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The TSH receptor in the pituitary and its clinical relevance

Brokken, L.J.S.

Publication date

2002

Document Version

Final published version

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Citation for published version (APA):

Brokken, L. J. S. (2002). The TSH receptor in the pituitary and its clinical relevance.

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Pituitary y

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Leonn J.S. Brokken

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TH€€ TSH R6C6PTOR IN TH€ PITUITflRV ANDD ITS CUNICflL R€L€VflNC€

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Colophon n

TheThe TSH receptor in the pituitary and its clinical relevance.

©© L.J.S. Brokken, Utrecht, The Netherlands, 2002.

ISBNN 90-9015742-5

Printing:: PrintPartners Ipskamp B.V., Enschede, The Netherlands.

Thiss thesis was prepared at the Department of Endocrinology & Metabolism, Academic Medicall Centre, University of Amsterdam.

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TH€€ T5H R€C€PTOR IN TH€ PITUITflflV fiNDfiND ITS CUNICfiL R€l€VflNC€

ACADEMISCHH PROEFSCHRIFT

terr verkrijging van de graad van doctor aann de Universiteit van Amsterdam opp gezag van de Rector Magnificus

prof.. mr. P.F. van der Heijden

tenn overstaan van een door het college voor promoties ingestelde commissie,, in het openbaar te verdedigen in de Aula der Universiteit

opp dinsdag 14 mei 2002, te 14.00 uur

door r

Leonn Joannes Stanley Brokken

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Promotiecommissie: :

Promotor: : Co-promotor: :

Prof.. Dr W.M. Wiersinga Drr M.F. Prummel Faculteitt der Geneeskunde

Overigee leden: Prof.. Dr J. Köhrle Prof.. Dr H.A. Drexhage Prof.. Dr D.F. Swaab Prof.. Dr E. Briët

Prof.. Dr C.J.F, van Noorden Prof.. Dr J.J.M, de Vijlder

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SapereSapere Aude!

'Dare'Dare to Think!', I. Kant 1784, inn 'Was istAufldarung?'

AanAan mijn ouders, voorvoor mijzelf.

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CONT€NTS CONT€NTS

CONTENTS S Chapterr 1

Introductionn 1

Chapterr 2

Expressionn of the thyroid stimulating hormone receptor in the folliculo-stellate

cellss of the human anterior pituitary 31

M.F.M.F. Frummel, L.J.S. Brokken, G. Meduri, M. Misrahi, O. Bakker, W.M. Wiersinga

Chapterr 3

Suppressionn of serum thyrotropin by Graves' immunoglobulins:

evidencee for a functional pituitary thyrotropin receptor 49

LJ.S.LJ.S. Brokken, J.W.C. Scheenhart, W.M. Wiersinga, M.F. Frummel

Chapterr 4

Graves'' immunoglobulins are responsible for long-time thyrotropin

suppressionn in euthyroid, treated Graves' disease patients 59

LJ.S.LJ.S. Brokken, W.M. Wiersinga, M.F. Frummel

Chapterr 5

Mousee pituitary folliculo-stellate cells express receptors for many,

butt not all, adenohypophyseal hormones 67

LJ.S.LJ.S. Brokken, M. Leendertse, O. Bakker, W.M. Wiersinga, M.F. Prummel

Chapterr 6

Functionall thyrotropin receptor expression in the pituitary folliculo-stellate

celll line TtT/GF 77

LJ.S.LJ.S. Brokken, O. Bakker, W.M. Wiersinga, M.F. Prummel

Chapterr 7

Generall discussion 97

Summaryy 123 Samenvattingg 127 Samenvattingg voor niet-ingewijden 131

Listt of abbreviations 135 Dankjulliewelwoordd 137

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1 1

Introduction n

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INTRODUCTION INTRODUCTION

1.11 REGULATION OF TSH SECRETION: AA CLASSIC FEEDBACK MECHANISM

Thyroidd stimulating hormone (TSH) is the major regulator of thyroid hormone synthesis and secretion.. It is a heterodimeric glycoprotein produced by the thyrotrophic cells of the anterior pituitaryy gland, consisting of two noncovalently linked subunits, a and P, with a total mass of 28-300 kDa (1). Differences in the molecular mass of TSH are primarily due to the

heterogeneityy of carbohydrate chains. The a subunit is common to two other pituitary glycoproteinn hormones, follicle stimulating hormone (FSH) and luteinizing hormone, as well ass to the placental hormone chorionic gonadotropin. The p subunit, however, is unique to eachh of the four hormones and confers specificity of action. Production of bioactive TSH involvess cotranslational glycosylation and folding that enables combination of the two subunits.. TSH is stored in secretory granules before it is released into the circulation. It binds too its receptor on the thyroid follicular cells where it stimulates the production and secretion off thyroid hormones, thyroxine (T4) and triiodothyronine (T3). Together with hypothalamic

thyrotropinn releasing hormone (TRH), thyroid hormones are the main regulators of TSH plasmaa levels (Figure 1.1).

TRHH stimulates TSH production by effects both at the transcriptional and

posttranscriptionall level and at the secretory level (2-4). On the other hand, T3 inhibits TSH production,, primarily at the transcriptional level on both the a and p-subunit genes (5,6). At thee posttranscriptional level, T3 decreases TSH bioactivity by reducing the addition of

oligosaccharidess (3). In addition, T3 acutely blocks the secretion of TSH (7).

Althoughh T3 is the bioactive thyroid hormone, both T3 and T4 provide a strong

negativee feedback on the thyrotrophs. The intracellular monodeiodination of T4 to bioactive

T33 is greater in the pituitary gland than in peripheral tissues, and contributes approximately halff to the intrapituitary T3 content (8). Due to the strong feedback by thyroid hormones on TSHH secretion there exists an excellent negative correlation between plasma free T4 and TSH

levelss (Figure 1.2) (9). Since TSH plasma levels fluctuate over a wider order of magnitude thann free T4 levels, TSH rather than free T4 is generally used to monitor thyroidal status.

Besidess TRH and thyroid hormones as the dominant determinants of TSH plasma levels,, there are additional positive and negative regulators of the system. Both the neuropeptidee somatostatin (SRIF) and the catecholamine dopamine inhibit basal and

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CHRPT€f>CHRPT€f> 1

Figuree 1.1. Anterior pituitary TSH secretion is stimulated by TRH released from the hypothalamus into the

portall system. Somatostatin (SRIF) inhibits pituitary TSH secretion by direct effects on the thyrotrophs as well ass diminishing the release of TRH. Dopamine (DA) also inhibits TSH secretion, although the net effect of the stimulatoryy actions on the hypothalamus is unclear. TSH binds to its receptor in the thyroid gland, and stimulates thee production and release of T4 and T3. In turn, the thyroid hormones provide a negative feedback loop at the levell of the pituitary as well as at the hypothalamus.

stimulatedd TSH secretion (10-13). SRIF may also have an inhibitory effect on TRH secretion att the hypothalamic level (14). Moreover, dopamine inhibits TSH a and (3-subunit gene transcriptionn by inhibition of adenylate cyclase resulting in decreased intracellular cAMP levelss (15,16). Furthermore, steroid hormones generally inhibit TSH secretion, but they are nott major determinants of TSH production (17). In healthy subjects, TSH is secreted in a pulsatilee manner, superimposed on a tonic secretion with a circadian pattern (18,19). Peak TSHH levels in humans occur between 23.00 and 05.00 hours and lowest levels occur at about

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INTRODUCTION INTRODUCTION

3000 500 850

Figuree 1.2. Relationship between serum TSH and free T4 (as reflected by free T4 index, FT4I) in 505 stable ambulatoryy patients with various states of thyroid function. The circles in the shaded area represent undetectable (<< 0.005 mU/L) TSH values. The solid lines represent the 95% confidence limits of the relationship (log TSH = 2.56-0.222 FT4I; r = -0.84, P < 0.001) (9).

1.22 REGULATION OF TSH SECRETION IN GRAVES' DISEASE

Graves'' hyperthyroidism is an autoimmune disease caused by stimulating autoantibodies directedd against the TSH receptor. These autoantibodies were first detected in a bioassay involvingg the ability of injected patient's serum to release radioiodine from guinea pig thyroidss in vivo (20) and in 1964 this long-acting thyroid stimulator was recognised as an immunoglobulinn G molecule (21,22). Nowadays they are either detected by their ability to competee with radiolabeled TSH for binding to the TSH receptor (TSH binding inhibiting immunoglobulins,, TBII), or by their stimulatory (TSH receptor stimulating antibodies, TSAb) orr inhibitory (thyrotropin stimulation blocking antibodies, TSBAb) effect on cAMP

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CH8PT€BCH8PT€B 1

productionn in cell lines transfected with the TSH receptor (23). It should be noted that the TBIII assay does not discriminate between TSAb and TSBAb. The large majority of these TSHH receptor antibodies is stimulatory, resulting in hyperthyroidism and goitre (24). A small percentagee blocks thyroid functions by inhibiting TSH-binding (25-28) or even without interferingg with TSH binding (29-31). An even smaller percentage is considered neutral and doess not affect TSH receptor activation, nor blocks TSH binding (32). Treatment of Graves' hyperthyroidismm with antithyroid drugs usually restores euthyroidism clinically and biochemicallyy (defined by normal free T4 and T3 levels) within 1 to 2 months (33). This is

typicallyy accompanied by a decrease in autoantibody titres.

Remarkably,, during adequate treatment of Graves' hyperthyroidism there is no consistentt correlation between free T4 levels and TSH levels once normal free T4 and T3

serumm concentrations have been restored. In clinical practice it is frequently observed that euthyroid,, treated Graves' disease patients with normal, or even low, thyroid hormone levels showw suppressed plasma TSH levels for months or even years after euthyroidism has been establishedd (33,34). The only explanation for this phenomenon provided to date has been a delayedd recovery of the thyrotrophs from a prolonged hyperthyroid state (35,36). However, experimentall evidence to support this theory has been lacking so far.

1.33 ULTRA-SHORT LOOP FEEDBACK IN THE ANTERIOR PITUITARY

Overr the last decade it has become clear that, in addition to central feed forward and peripherall feedback control, additional autocrine and paracrine mechanisms within the anteriorr pituitary also influence pituitary hormone secretion (37-39). This offers the possibilityy of fine regulation of hormone secretion at the pituitary level. By analogy with a modernn central heating system that measures and regulates its own heat production by a centrall thermostat (Figure 1.3), the pituitary could harbour such a central thermostat. The animall body is, after all, a machine, a device that utilises energy to produce or execute certain functions.. Monitoring the production of its own secretagogues might enable the pituitary to fine-regulatee hormone secretion more efficiently.

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INTRODUCTION INTRODUCTION

Figuree 1.3. Modern central heating systems

fine-regulatee their heat production not only throughh monitoring the temperature with a locall room thermostat (RT) but also by directlyy assessing the heat production with aa central thermostat (CT).

Ultra-shortt loop regulation of PRL secretion

Autocrinee and paracrine regulation of pituitary hormone secretion has been studied most extensivelyy in lactotroph cells. Numerous peptides and other molecules that are synthesised byy lactotrophs, gonadotrophs, corticotrophs, and folliculo-stellate cells are known to be capablee of modulating prolactin (PRL) secretion (39-41). It is well established that PRL can inhibitt its own secretion by activating dopaminergic neurons in the hypothalamus (42-44). However,, PRL also acts directly at the lactotroph through an ultra-short loop feedback. PRL receptorr mRNA expression has been demonstrated in rat and human lactotrophs (45,46) and PRLL inhibits its own release in an autocrine/paracrine manner, in both human and rat pituitary (47-49).. Interestingly, in certain physiological conditions, such as lactation or prolactinoma, thee lactotrophs appear to lose their autoinhibitory response to prolactin (50,51). Strong evidencee indicates that the ultra-short loop inhibition by prolactin is related to a particular isoformm of the hormone, and that impairment in the production of this isoform is responsible

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CHRPT6RCHRPT6R 1

forr the loss of ultra-short loop feedback. For an extensive review on the complex regulation of PRLL secretion, see Ref. (52).

Prolactinn secretion is also modulated by folliculo-stellate cells. In the anterior pituitary thesee cells are the only source of interleukin-6 (IL-6) (53). This cytokine has been shown to stimulatee prolactin secretion, both in vitro and in vivo (54,55). However, folliculo-stellate cellss suppress the prolactin-secretory response to angiotensin II and TRH when they are culturedd with lactotrophs as cell aggregates. Intimate contact between folliculo-stellate cells andd lactotrophs is not required for this inhibition, since it is still observed after dispersion of thee co-aggregates into single cells (41). This suggests that a diffusable inhibitory factor, differentt from IL-6, is secreted by the folliculo-stellate cells and suppresses prolactin secretion.. However, the identity of this putative paracrine factor is unknown.

Further,, PRL-releasing peptide was long believed to be released by the hypothalamus only,, but the detection of PRL-releasing peptide as well as its receptor in the human anterior pituitaryy also suggests a paracrine or autocrine role for this peptide (56).

Ultra-shortt loop regulation of GH secretion

Ultra-shortt loop regulation within the pituitary has also been suggested for growth hormone (GH)) secretion. Using double immunocytochemistry, Mertani and co-workers (57) showed thatt the GH receptor is expressed in the human anterior pituitary by somatotrophs,

lactotrophs,, and gonadotrophs but not in the thyrotrophs or corticotrophs. In the rodent pituitary,, GH receptor and/or binding protein expression has also been demonstrated in somatotrophss as well as in other hormone producing cells (58-61). Furthermore, Asa and

co-workersworkers (62) found that somatotrophs in transgenic mice expressing a GH antagonist become hyperactivee as observed by increased synthetic and secretory activity at the

electron-microscopicall level. Since this stimulatory effect on the activity of the somatotrophs was even strongerr in transgenic mice that were devoid of the GH receptor, these findings are consistent withh a direct inhibition by GH on pituitary somatotrophs. A direct negative feedback by GH onn its own secretion was also demonstrated in intact rainbow trout pituitary using an in vitro perifusionn system (63). Pituitaries exposed to increasing doses of ovine GH, before being returnedd to a GH-free perifusion, indeed showed a dose-dependent inhibition of GH secretion. However,, Veldhuis and co-workers (64) showed that, in addition, GH feeds back on the

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INTRODUCTION INTRODUCTION

somatostatinn release and inhibits GH-releasing hormone outflow, ultimately decreasing GH releasee from the anterior pituitary. This offers an additional explanation for the observation by Asaa and co-workers. For an extensive review on the complex regulation of GH secretion in humanss and rodents see Giusina and co-workers (65).

Ultra-shortt loop regulation of TSH secretion

Indicationss for autoregulation of TSH secretion were already noted in 1971 by Leoni and co-workerss (66) in order to explain their results obtained by in vitro superfusion experiments withh porcine anterior pituitaries. Remarkably, TSH levels in the incubation medium never exceededd an average concentration of 137 mU/ml/g dry weight. Adding exogenous TSH at 3000 mU/ml completely abolished 3H-TSH release from the pituitaries. Thus, it is tempting to postulatee that TSH secretion is autoregulated within the anterior pituitary. For a short-loop controll of TSH secretion to be operational, a specific pituitary cell type should be able to monitorr the secretion of TSH by the thyrotrophs. One way of achieving this is by the expressionn of the TSH receptor. This monitoring role could be performed either by the thyrotrophh itself, in an autocrine fashion, or by one or more intermediate cell types in a paracrinee fashion. Thus, TSH binding and TSH receptor activation will either directly down regulatee TSH secretion in the thyrotrophs, or activate an intermediate cell type to inhibit TSH secretion,, either directly by cell-cell contact or via certain specific agents released in the intercellularr space. We therefore hypothesised that TSH secretion is also regulated at the pituitaryy level in an autocrine or paracrine manner.

Thiss hypothesis was developed on two grounds. First, in our experience, suppressed plasmaa TSH levels are more frequently observed in euthyroid patients treated for autoimmune Graves'' hyperthyroidism than in euthyroid patients treated for other forms of

hyperthyroidism,, such as toxic nodular goitre. Second, when euthyroidism is restored by antithyroidd drug treatment, this is usually accompanied by a concomitant decline of the TSH receptorr antibody titres. However, in many patients these antibodies remain detectable in the bloodd for years (33,34). In contrast to the central nervous system, the pituitary resides outside thee blood-brain barrier and is thus well accessible for high molecular weight proteins such as antibodiess (67). In our theory, these antibodies will mimic the action of TSH resulting in downn regulation of TSH secretion by binding to the pituitary TSH receptor (Figure 1.4).

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CHRPTSFICHRPTSFI 1

Figuree 1.4. Regulation of TSH secretion during Graves' disease. Before treatment (left panel) TSAb stimulate

thee thyroid resulting in hyperthyroidism. Increased thyroid hormone feedback at the level of the hypothalamus andd pituitary results in suppressed serum TSH levels. After treatment (right panel) with antithyroid drugs (MMI) andd supplementation with T4, the patient is rendered euthyroid as defined by normal thyroid hormone levels. Thiss should restore the normal feedback on TSH secretion. However, the continuing presence of TSAb suppressess TSH secretion through its action on the putative TSH receptor in the pituitary.

Inn fact, this hypothesis may also explain the intriguing fact that a relapse of Graves' disease afterr discontinuation of antithyroid drugs occurs more often in patients with high TBII titres (68-70),, but also in those who continue to have suppressed TSH levels in the absence of detectablee TBII titres (71). These suppressed TSH values might result from biologically active TSHH receptor antibodies below the detection limit of routine TBII assays. The widely used TRAKK assay measuring TBII only has sensitivity for Graves' disease of 83.5% (72).

1.66 EXTRATHYROIDAL TSH RECEPTOR EXPRESSION

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INTRODUCTION INTRODUCTION

thyroid.. However, results obtained over the last decade have demonstrated that TSH receptor expressionn is not at all restricted to the thyroid. Already in 1964, it was found that TSH could stimulatee lipolysis and glucose metabolism in isolated fat cells (74). Later, binding studies usingg radiolabeled TSH not only suggested TSH receptor expression in guinea pig fat tissue (75),, but also in human peripheral lymphocytes (76), human fat, adrenal and testis tissue (77).

Thee molecular cloning of the TSH receptor from dog (78), human (79-81) and rat (82) finallyy offered the tools to confirm the presence of TSH receptor mRNA to be responsible for thee observed radio-labelled TSH binding in adipocytes (83,84). Moreover, the demonstration off TSH receptor protein by Western blot analysis in rat adipose tissue indicated that indeed thee message was translated into protein (84). TSH receptor mRNA expression was also detectedd after amplification by reverse transcription-polymerase chain reaction (RT-PCR) in humann peripheral lymphocytes (85), retro-orbital tissue (86) and retro-ocular fibroblasts in culturee (87). However, care should be taken in the interpretation of these results. First, the extremee sensitivity of the method allows for detection of rare transcripts in complex mixtures off mRNA. The demonstration of these rare transcripts does not necessarily mean that a functionall protein is expressed. In a study to establish the physiological significance of extrathyroidall TSH receptor mRNA expression, Aust and co-workers (88) were indeed able to amplifyy unexpected transcripts for TSH receptor as well as thyroperoxidase, thyroglobulin, FSHH receptor and insulin not only in thyroid tissue but also in typically non-expressing tissues andd cell types. Second, the RT-PCR used to demonstrate TSH receptor mRNA in

lymphocytess was performed using primers complementary to sequences within one exon. Thiss can easily result in amplification of genomic DNA.

Moree conclusive evidence regarding legitimate extrathyroidal TSH receptor mRNA expressionn has been obtained by direct cloning and sequencing of the full length TSH receptor (Tablee 1). In 1995, Endo and co-workers (89) cloned and sequenced TSH receptor cDNA fromm a rat epididymal fat cell cDNA library. Furthermore, CHO cells transfected with this TSHH receptor cDNA responded to TSH with an increase in cAMP. Northern blot analysis confirmedd the expression of TSH receptor mRNA in rat epididymal fat (89), guinea pig retro-orbitall fat (83) and in orbital fat from a Graves' disease patient (90). Thus, it can be concluded thatt a functional TSH receptor is indeed expressed by certain fat cells.

Off particular interest is the expression of TSH receptor in retro-orbital tissues. During Graves'' disease, a vast proportion of the patients develops Graves' ophthalmopathy,

characterisedd by increased size of the extraocular muscles and retrobulbar fat. This swelling is

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INTRODUCTION INTRODUCTION

largelyy attributable to excessive secretion of glycosaminoglycans by orbital fibroblasts. Althoughh the eye changes are well understood in a mechanistical sense, the nature of the autoantigenn in Graves' ophthalmopathy is still under debate (104). Since TSH receptor antibodiess are the immediate cause of Graves' hyperthyroidism, it was expected that the TSH receptorr would also be the autoantigen in orbital tissues. After its first discovery in healthy andd Graves' retro-orbital tissue by RT-PCR in 1993 (86), TSH receptor expression has been confirmedd in orbital fibroblasts from patients with Graves' ophthalmopathy at the mRNA as welll as protein level (92,93,105). Furthermore, this preadipocyte fibroblast TSH receptor is functional,, as evidenced by the up regulation of TSH receptor (106) and p70 S6 kinase (a serine/threoninee kinase, known to be a down stream target of the TSH receptor in thyroid cells)) (107) by TSH in cultured Graves' orbital fibroblasts. In a recent study, Wakelkamp and co-workerss (108) showed that TSH receptor mRNA expression in the orbit is restricted to the activee stage of the inflammatory process. It now seems that preadipocyte orbital fibroblasts indeedd express the TSH receptor when stimulated to differentiate into mature adipocytes (109,110). .

Thee TSH receptor is also expressed in the brain. Full length TSH receptor cDNA was clonedd from an ovine hypothalamic cDNA library (99). In rat astroglial cells in primary culture,, the TSH receptor was demonstrated using RT-PCR (111). TSH stimulates type-II iodothyroninee 5'-deiodinase activity in these cells, which suggests a role in the local regulationn of T3 concentrations in the central nervous system. Furthermore, Tournier and co-workerss (112) showed that TSH also stimulates mitogen-activated protein kinase (MAP kinase)) in these cells. However, a mitogenic effect of TSH was not found. More convincing evidencee for the expression of a brain TSH receptor was provided recently by Crisanti and co-workerss (100), who showed TSH receptor expression in astrocytes and neurons in rat brain tissuee by in situ hybridisation as well as by immunohistochemistry.

TSHH receptor expression has also been demonstrated by northern blot analysis in cardiacc muscle cells and TSH stimulation of cultured mouse AT-1 cardiomyocytes elevated thee levels of intracellular cAMP (96). Furthermore, Selliti and co-workers (113) demonstrated byy RT-PCR that TSH receptor mRNA expression in porcine heart varies regionally, and suggestedd that areas of high expression (coronary arteries, adipose tissue, right atrium) are potentiall sites for actions of TSH.

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CHRPT€RCHRPT€R 1

Thus,, from these data it can be concluded that the TSH receptor is functionally expressedd throughout the body in a variety of tissues and cell types. Extrathyroidal TSH receptorr expression in the anterior pituitary should therefore seriously be considered.

1.77 INTRACELLULAR SIGNALLING

TSHH receptor structure

Thee TSH receptor belongs to the serpentine receptors and is a membrane protein consisting of aa single polypeptide chain of 764 amino acids that includes a 20-amino acid signal peptide (80).. It forms a long extracellular amino terminal domain, three extracellular loops, seven hydrophobicc transmembrane-spanning helices, three intracellular loops and a short intracellularr carboxyl domain (114). The extracellular domain as well as part of the transmembranee domain contributes to TSH binding and the three intracellular loops of the transmembranee segments are important for signal transduction (115).

GG protein-coupled receptors

Thee TSH receptor belongs to the large and diverse superfamily of guanidine nucleotide-bindingg protein (G protein) coupled receptors, which signal to diverse groups of effector units (e.g.. adenylate cyclases, phospholipases, and various ion channels) through a family of nearly 200 heterotrimeric G proteins which act as transducers and signal amplifiers. These G proteins aree members of a superfamily of GTPases that are fundamentally conserved from bacteria to mammalss and are composed of a, (3 and y subunits (116,117). To date, 23 distinct a subunits aree cloned, including several splice variants. According to homologies in sequence and functionn of their a subunits, the G proteins are subdivided in four major subfamilies that includee stimulatory (Gs), inhibitory (Gj), Gq and G]2 a subunits (118). Five P subunits and 12

yy subunits have so far been identified (119). Binding of ligands of various chemical natures, suchh as hormones, neurotransmitters, chemokines, lipids, nucleotides, ions and proteases, activatess G protein-coupled receptors by inducing or stabilising a new conformation in the receptorr (Figure 1.5).

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INTRODUCTION INTRODUCTION

... . >> effector

* effector

Figuree 1.5. Schematic representation of G protein activation and signalling. Ligand binding to the heptahelical G

protein-coupledd receptor causes activation of G proteins by facilitating the exchange of GTP for GDP on the a subunitt of the trimeric G protein. The a subunit now has a reduced affinity for the Py-dimer and the receptor, and dissociates.. The a and Py subunits are now free to activate downstream effectors. Shutdown of the signal is dependentt on the intrinsic GTPase activity of the a subunit. Hydrolysis of subunit-bound GTP to GDP reconstitutess the heterotrimer, ready for another round of activation.

Changess in the intracellular loops are thought to uncover previously masked G protein-bindingg sites on the intracellular loops and lead to increased coupling to the heterotrimericc G protein resulting in GDP release from its binding site on the Ga subunit. GTPP binds Ga, which then has a reduced affinity for the GPy-dimer and receptor. Both Ga andd G(3y are then free to activate downstream effectors, such as enzymes and ion channels (Tablee 1.2).

Hydrolysiss of bound GTP to GDP allows the a-subunit to reassociate with the Py-dimer,, ready for another round of receptor-regulated activation. The intrinsic GTPase activity off Ga and its association with the Py-subunits are both regulated by accessory proteins. These 'regulatorss of G protein signalling', or RGS proteins, may bind to Ga and accelerate the rate off GTP hydrolysis (117). The Py-subunits are regulated by phosducin, a protein that binds tightlyy to the subunits, thus preventing their interaction with Ga and effectors (122-124).

.NH2 2

inactivee + active e

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CHRPT€RCHRPT€R 1

Tablee 1.2. Families of G proteins are classified based upon amino acid sequence similarity of their a subunits,

andd include stimulatory (Gs), inhibitory (G:), Gq and Gi2 a subunits.a Downstream effects are shared among the differentt members in one family. AC, adenylate cyclase; PDE, phosphodiesterase; PLCp, phospholipase Cp (119-121). .

Family Family Members Members TissueTissue distribution DownstreamDownstream effects"

a, , Wide e Brain n Increasedd AC activity, Ca2++ channel opening, K++ channel closing G, , a,.|,a,.i,, a, 3 ao-i,, ao-2 att i. a,-2 gUM M Wide e Brain n Retina a Tastee buds Wide e Reducedd AC activity, Ca"++ channel closing, K++ channel opening, increasedd PDE activity

an n at4 4 ai5,, a16 Wide e Wide e Stromal/epithelial l Myeloid d Increasedd PLCp activity

ÖI2,, an Wide e Na+/H++ exchange

Adenylatee cyclase/cAMP and IP^Ca2* pathway

Inn human thyrocytes, the TSH receptor interacts with multiple G proteins. Best studied is its associationn with Gs and Gq/n (125). Activation of Gs activates adenylate cyclase which

catalysess the formation of cAMP from the substrate Mg2+-ATP (126,127). At higher

concentrationss TSH also interacts with Gq/n, stimulating phospholipase C which results in the

hydrolysiss of phosphatidyl inositol 4,5-Diphosphate into inositol 1,4,5-triphosphate (IP3) and diacylglyceroll (128,129). IP3 binds to its receptor on the endoplasmic reticulum resulting in a releasee of Ca2+ from intracellular stores. Activation of these two pathways leads to the activationn of cAMP-dependent protein kinase A and Ca2+-dependent protein kinase C. Protein phosphorylationn causes an allosteric change in the protein's conformation, which either increasess or decreases its activity, and induces a cascade of signal transduction events both intracytoplasmicallyy and within the nucleus. Protein phosphorylation is reversible because of thee abundant phosphatases present in the cytosol. When protein kinases are turned off, these phosphatasess remove the phosphate from the phosphorylated enzyme, and the enzyme activity

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INTRODUCTION INTRODUCTION

Phospholipasee A2/arachidonic acid pathway

Inn primary cultured rat astroglial cells, however, TSH receptor neither signals through adenylatee cyclase nor phospholipase C. Instead, cytosolic phospholipase A2 activity is

stimulated,, resulting in the release of arachidonic acid (AA) from phosphoglycerides (111). Thee ability of the TSH receptor to couple to phospholipase A2 after activation by TSH and/or Graves'' immunoglobulins is supported in several studies using rat FRTL-5 thyroid cells (130,131),, human thyroid cells (132) and in CHO cells transfected with the human TSH receptorr (133). In porcine thyroid cells, however, this pathway appears to be activated by TSAbb only, and not by TSH (134). It remains to be elucidated which G protein is implicated inn activation of phospholipase A2 by the TSH receptor. Studies in the retina suggest that

phospholipasee A2 is activated through Gpy subunits (135).

JAK/STATT pathway

GG protein-coupled receptors can physically associate with intracellular proteins other than G proteins.. Recent evidence indicates that in this way, they might also signal through activation off the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathway.. This pathway was originally discovered through the study of interferon-induced intracellularr signal transduction. Four members of the JAK family have been identified (JAK1,, JAK2, JAK3 and Tyk2). They form a unique class of tyrosine kinases that are typicallyy associated with cytokine receptors. JAK1, JAK2 and TYK2 appear to be

ubiquitouslyy expressed. JAK3 expression, however, is predominantly restricted to cells of the hematopoieticc lineage and plays an important role in immunoregulation (136,137).

Uponn ligand binding, the dimerisation of cytokine receptors results in

transphosphorylationn and activation of the JAKs at tyrosine residues within the kinase domain (Figuree 1.6). These JAKs then phosphorylates tyrosine residues in the cytoplasmic domain of thee receptor. These phosphorylated sites serve as docking sites for various members of the STATT family. Finally, JAKs activate the bound STATs through phosphorylation on a single tyrosinee residue. Growth factor receptors with intrinsic tyrosine kinase activity can also activatee STATs directly. Tyrosine phosphorylation allows the activated STATs to form

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CHRPTSRCHRPTSR 1 CK K O O

extracellular r

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mmimmmmMmmmmxmmmmmmmmmm mmimmmmMmmmmxmmmmmmmmmm

U U

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intracellular r

STAT T

ucleus s

Figuree 1.6. The JAK/STAT pathway in cytokine signalling. Signalling is triggered when a cytokine (CK) binds

to,, and induces dimerisation of, its receptor (1). The JAKs are transphosphorylated (P) (2) and this activates themm to tyrosine phosphorylate the receptor chains (3). The STAT proteins then bind to the phosphorylated receptorss (4) and the STATs are tyrosine phosphorylated by the JAKs (5). Once the STATs are tyrosine phosphorylatedd they dissociate from the receptor (6), dimerise, and the homo- or heterodimers migrate to the nucleuss (7) where they associate with specific sequences in the promoter regions of target genes and activate genee transcription (8) (140).

and/orr heterodimers, translocate to the nucleus, bind to specific response elements in promoterr regions of target genes, and transcriptionally activate these genes (138). So far, sevenn mammalian STAT proteins have been identified as STAT1, STAT2, STAT3, STAT4, STAT5a,, STAT5b, and STAT6. STAT3, STAT5a and STAT5b are activated by a variety of cytokines,, whereas each of the other five members is activated by specific cytokines (139). Unlikee many single membrane growth factor receptors (e.g., epidermal growth factor receptor andd platelet-derived growth factor receptor), the G protein-coupled receptors as well as type I andd type II cytokine receptors lack cytoplasmic tyrosine kinase domains. Cytokine receptors thereforee recruit STAT proteins that are constitutively associated to the receptor, while in G protein-coupledd receptor signaling JAK association is mediated by ligand binding. Although it appearedd that G protein-coupled receptors could activate STATs independently of the

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INTRODUCTION INTRODUCTION

thee trimeric G protein that provides the missing link between the classic G protein-coupled receptorr and signalling proteins activated by tyrosine phosphorylation (141).

Thee first G protein-coupled receptor that was reported to activate JAK/STAT pathway wass the angiotensin II ATI receptor (142). Stimulation of this receptor leads to the activation off JAK2 and its substrates STAT1, STAT3 and STATS. Two other peptide molecules that signall through the JAK/STAT pathway are a-melatonin stimulating hormone (aMSH) and serotoninn (143,144). aMSH activates JAK2/STAT5 in human cultured lymphocyte (EM-9) cellss and in mouse L-cells stably expressing the G protein-coupled human MC5 receptor. The serotoninn receptor signals though JAK2/STAT3 in myoblasts. Recently, Park and co-workers (145)) demonstrated that also the TSH receptor can signal through this intracellular

phosphorylationn pathway. In rat FRTL-5 cells as well as in CHO cells transfected with the humann TSH receptor, TSH induces the phosphorylation of JAK 1 and JAK2 and JAK substratee STAT3 is rapidly tyrosine phosphorylated by TSH. It was also shown that there is a directt association of JAK1, JAK2 and STAT3 with TSH receptor (145).

TSHH receptor interacts with multiple G proteins

Furthermore,, as was demonstrated in human thyroid membranes, the TSH receptor is also coupledd to several subtypes of Gj and Go as well as to G12 and Gn (146). In contrast to the rolee of the Gs-cAMP cascade in TSH signalling, the role of pathways through Gq/n, Guo and

G12/133 proteins remains to be elucidated. Activation of Gj-dependent pathways may lead to a decreasee in adenylate cyclase activity. In neuronal and neuroendocrine tissues Go mediates inhibitionn of voltage-gated Ca2+ channels (147). But the expression of Ca2+ channels has not beenn demonstrated in normal human thyrocytes yet. Instead, the authors suggest a role for Go inn the secretory processes of the thyrocyte. Little information is available regarding G12/13. Bothh G proteins have oncogenic potential in mouse NIH 3T3 fibroblasts and appear involved inn processes of differentiation and organogenesis (148), and show the ability to stimulate Na+/H++ exchangers (149). In dog thyroid membranes it was even demonstrated that TSH receptorr could couple to 11 different G protein subtypes belonging toGj/o, Gs and Gq/i 1

subfamiliess (150). However, in intact dog thyroid cells in primary culture, the authors did not observee an effect on Gq/i 1 as indicated by a lack of IP3 accumulation. Nevertheless, it thus

seemss that, at least in the thyroid, the TSH receptor is a general G protein-activating receptor.

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CHf¥V€RCHf¥V€R 1

1.88 SUMMARY

Itt can be concluded that extrathyroidal TSH receptor expression is not at all a far-fetched idea sincee the TSH receptor has been demonstrated in a variety of tissues. Furthermore, autocrine regulationregulation of PRL secretion is well described and evidence that also GH inhibits its own releasee through a pituitary GH receptor is accumulating. Thus, an ultra-short loop mechanism modulatingg TSH secretion within the anterior pituitary can be envisioned. Activation of the TSHH receptor classically stimulates adenylate cyclase and phospholipase C, resulting in the formationn of cAMP and IPv/Ca"+. However, as has been shown recently, multiple signalling pathwayss can be activated by the stimulation of the TSH receptor.

1.99 SCOPE OF THE THESIS

Inn order to evaluate that an ultra-short loop feedback, mechanism controlling TSH secretion at thee pituitary level is responsible for the suppression of TSH levels in euthyroid treated Graves'' disease patients, we hypothesised that the pituitary would express a TSH receptor.

Inn chapter 2 the TSH receptor localisation in the human anterior pituitary is described. Screeningg of a human pituitary cDNA library with a TSH receptor specific cDNA probe producedd two positive clones. One clone was sequenced and encoded the full-length human TSHH receptor. Combined in situ hybridisation and immunocytochemistry, and double-immunocytochemistryy confirmed the presence of TSH receptor mRNA as well as TSH receptorr protein in distinct pituitary cells phenotypically identified as a subset of folliculo-stellatee cells.

Inn chapter 3 and 4 we examined if this pituitary TSH receptor is indeed involved in a

negativee feedback mechanism on TSH secretion. If so, TSH receptor activation should be able too suppress TSH secretion independently from the classical feedback loop via thyroid

hormones.. To investigate this we developed a modified version of the LATS (long-acting thyroidd stimulator) bioassay (chapter 3). Biochemically thyroidectomised rats supplemented withh L-thyroxine to maintain euthyroidism showed lower plasma TSH levels after injection withh stimulatory human autoantibodies against the TSH receptor as compared to rats injected withh normal human IgG. Chapter 4 describes that in euthyroid treated Graves'

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titre.. Furthermore, TSH levels are quantitatively correlated to TBII titres and not to thyroid hormonee levels in the serum. Both studies strongly support our hypothesis. However, they do nott provide evidence regarding the mechanism by which FS cells do regulate TSH secretion.

Inn chapters 5 and 6 we aimed to clarify how the folliculo-stellate cells might be involvedd in the regulation of TSH secretion. First, we characterised a mouse folliculo-stellate celll line (clone TtT/GF) in terms of adenohypophyseal hormone receptor expression (chapter 5).. Remarkably, the cell line expresses receptors for TSH, growth hormone and

adrenocorticotropicc hormone. No expression of the gonadotropic hormone receptors or PRL receptorr was detected. Chapter 6 then describes postreceptor effects of TSH in this cell line. TSHH receptor activation did not signal through adenylate cyclase/cAMP, IP3/intracellular

calciumm or the JAK/STAT3 cascade, but probably signals through STAT5a. The possible role off TGF-p2 as a paracrine factor involved in the cross talk between folliculo-stellate cells and thyrotrophss is discussed.

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