin can decrease TSH production. It is therefore paradoxical that chronic administration of dopamine agonists or somatostatin analogues does not produce central hypothyroidism. It is likely that the exquisite sensitivity of TSH production to small changes in thyroid hormone levels may result in a correction for alterations induced by these analogues. As was outlined earlier, leptin derived from fat cells does circulate in plasma and has a variety of effects on the hypothalamus, one of which is to increase TRH production.

The serum levels of TSH in hypothalamic hypothyroidism may be low, normal, or even minimally elevated.²⁶² The paradoxical finding of low serum thyroid hormone levels in association with normal or elevated basal TSH levels suggests that circulating TSH in hypothalamic hypothyroidism is biologically defective.²⁶⁵ In fact, TRH deficiency has been associated with differences in glycosylation patterns of the TSH molecule, which result in decreased TSH receptor binding and activation. The 24 hour secretory profile of TSH in patients with hypothalamic hypothyroidism is also abnormal.⁷⁵ The frequency of TSH pulses is the same as for euthyroid controls, but the amplitude of the pulses is decreased, particularly at nighttime, resulting in loss of the normal nocturnal surge (see Fig. 2-4, bottom panel).

Pituitary Disorders: Congenital TSH Deficiency

Increases or decreases in pituitary TSH production can result directly from abnormalities in the pituitary due to congenital or acquired causes. Congenital hypothyroidism due to decreased TSH production generally is inherited as an autosomal recessive disorder, and affected individuals have severe mental and growth retardation. The molecular basis for isolated TSH deficiency has usually involved mutations in the TSHβ gene (Table 2-2). For example, a single base substitution in one family at nucleotide position 145 of the TSHβ gene altered the CAGYC region,²⁶⁶ a critically important contact point for the noncovalent combination of the TSHβ and α subunits. In other kindreds, a single base substitution introduced a premature stop codon, resulting in a truncated TSHβ subunit, which included only the first 11²⁶⁷ amino acids. Another type of mutation involves a nonsense 25 amino acid protein that results from mutation of a donor splice site and a new out-of-frame translational start point.²⁶⁸ In other cases, the disorder involves the production of biologically inactive TSH with loss of cysteine105 that disrupts the disulfide bridge formation important in “seatbelt” stability^269,270 and is perhaps the most common of the TSHβ mutations; the less common mutation at cysteine85 disrupts the cysteine knot that is important for heterodimer formation and TSH receptor binding,^268,271 resulting in a similar phenotype, except that in some cases circulating TSH was detectable.

A more common cause of congenital TSH deficiency arises not as the result of a mutation in the TSHβ gene, but rather following defective production of a key transcription factor necessary for TSHβ gene expression. This occurs in combined pituitary hormone deficiency (CPHD), in which subjects have congenital hypothyroidism and growth retardation secondary to TSH, GH, and prolactin deficiencies.272,273 The genes for all three of these proteins are dependent on the pituitary-specific transcription factor Pit-1 for their expression. Mutations in the coding region of the Pit-1 gene alter the function of the Pit-1 protein or completely disrupt its structure. The absence of Pit-1 prevents normal pituitary development, resulting in hypoplasia of the pituitary. In heterozygotes, in which a normal allele is present, the abnormal Pit-1 protein can bind to DNA but may not be able to effect transactivation, interfering with the function of the normal Pit-1 (dominant negative mechanism). It is interesting to note that a similar combined hormone deficiency syndrome has been reported in two murine models in which the Pit-1 gene is defective: a point mutation found in the Snell dwarf (dw)⁸ and a major deletion in the Jackson dwarf (dwJ).²⁷⁴

An even more frequent cause of CPHD has been delineated by the discovery of a pituitary-specific transcription factor called “prophet of Pit-1” (PROP-1). This factor is a paired-like homeodomain protein in which a mutation in the murine species causes the Ames dwarf (df) mouse phenotype.²⁷⁵ Subsequently, patients with CPHD were found to have mutations in the PROP-1 gene.^276,277 More than 50% of families with CPHD have been shown to contain mutations in the PROP-1 gene,²⁷⁸ far exceeding the prevalence of mutations in the Pit-1 gene as a cause for CPHD. The mutations all are found in the homeodomain part of the molecule. It is interesting to note that the phenotype of patients with PROP-1 mutations includes deficiencies not only of GH, prolactin, and TSH, but also of LH and FSH. Furthermore, the development of hormone deficiencies may not be neonatal but rather may occur progressively up to the age of adolescence. ACTH deficiency has been reported as a late consequence in CPHD.²⁷⁹

Pituitary Disorders: Acquired TSH Deficiency

The acquired causes of pituitary TSH deficiency generally relate to destructive processes in the anterior pituitary or hypothalamus. These can include infiltrative or infectious disorders, compression secondary to neoplastic processes, or active ischemic and hemorrhagic processes involving the pitutary gland. The most common cause of acquired pituitary TSH deficiency is neoplastic destruction or compression of normal anterior pituitary cells by a primary pituitary neoplasm, a craniopharyngioma, or infiltrating metastatic disease to the pituitary. Likewise, these same processes can extend into the hypothalamus, thus interrupting normal TRH production. In acquired pituitary TSH deficiency, multiple pituitary deficiencies are concomitantly associated; acquired isolated TSH deficiency is rarely, if ever, seen. When hypothyroidism is due to a pituitary disorder, the disease is called secondary hypothyroidism, and when it is due to a hypothalamic disorder (see “Hypothalamic Disorders”), it is called tertiary hypothyroidism. Most patients with acquired pituitary TSH deficiency have symptoms of hypothyroidism, as well as symptoms of LH, FSH, GH, and usually ACTH deficiency. Serum free T₄ and T₃ levels are low in association with a low or low/normal basal TSH level. The distinction between secondary and tertiary hypothyroidism can be challenging. A completely absent TSH and prolactin response to TRH favors secondary hypothyroidism, whereas a mildly elevated basal prolactin level (due to disrupted hypothalamic dopamine production) and normal or elevated basal TSH levels (with subnormal bioactivity) favor tertiary hypothyroidism.

Pituitary Disorders: Increased Pituitary TSH Production

Most cases of elevated serum TSH levels are a result of primary thyroidal disease, not primary pituitary disease. However, two important causes of elevated TSH levels originating from pituitary disorders have been described.