Progress in Understanding the Etiology of Thyroid Autonomy1

Knut Krohn and Ralf Paschke

Third Medical Department (K.K., R.P.) and Interdisciplinary Center for Clinical Research (K.K.), University of Leipzig, D-04103 Leipzig, Germany

Address all correspondence and requests for reprints to: R. Paschke, M.D., Third Medical Department, University of Leipzig, Ph. Rosenthal Strasse 27, D-04103 Leipzig, Germany. E-mail: pasr{at}server3.medizin.uni-leipzig.de

The etiology of thyroid autonomy and autonomously functioning thyroid nodules has been reviewed, with emphasis on histological (1, 2), molecular (3), and clinical aspects (4) as well as transgenic animal experiments that model thyroid autonomy (5). Somatic TSH receptor (TSHR) mutations have been discovered as the most prevalent molecular event in the etiology of autonomously functioning thyroid nodules (6). However, the mechanisms that induce TSHR mutations or determine their variable clinical phenotype are currently unknown. The purpose of this review is therefore to integrate new findings concerning the early molecular etiology of thyroid autonomy with previous molecular, histological, and in vitro results and to examine their possible clinical and therapeutic consequences.

Diagnosis of thyroid autonomy

Autonomously functioning thyroid tissue is characterized by the ability to function without TSH. For clinical diagnosis, radioiodine or 99mTc-pertechnetate imaging of the thyroid gland is performed to determine increased radionuclide uptake of autonomous thyroid epithelia compared with surrounding parenchyma (7, 8). In patients without complete scintigraphic suppression of the surrounding parenchyma, thyroid hormones are administered before suppression scintigraphy to differentiate autonomous from normal thyroid tissue. Histological examinations of tissue corresponding to scintigraphically autonomous areas show different architectural patterns and histological features of follicular cells compared with normal quiescent tissue (1, 9, 10, 11). Thyroid autonomy is most frequently found in toxic multinodular goiters, a heterogeneous clinical disorder that includes the presence of autonomously functioning nodules in a goiter with or without additional nodules. Additional nodules can show normal or decreased (cold nodules) uptake on scintiscan. The variable scintigraphic pattern includes circumscript areas of increased uptake with suppression of the surrounding thyroid tissue or an uneven (patchy) uptake with areas of increased uptake but no suppression of the surrounding parenchyma. Epidemiological studies (12) suggest that toxic multinodular goiters mostly originate from euthyroid goiters in iodine-deficient regions (for details, see below). Three distinct categories of thyroid autonomy have been described: 1) benign thyroid tumors with a follicular differentiation presenting as adenoma (encapsulated tumor) or adenomatous nodules (not encapsulated but circumscribed), 2) malignant thyroid epithelial tumors causing hyperthyroidism, and 3) microscopic areas in euthyroid goiters that show strong 125I labeling under TSH suppression (13). This latter form of autonomy might also be the histological equivalent of disseminated autonomy.

Thyroid epithelial cell function

In normal thyroid tissue TSH stimulates all aspects of thyroid physiology, i.e. iodine uptake and metabolism, thyroid hormone synthesis, and release (14). In the human thyroid TSH signaling is mediated through cAMP and phospholipase C (15). To date, the cAMP aspect of TSH signaling is much better studied and understood. Early work by Pisarev et al. demonstrates that cAMP produces goiter (16). Moreover, in contrast to a previous report (17), cAMP stimulates the proliferation of many endocrine tissues, including thyroid epithelial cells in vitro (18, 19, 20). Studies with goitrogen-treated rats exhibiting elevated TSH levels also suggested a link between proliferation and TSH signaling in vivo (21). More recently, the introduction of transgenic models has added the aspect of chronic in vivo stimulation of the cAMP cascade to study thyroid epithelial cell proliferation in vivo (22, 23, 24). Targeted expression of a dominant negative cAMP response element-binding protein to the thyroid glands of transgenic mice provide the first evidence that the cAMP effect on thyroid epithelial cell proliferation in vivo is a direct one (25).

Studies of interactions between TSH and growth factors have been focused especially on insulin and insulin-like growth factors (IGFs) (26). Numerous in vitro and in vivo experiments in different species show a permissive effect of insulin/IGF-I on TSH signaling (27, 28, 29, 30). Moreover, a cooperative interaction of TSH and insulin/IGF-I is very likely required for the signaling of both TSH and insulin/IGF-I in thyroid tissue (31). IGF-I is an autocrine factor in the thyroid that is regulated by TSH in the level of expression, receptor signaling, and intracellular signal transduction (32, 33, 34, 35). Therefore, local autocrine regulation through IGF-I signaling most likely acts in combination with endocrine regulation through systemic TSH/thyroid hormones to control thyroid epithelial cell growth and function. There are several reasons for maintaining a local regulatory system (IGF-I signaling) in addition to the endocrine control of thyroid epithelial cells. 1) A second system would allow the regulation of other cells in the thyroid that do not respond to TSH signaling (e.g. fibroblasts), but need to be regulated with respect to thyroid epithelial cell growth and function in concert with thyroid epithelial cells. 2) It would allow the integration of multiple mitogenic signals. 3) A local system would also allow accumulation of the endocrine signal so that a cooperative response by a number of epithelial cells is necessary to induce growth stimulation. This accumulation of local growth factors [e.g. IGF-I or other growth factors produced in thyroid epithelial cells, such as transforming growth factor-{alpha} (36)] in addition to an endocrine signal has been suggested by Dawson and Wynford-Thomas (37) to be the basis for a mechanism that protects against tumor development in the thyroid (for details see below).

Constitutive activation of the cAMP cascade

In 1989, Dumont et al. (38) hypothesized that relative overactivity of the cAMP system could account for some of the benign thyroid tumors and that molecular defects resulting in signaling through the cAMP cascade might be responsible for autonomous function of thyroid epithelial cells. The first clinical evidence for this reasoning came from the identification of somatic mutations in the Gs{alpha} protein that conferred constitutive activation to the cAMP cascade resulting in GH-producing pituitary adenomas (39). However, these mutations only provided a molecular explanation for a limited number of benign thyroid tumors (40). Therefore, signaling proteins other than Gs{alpha} had to be considered. Screening for the molecular etiology of thyroid tumors was stimulated by in vitro mutagenesis studies. Constitutive activation (independent of the ligand) of the ß-adrenergic receptor after substitution of a single amino acid established a new model for G protein-coupled receptor activation (41). In vivo, a constitutive activation of the TSHR as the result of somatic point mutations was subsequently identified in hyperfunctioning adenomas of the thyroid (42). Moreover, the clinical relevance of TSHR mutations has been demonstrated in the context of germline mutations causing autosomal dominant nonautoimmune hyperthyroidism (43). Whereas a hyperfunctioning thyroid adenoma harboring a constitutively activating somatic mutation of the TSHR develops over a long period and generally rather late in life, the same mutation present in the germline expressed in every thyroid cell results in severe neonatal hyperthyroidism and goiter with frequent relapses during antithyroid therapy and after partial thyroidectomy (reviewed in Refs. 6 and 44). The principle of constitutively activating mutations was expanded to a number of G protein-coupled receptors as the genetic basis of endocrine disease (reviewed in Refs. 45 and 46).

Frequency of TSHR or Gs{alpha} mutations

Studies investigating the prevalence of TSHR or Gs{alpha} mutations in autonomously functioning thyroid nodules have reported variable results, from 8–82% for activating TSHR mutations (47, 48, 49, 50, 51, 52, 53). Similarly, the gsp mutation frequency ranges from 8–75% (40, 49, 50, 51, 54). A direct comparison of these studies is difficult due to differences in the extent of mutation detection. Reports of a low prevalence of TSHR mutations are mostly based on a partial screen of the TSHR gene (47, 48, 49, 55). However, a partial screen of the TSHR in these studies does not completely explain the low frequency of TSHR mutation detection compared with studies based on a full screen of the receptor for the following reason; less than 25% of all known mutations are located in the parts of the TSHR gene not screened in these studies [see TSH Receptor Database at http://www.uni-leipzig.de/~innere/TSH (56)]. Therefore, these studies miss statistically only 25% of all TSHR mutations located in the full coding sequence. Besides different mutation frequencies in different cohorts, which is discussed below, technical limitations are likely to have compromised some mutation detections (57). Different screening methods have been used, e.g. direct sequencing (47, 48, 50, 51, 52) or single strand conformation polymorphism (55), which is less sensitive in detecting point mutations (58). Moreover, the quality of the examined tissue and, hence, the integrity of the DNA were highly variable, as DNA extracted from paraffin-embedded tissue (54, 55) is highly degraded (59) and therefore more difficult to examine than DNA extracted from shock frozen tissue (47, 48, 50, 51, 52, 53, 60). Nevertheless, even studies comparable for their mutation detection methods and screening at least exon 10 of the TSHR, which encodes the entire transmembrane region, have reported frequencies ranging from 20–82% in cohorts similar with respect to iodine supply (50, 51, 52, 53, 60). Therefore, the tissue identification and tissue asservation should be considered. Specimens collected from autonomously functioning thyroid nodules rarely consist of pure adenomatous tissue. Due to contamination with blood, connective and surrounding healthy tissue, as well as degenerate areas within the nodule, the amount of mutated allele may be decreased to less than 50%, making the detection of a somatic mutation difficult by fluorescent or radioactive direct sequencing (61). A comprehensive study of our group using the more sensitive denaturing gradient gel electrophoresis (61, 62, 63) to screen for TSHR mutations in 75 consecutive toxic thyroid nodules, including specimens that have been studied with direct sequencing previously (52, 53) revealed a constitutive TSHR mutation in six cases that were mutation negative with direct fluorescent sequencing (64). The frequencies of TSHR mutations and Gs{alpha} mutations in this latter study were 57% and 3%, respectively. Most studies on somatic mutations in autonomously functioning thyroid nodules (40, 42, 49, 50, 51, 54) have concentrated on thyroid adenomas, which are characterized by a capsule of connecting tissue around the thyroid lesion (65, 66). However, a large number of toxic thyroid nodules do not share the feature of encapsulation, which does not necessarily imply a different etiology. Indeed, TSHR and Gs{alpha} mutations have also been detected in adenomatous thyroid nodules (52, 60), which further underlines that clonality of the lesion is the definite criterion and that histological analysis alone does not allow identification of the clonal origin. However, the investigation of autonomously functioning adenomatous thyroid nodules for somatic mutations requires special attention to avoid sampling errors. It is necessary to clearly identify the autonomously functioning nodule macroscopically by ultrasound and scintigraphy and to histologically evaluate the tissue used for mutation detection.

Likelihood of other somatic mutations

A frequency of 60% for somatic mutations in autonomously functioning thyroid nodules detected with denaturing gradient gel electrophoresis and direct sequencing (64) raises the question of the molecular etiology of mutation-negative nodules. Mutation-negative autonomously functioning thyroid nodules could comprise polyclonal lesions that do not evolve from a single mutated cell. Heterozygous polymorphisms in X-chromosome-linked markers (67) can be used to decide the clonal origin of thyroid tissue from female patients. Using a PCR approach to amplify the X-linked human androgen receptor, 50% of mutation-negative cases from female patients show a monoclonal origin when tested for X-chromosome inactivation (64, 68). However, as discussed for the detection of somatic mutation (see above), monoclonal origin could be concealed by contamination with blood and connective and surrounding healthy tissue. As the phenotype of thyroid autonomy (adenoma and hyperthyroidism) seems to be mainly regulated by constitutive cAMP activation (69), it is likely that further candidate genes for the development of toxic thyroid nodules are located in this signal cascade. This hypothesis is also supported by the finding of resistance to TSH without the presence of loss of function mutations in the TSHR (70). Different components of the complex G protein-signaling pathway have been cloned in the past years and include 20 different mammalian G protein {alpha}-subunits, 4 ß-subunits, and 6 {gamma}-subunits (71, 72), as well as 16 phospholipases C (73), 8 adenylyl cyclases (74), and more than 20 phosphodiesterases (75, 76, 77). Despite the heterogeneity in putative signaling proteins, components downstream of the TSHR have only been defined at the G protein level to date (78, 79). In addition to activating mutations in {alpha}-subunits of G proteins, mutations could occur in adenylyl cyclases or more downstream effector molecules, such as protein kinase A and its substrates or in molecules activating other signaling cascades (e.g. tyrosine kinase cascade through IGF-I), which can act synergistically with cAMP accumulation. Alternatively, overexpression of signaling proteins such as the TSHR, Gs{alpha}-subunits, adenylyl cyclases, or downstream effector molecules could also be a cause of autonomously functioning thyroid nodules. The possible pathophysiological significance of the degree of Gs{alpha} expression in pathological conditions has recently been demonstrated in animal models. In mice, for example, long-term cardiac overexpression of the Gs{alpha}-subunit leads to myocardial hypertrophy (80), whereas blockade of the Gq{alpha} protein signaling pathway resulted in a reduction of myocardial hypertrophy (81). Regarding the level of adenylyl cyclase activity, the overexpression of adenylyl cyclase VI has been shown to result in a proportional increase in ß-adrenergic receptor-dependent cAMP accumulation in embryonic cardiac rat myocytes (82). However, thyroid tissue studies quantifying adenylyl cyclase activity in autonomously functioning thyroid nodules are controversial (83, 84, 85). In addition to increased cAMP synthesis, impaired cAMP degradation could be the cause of the higher cAMP accumulation. However, down-regulation of the cAMP inhibitory pathways, e.g. by inactivating mutations in the phosphodiesterases, have not been reported in autonomously functioning thyroid nodules. In contrast, the activities of different phosphodiesterase isoforms have been found to be dramatically induced in autonomously functioning thyroid nodules harboring a constitutively activating mutation (86). As for cAMP metabolism, an altered turnover of the TSHR could also play a role in aberrant TSH signaling, leading to increased cAMP accumulation. Therefore, defects in receptor desensitization and internalization (G protein receptor kinases, ß-arrestins) may have to be considered. Changes in the expression and activity of the receptor kinases have been associated with various disorders, including the development of cardiomyopathy (87, 88, 89, 90) and rheumatoid arthritis (90). These examples underline the importance of components of the desensitization apparatus for the function of the corresponding G protein-coupled receptor. The TSHR G protein-coupled receptor kinases 2 and 5 as well as ß-arrestin-1 have been suggested to play a key role in TSHR desensitization in rat FRTL5 cells (91, 92). However, in autonomously functioning thyroid nodules increased ß-arrestin-2 expression is predominant compared with that in surrounding tissue (93). Moreover, G protein-coupled receptor kinase 3 and 4, but not G protein-coupled receptor kinase 2, expression is significantly increased in autonomously functioning thyroid nodules (94). Further investigations are therefore mandatory to evaluate the role of signaling proteins downstream of the TSHR as candidate genes for the development of autonomously functioning thyroid nodules.

Somatic TSHR mutations in toxic multinodular goiter

Similar to solitary toxic thyroid adenomas and adenomatous nodules somatic mutations in the TSHR have also been detected in hot nodules of toxic multinodular goiters (60, 95, 96, 97). Scintigraphically, these toxic multinodular goiters showed hot nodules, with suppression of the surrounding thyroid tissue. In contrast, Gabriel et al. (98), studying 24 cases of toxic multinodular goiters, did not detect any somatic mutation in the transmembrane and C-terminal regions of the TSHR. Toxic multinodular goiter is a clinical disorder rather than a disease entity. Therefore, different pathophysiologies of the clinical disorder toxic multinodular goiter may, in fact, reflect different and/or overlapping etiologies, possibly without common molecular features (e.g. TSHR mutations). Moreover, wrong classification due to insufficient TSH binding-inhibiting Ig assay sensitivity as another possibility is illustrated by the detection of TSHR antibodies with sensitive in vitro assays in hyperthyroid patients that show a scintiscan compatible with toxic multinodular goiters (99, 100). Therefore, the lack of somatic TSHR mutations (98) might also be explained in the context of different etiologies of toxic multinodular goiters. However, especially in areas with iodine sufficiency, it raises concern about the exclusion of Graves’ disease, the quality of sampling in terms of tissue identification, and tissue asservation in patients who show scintigraphic patterns similar to those initially presented by Kraiem (99). To identify the molecular events leading to toxic multinodular goiters requires meticulous dissection of both functional and morphological aspects of the tissue under investigation before tissue sampling. Without ultrasound and histological examination, circumscribed areas of a scintiscan in toxic multinodular goiters do not necessarily correspond to well defined nodular lesions. This can severely compromise the outcome of a mutation screening. In this context, the finding of the absence of somatic TSHR mutations in toxic multinodular goiter from iodine-sufficient areas (98) does not necessarily suggest a different genetic cause from those in iodine-deficient regions, but needs to be interpreted with caution. This is also true for a germline genetic variation in codon 727 of the TSHR gene (101), which has been associated with toxic multinodular goiter (98). Similar studies in geographic areas with iodine deficiency did not reveal such an association (102, 103). Moreover, the putative predisposition for toxic multinodular goiter was based on functional analysis of the TSHR codon 727 variation, which revealed a higher cAMP response compared with the wild-type receptor. However, this in vitro finding was not confirmed in a recent study (102).

TSHR mutations in hot microscopic areas of iodine-deficient euthyroid goiters

Thyroid autonomy is very prevalent in geographic regions with iodine deficiency (47% of all cases with hyperthyroidism) and rarely occurs in iodine-sufficient regions (6% of all cases with hyperthyroidism) (12). Moreover, studies that compare the frequency of different thyroid pathologies before and after iodine supplementation (104) suggest that the level of dietary iodine is an important etiological factor in thyroid autonomy. Baltisberger et al. (104) demonstrated that iodine deficiency is associated with a significantly increased frequency of thyroid autonomy, whereas iodine supplementation leads to a significantly reduced frequency of thyroid autonomy. These studies tempted us to speculate that iodine deficiency is probably involved in the generation or propagation of somatic TSHR mutations. If this is correct, early stages of thyroid autonomy already detectable in euthyroid goiters in iodine-deficient regions should contain somatic TSHR mutations. Scintigraphically, nonsuppressible thyroid areas have been demonstrated in 40% of euthyroid goiters in iodine-deficient regions (8). Moreover, microscopic foci of autoradiographically increased uptake of 125I have been demonstrated in euthyroid goiters of previously iodine-deficient areas (1, 13, 105, 106). Therefore, we recently studied microscopic autonomous areas with increased 125I uptake in euthyroid goiters studied by autoradiography 20 yr ago (105) for the occurrence of somatic TSHR mutations. The detection of constitutively activating TSHR mutations in autoradiographically hot areas of euthyroid goiters, as shown in Fig. 1Go (107), marks an important step in understanding the etiology of thyroid autonomy in endemic goiters. Minute areas in euthyroid goiters were thus identified as likely starting foci, probably leading to toxic thyroid nodules in iodine-deficient goiters. Therefore, iodine deficiency leads not only to euthyroid goiter, but also to thyroid autonomy. Our finding also elucidates one possible molecular basis for the long-discussed follicular heterogeneity in euthyroid goiters, although it does not explain why heterogeneity, as defined by morphological or immunohistochemical criteria, is also present without detectable autonomy or nodular alterations (1, 10, 13, 106, 108).



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Figure 1. Autoradiograph showing 125I-labeled thyroid tissue from a euthyroid goiter counterstained with hematoxylin-eosin. Patients received 125I 17 h preoperatively. A, Low magnification (x4) photomicrograph showing areas with high, medium, and low iodine incorporation. DNA was extracted from thyroid tissue with high (area I) and low (area II) iodine incorporation. After PCR amplification, part of the TSHR was sequenced. A mutation in the TSHR (F631L) was detected in area I. B, Higher magnification (x18) of the section in A showing a number of small follicles highly labeled with 125I. Reproduced with permission from the Journal of Pathology (107 ).

 
Propagation of TSHR mutations

TSHR mutations have been detected with a frequency of up to 80% in toxic thyroid nodules (69). Our screening for TSHR mutations suggests a much lower frequency in hot microscopic areas from euthyroid goiters (107). These results imply that autoradiographically labeled microscopic areas with constitutively activating TSHR mutations preferentially develop into autonomously functioning thyroid nodules. A likely explanation for this finding could be a process of extreme clonal selection and expansion driven by a constitutively activated TSHR. A constitutively activated TSHR overrides the normal feedback control that limits thyroid epithelial cell growth and function. TSHR mutations in hot microscopic areas from euthyroid goiters demonstrate that TSHR mutations coincide with thyroid hyperplasia. Indeed, TSHR mutagenesis might actually be initiated by thyroid hyperplasia. This process is likely to include the following sequence of events (see also Fig. 2Go). 1) Iodine deficiency results in impaired thyroid hormone synthesis. In animal models of iodine deficiency a significant reduction in T4 levels is detectable (109, 110). Impaired thyroid hormone synthesis causes thyroid hyperplasia (111). 2) The resulting increase in thyroid cell mass compensates for impaired thyroid hormone synthesis. 3) The increase in mitotic activity and proliferation of thyroid epithelial cells increases the chance of mutagenesis due to an increase in the number of cell divisions and DNA replications, which leads to TSHR mutations. Alternatively, iodine deficiency might increase radical generation and the mutation rate by excessive H2O2 production (112, 113). 4) Hyperplasia induces cell proliferation, which generates small thyroid epithelial cell clones. A small cell clone from a progenitor cell containing an activating TSHR mutation might proliferate even without paracrine stimulation, because constitutive activation of the cAMP cascade causes production of autocrine factors [e.g. IGF-I (32, 33, 34, 35)]. In contrast to a single constitutively activated cell in which production of an autocrine factor might not be sufficient to cause self-stimulation, a small cell clone might produce enough autocrine factors for self-sustained proliferation. The constitutively active clone might therefore overcome inhibitory paracrine influences (e.g. lack of coproduction of an activating factor) from surrounding normal cells, as suggested by Dawson and Wynford-Thomas (37). In contrast to iodine deficiency, in areas with adequate iodine supply where hyperplasia is not common, the lack of coproduction of autocrine factors through surrounding nonproliferating cells could prevent clonal expansion of thyroid cells with activating TSHR mutations. Moreover, the frequency of TSHR mutations might be lower in iodine sufficiency vs. iodine deficiency due to a lower number of cell divisions. However, thyroid hyperplasias caused by goitrogens [e.g. propylthiouracil in animal experiments (114) or goitrogens in food (115) and in drinking water (116)] instead of iodine deficiency could also initiate the suggested line of events and lead to autonomously functioning thyroid nodules that are diagnosed in iodine-sufficient areas.



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Figure 2. The etiology of thyroid autonomy. The starting point for the development of thyroid autonomy is hyperplasia induced by iodine deficiency. Iodine deficiency increases mutagenesis directly (production of H2O2/free radicals) or indirectly (proliferation and increased number of cell divisions). Subsequently, hyperplasia forms cell clones. Some of them contain somatic mutations leading to cold (blue dots) or hot (red dots) thyroid nodules.

 
Etiology of nodular transformation

Nodular transformation could be the result of neoplastic growth, defined as clonal expansion caused by an advantageous mutation. This assumption is supported by data demonstrating a predominant monoclonal origin of benign thyroid adenomas or adenomatous nodules (64, 68, 117, 118, 119, 120). Moreover, a systematic study in multinodular goiters demonstrates a larger number of nodules of monoclonal vs. polyclonal origin (121). All of these studies suggest a chain of events leading to clonal lesions that is detailed here. For autonomously functioning thyroid nodules the advantageous mutation (e.g. in the TSHR or Gs{alpha} protein) leads to constitutive activation of the cAMP cascade, which stimulates growth and function. However, a differential growth advantage of affected cells is necessarily the result of increased cAMP signaling over that of unaffected cells. This differential growth advantage of cells with TSHR or Gs{alpha} mutations could be increased by a relatively lower cAMP level in cells with wild-type TSHR or Gs{alpha} protein. Currently, long-term changes in TSH levels during goiter formation induced by iodine deficiency are not well investigated. Animal models with a severe reduction of iodine in the diet show a tremendous increase in TSH. This has also been shown in humans living in areas of extreme iodine depletion (122). In newborn populations in Europe, there is an inverse relationship between the urinary iodine concentration and the frequency of serum TSH levels above 50 µU/mL, which is the recall level for the suspicion of congenital hypothyroidism (123). In contrast, studies by Brabant et al. (124) suggest a short-term reduction of serum TSH in adults in response to a moderate decrease in iodine supply. Barbant et al. (124) hypothesize that changes in sensitivity to TSH explain thyroid hypertrophy and a more effective release of thyroid hormones. A similar conclusion has been drawn from experiments in rats fed a low iodine diet (125). Therefore, differences in the serum TSH response to a decrease in iodine supply are probably modulated by the severity of iodine depletion, as higher depletion leads to an increase in TSH (reviewed in Refs. 126 and 127). The TSH response to low iodine is likely to be different for newborns and children (high increase) compared with adults or adolescents (low increase to decrease), as studies of extreme iodine depletion in Zaire also demonstrate an inverse relationship of the TSH level with the age of the patients (122). Moreover, changes in serum TSH are likely to depend on the duration of the iodine depletion, as older people (long duration) show lower TSH levels. Finally, the work of Fenzi et al. (128) and Berghout et al. (129) suggests that the serum TSH level decreases with goiter size and is below normal in patients with large goiters. This decrease in TSH is probably the consequence of increasing amounts of autonomous tissue.

In a state with normal or reduced serum TSH (e.g. large goiter or long duration of iodine deficiency), a mutation affecting the TSH signaling pathway (i.e. cAMP cascade) resulting in TSH-independent stimulation (e.g. through a mutation in the TSHR or Gs{alpha} protein) could induce differential growth stimulation and induce clonal expansion. This clonal expansion of thyroid cells beyond a certain cell mass would result in hyperthyroidism. It is therefore very likely that endemic goiter in iodine-deficient areas is the starting point for thyroid autonomy (129). Nodular transformation is very likely the result of a long process of clonal expansion, as the frequency of nodular goiters increases with age (130). Moreover, subclinical hyperthyroidism characterized by a reduced TSH level probably further supports growth of the nodule, because the difference between lack of TSH stimulation in normal thyroid tissue (decreased TSH level) and growth stimulation by an advantageous somatic mutation (e.g. causing constitutively activated cAMP) is increased. Nodular transformation could therefore be the result of early thyroid hyperplasia (e.g. as the result of iodine deficiency or other goitrogens), which increases mutagenesis, followed by selection of advantageous mutations (e.g. constitutively activated TSHR or Gs{alpha} protein). The process of clonal expansion and thyroid nodule formation is likely to require a longer time period if the differential growth advantage of mutated cells is small. To date, an estimate of the differential growth advantage is difficult because the results of in vitro studies of TSHR activation are difficult to extrapolate to in vivo conditions (131). Finally, our hypothesis that nodular transformations and growth are supported by low TSH levels is also supported by the observation that thyroid hormone therapy does not lead to a significant reduction of nodule size (132, 133).

Iodine deficiency, hyperplasia, mutagenesis, and selection of clones could also explain cold thyroid adenomas or adenomatous nodules (see Fig. 2Go) by advantageous mutations that only initiate growth, but not function, of the affected thyroid cells (e.g. ras mutations). Moreover, nodular transformation of thyroid tissue after resection of TSH-secreting pituitary adenomas (134), nodular transformation of thyroid tissue in Graves’ disease (135), as well as goiters in patients with acromegaly (136) could follow a similar mechanism (see Fig. 2Go). The etiology of the nodular thyroid pathology in these patients is characterized by thyroid hyperplasia, which is also induced by iodine deficiency.

Proliferation in toxic thyroid nodules

If TSHR or Gs{alpha} mutations trigger a process of increased proliferation leading to the manifestation of autonomously functioning thyroid nodules and hyperthyroidism, a differential growth advantage should be detectable as increased proliferation in late stages of thyroid nodules. We and others have addressed this issue, studying proliferation markers in nodular and surrounding thyroid tissue (137, 138). In general, the expression of these proliferation markers is significantly increased in most autonomously functioning thyroid nodules compared with that in normal thyroid tissue. These findings indicate that an increase in cellular proliferation is still detectable in late stages of thyroid nodules. However, the 2- to 3-fold increase in proliferation in toxic thyroid nodules over surrounding tissue (137) or a slightly higher increase over surrounding tissue when comparing with the area of highest density of the marker within the nodule (138) does not seem to be sufficient to explain the formation of autonomously functioning thyroid nodules. It has been reported that human adult thyroid cells divide about 5 times during their life (139, 140), and that mitotic activity decreases in the adult rat (141). A 2- to 3-fold increase in thyroid epithelial cell proliferation would lead to 7 additional cell divisions, which for a single cell with a somatic mutation would translate into a cell mass of about 128 cells given that the life span of the cell does not change. Therefore, the average increase in proliferation markers in autonomously functioning thyroid nodules vs. surrounding tissue does not seem to be sufficient to generate an autonomously functioning thyroid nodule with thousands of cells. However, peak stimulation of thyroid cells and, hence, proliferation might be much higher and is very likely reached long before the patient becomes hyperthyroid. Indeed, a decline of mitotic activity in thyroid tissue has been found after prolonged TSH stimulation in a rat model of prolonged goitrogen administration (142). Such a decline in TSHR signaling could be the result of receptor desensitization [e.g. through activation of G protein-coupled receptor kinases as well as ß-arrestin (91, 92, 93)] or uncoupling of TSHR signal transduction.

Therapeutic implications

Our hypothesis concerning nodular transformation also affects strategies of thyroid surgery for hyperthyroidism and strategies of radioiodine therapy for thyroid autonomy or euthyroid goiters. Even very small nodular structures that contain cell clones with an advantageous mutation left behind during partial resection of the thyroid could cause a recurrence. Indeed, a lower rate of recurrence has been reported for resections of the thyroid that completely removed nodular tissue (143) compared with partial resection (reviewed in Ref. 144). It is therefore necessary to remove all nodular thyroid tissue. This is especially true for the hilar region of the thyroid that is not removed in subtotal thyroidectomy (144). Moreover, total resection of nodular tissue is also the therapy of choice to deal with possible foci of malignant thyroid tissue within areas of nodular transformation (145). A reliable removal of all nodular tissue requires precise ultrasound and scintigraphy before surgery and careful morphological evaluation of the tissue during surgery. With regard to radioiodine treatment of thyroid autonomy, our hypothesis offers explanations for reports describing difficulties to equally determine the volume of thyroid autonomy in toxic multinodular goiters as a basis for the calculation of appropriate radioiodine doses (146). Finally, it is one explanation for the volume reduction of euthyroid goiters by radioiodine therapy (147).

Conclusion

Based on the identification of TSHR and Gs{alpha} mutations in autonomously functioning thyroid nodules and especially in microscopic areas of euthyroid goiters as well as on aspects proposed and reviewed recently (2, 4, 127), the sequence of events that very likely lead to thyroid autonomy in iodine-deficient areas are as follows (1). Early changes in response to iodine deficiency induce diffuse thyroid hyperplasia in children (130) (2). Due to increased proliferation during this stage of thyroid hyperplasia, mutagenesis is increased, thus resulting in a higher number of cells bearing a mutation. Some of these mutations confer constitutive activation of the cAMP cascade, e.g. TSHR and Gs{alpha} mutations (3). In a proliferating thyroid most cells divide and form small clones. The same is true for cells with a constitutively activated cAMP cascade. After reduction of TSHR stimulation by suppressed TSH (e.g. as the result of increased thyroid mass), small clones with constitutively activated cAMP cascade gain increased growth advantage due to their TSH-independent cAMP stimulation. They could thus form small hot foci that will develop into solitary autonomously functioning thyroid nodules or nodules within a multinodular goiter. A similar mechanism, however, with mutations in genes that favor dedifferentiation (e.g. ras oncogene) could be the origin of cold thyroid nodules. Indeed, ras mutations are very common in morphologically characterized thyroid adenomas or adenomatous nodules (148). However, TSHR mutations are exclusively found in hot thyroid nodules, whereas ras mutations are exclusively found in cold thyroid nodules (49, 149, 150).

Hyperplasia is very likely a response to cope with impaired thyroid hormone synthesis and to avoid hypothyroidism as long as possible. However, as outlined above, it probably leads to nodular transformation later in live. As suggested by Dumont et al. (127), hypertrophy, including higher blood flow and enhanced iodide trapping, would be an alternative response to economize iodine utilization and avoid hypothyroidism. As goiter is not common to all people in iodine-deficient areas, this alternative response might occur and go undetected. According to our hypothesis, such a response should prevent thyroid autonomy and nodular transformation. However, as both responses avoid hypothyroidism, and thyroid autonomy and nodular transformation are pathologies that develop later in life and have no obvious effect on reproduction, evolution was very likely not required to decide between the two alternatives.

Footnotes

1 This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG/Pa423/10-1), Deutsche Krebshilfe (10-1575-Pa1) and IZKF Leipzig, BMB-F, Interdisciplinary Center for Clinical Research at the University of Leipzig 9504 (01KS 9504, projects B5, B10 and B14). Back

Received November 10, 2000.

Revised February 26, 2001.

Accepted March 5, 2001.

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