321 REVIEW

Growth factors and goitrogenesis

S P Bidey1, D J Hill2 and M C Eggo3 1Endocrine Sciences Research Group, Department of Medicine, University of Manchester, Manchester, UK 2Lawson Research Institute, London, Ontario, Canada 3Department of Medicine, University of Birmingham, Birmingham, UK (Requests for offprints should be addressed to M C Eggo, Department of Medicine, Queen Elizabeth Hospital, Birmingham B15 2TH, UK)

Abstract By combining data from studies of multinodular non-toxic thyroidal vascular bed through angiogenesis is closely goitre (MNTG) with data from rat models of goitre co-ordinated with follicular cell expansion and folliculo- induction and in vitro models, a map of the growth factors neogenesis, and while the integrated paracrine actions of involved in goitrogenesis has been constructed. We have fibroblast growth factors, vascular endothelial growth addressed the roles of the insulin-like growth factors, factor and endothelin probably play central roles, add- transforming growth factors, fibroblast growth factors, itional, as yet elusive, factors are probably involved. The endothelins, etc. We hypothesise that an imbalance in the combination of in vitro and in vivo approaches, designed to interactions between the various growth factor axes exists address specific questions, will undoubtedly continue to in MNTG which favours cell replication. Thyrotrophin, prove invaluable in dissecting further the complex inter- although not significantly elevated in MNTG, exerts actions that exist between these growth factors, their critical effects through interactions with autocrine and binding proteins and receptors in goitrogenesis. paracrine factors and their receptors. Expansion of the Journal of Endocrinology (1999) 160, 321–332

Introduction expansion, and several studies have shown that poly- clonal and monoclonal nodules coexist within goitrous The homoeostasis of growth in differentiated epithelia thyroid tissue (Thomas et al. 1988, Kopp et al. 1994). reflects a critical balance between the promotion and Pioneering studies by Studer and colleagues have suppression of cell division. In accord with this generalised provided evidence that the onset and progression of model, the follicular epithelium of the normal adult MNTG reflect the expansion of follicular cells and follicles thyroid gland is essentially quiescent, and the loss of that have acquired different patterns of growth factor thyroid follicles and cell death is closely balanced against responsiveness, with individual nodules typically display- follicular neogenesis (Coclet et al. 1989). However, the ing different rates of cell division (Studer et al. 1992). An thyroid gland is among the most common sites of epithelial explanation for the high incidence of nodules (>50%) and hyperplasia, affecting up to 15% of the adult population, possibly micropapillary carcinoma (6–28·4%) found at and frequently requiring surgical intervention to relieve autopsy (Wheeler 1994) is that the stem cell population of compression of the trachea and laryngeal nerves. Typically the thyroid gland has innate growth potential. Accelerated presenting as ‘sporadic’ or multinodular non-toxic goitre by fluxes of growth stimulation, the localised expansion of (MNTG), the hyperplastic gland usually contains well- the follicular cell population will thus, given adequate defined nodules of varying size, surrounded by a normal time, give rise to focal hyperplasia (Studer et al. 1995). epithelium. Patients with MNTG are invariably euthy- While our understanding of the cellular and humoral roid, with thyrotrophin (TSH) levels within the normal mechanisms underlying MNTG remains incomplete, dys- physiological range (Gutekunst & Scriba 1989), and may regulation in the bioavailability of key growth regulators be contrasted with those with endemic (iodide-deficiency) has become an intensive area of research. Progress in such goitre, in whom thyroidal enlargement reflects an increase studies has, to date, revealed evidence for an involvement in cell proliferation as a consequence of dietary iodine of several autocrine growth stimulators and their receptors deprivation. Within the thyroid gland affected by MNTG, in the progression of MNTG. Prominent among these are individual nodules typically display different rates of the insulin-like growth factors (IGFs; Minuto et al. 1989),

Journal of Endocrinology (1999) 160, 321–332  1999 Society for Endocrinology Printed in Great Britain 0022–0795/99/0160–321 Online version via http://www.endocrinology.org

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fibroblast growth factors (FGFs; Thompson et al. 1998), induced rapidly following goitrogen treatment in combi- transforming growth factor â1 (TGFâ1; Gru¨beck- nation with a low iodine diet. Thyroid hypertrophy is Loebenstein et al. 1989, Perlino et al. 1996), hepatocyte evident after 1 week and hyperplasia at 2 weeks. While growth factor (HGF; Eccles et al. 1996), and the paracrine growth continues to 12 weeks, growth velocity declines. angiogenic factors, vascular endothelial growth factor However, despite the usefulness of this model, the danger (VEGF), placental-derived growth factor (PlGF) (Viglietto of extrapolating data from rats to humans should not be et al. 1997) and endothelin (Sellitti & Hughes 1990). underestimated. Furthermore MNTG is not synonymous with iodide-deficiency goitre and the evolution of these two diseases may differ. Notwithstanding the inherent Experimental systems limitations of both in vivo and in vitro models, it is clear that this experimental approach has afforded considerable in- Much of our understanding of the growth factor- sight into the roles that autocrine, paracrine and endocrine dependent regulation of thyroid cell proliferation and factors play in goitrogenesis of the human thyroid gland, differentiation is derived from studies with thyroid and in this review we shall attempt to integrate data from follicular cells in primary culture which, while retaining clinical studies, thyroid cell culture models and rat models a high level of differentiation, express and respond to of goitre induction to delineate possible underlying numerous growth factors and cytokines (Eggo et al. 1989, mechanisms. Bidey 1990, Dumont et al. 1991, Eggo & Sheppard 1994). Such models, which can be maintained and manipulated under closely controlled conditions, have facilitated IGFs, their binding proteins and their receptors detailed studies of the interactions of TSH with growth factors and their respective receptors with regard to Acromegaly, characterised by elevated levels of serum follicular cell growth and function, and have furnished IGF-I, has long been associated with goitre (>70% inci- clues to the roles of these factors in the goitrogenic process. dence; Wuster et al. 1991). Minuto et al. (1989) described It is important to recognise at the outset, however, the a 2-fold increase in immunoreactive IGF-I in patients with fundamental differences between in vitro and in vivo MNTG compared with controls, but IGF-I levels were models, which should be borne in mind when extrapolat- not elevated in goitres associated with Graves’ disease. ing effects observed in cell cultures to the intact thyroid Whether the incidence of goitres is increased in hyper- gland. The first drawback is that cell cultures condition insulinaemic states such as polycystic ovary syndrome has their medium with autocrine growth-regulating factors, not yet been established, although one study showed that which may attain far greater concentrations than those 28 children with MNTG all had grossly elevated insulin found within the intact thyroid gland in vivo. The levels levels (Abdullaeva et al. 1987). A more recent study of thyroid hormones released from endogenous stores of showed that the elevated levels of IGF-I in MNTG were iodinated thyroglobulin can also be very high and can located in the follicular cells (Maiorano et al. 1994). Sheep persist for a considerable time in culture (Becks et al. thyroid follicles in primary culture provided the first 1987). Accordingly, the possibility has to be considered in vitro model of the thyroid shown to express IGF-I and that regulation of cell growth and function in vitro may be IGF-II (Mak et al. 1985, Bachrach et al. 1988), a finding deranged by the local concentrations of cell-derived factors later confirmed in follicular cell cultures from other in the medium. Furthermore, since primary cultures may species, including humans (Ollis et al. 1989, Tode et al. be ‘contaminated’ to variable degrees with endothelial 1989) and pigs (Beere et al. 1991). Furthermore, prolifer- cells, C cells and fibroblasts, determination of which cell ation of follicular cells from a solitary nodule was found to type in the thyroid produces a given growth factor is be independent of exogenous IGF-I, confirming the difficult (Eggo 1998). In relation to the above, however, a potential autocrine role of IGF-I in proliferative thyroid further critical difference deserving special attention is disease (Williams et al. 1988). Cell culture studies of that, unlike follicular cells in culture, the intact thyroid follicular cells from several species have shown that IGFs follicle is closely associated with a fibroblast-containing synergise with, and are obligatory for, the stimulatory mesenchyme. In addition to synthesising a variety of effects of TSH on both thyroid growth and function (Eggo matrix molecules which may moderate follicular cell et al. 1985, 1990, Ollis et al. 1989, Williams et al. 1988), function and growth, this cell layer will also contribute to whereas TSH alone is ineffective. The requirement for the pool of growth factors to which follicular cells are both TSH and IGF-I for follicular cell growth in vitro has exposed, which may also contribute to the adjustment of subsequently been confirmed in vivo. Thus, in adults with growth control in follicular hyperplasia. The matrix itself hypopituitarism, increases in serum IGF-I effected by may also, as detailed below, serve to sequestrate specific growth hormone replacement are not associated with an cell-derived growth factors and regulate their subsequent increased thyroid mass in the absence of TSH over a availability and cellular action. Rat models of goitrogenesis 6 month period (Cheung et al. 1996). The mechanism obviate the first of these problems, and these may be of interaction between TSH and IGF-I has been

Journal of Endocrinology (1999) 160, 321–332

Downloaded from Bioscientifica.com at 09/28/2021 08:55:23AM via free access Growth factors and goitrogenesis · SPBIDEYand others 323 extensively investigated and remains a major focus for study (Santisteban et al. 1987, 1992, Brenner-Gati et al. 1988, Saji & Kohn 1991, Liu et al. 1996, Yamamoto et al. 1996, Ortiz et al. 1997). In cultures of human thyroid follicular cells, Tode et al. (1989) found evidence for an upregulation by TSH of IGF-I expression, and a study of IGF-I mRNA levels in pig follicles maintained in culture (Hofbauer et al. 1995) supports this finding. Thyroid follicular cells express the type I IGF receptor, but, although many of the data on IGF receptor regulation have been obtained using the rat thyroid cell strain FRTL-5 (Tramontano et al. 1987, Perrotti et al. 1989, Takahashi et al. 1991, Saji et al. 1992), caution should be exercised in extrapolating such findings to thyroid follicular cells generally. In dog thyroid cells for example, TSH does not regulate IGF receptor expression, but does regulate the functionally similar insulin receptor (Burikhanov et al. Figure 1 Secretion (solid arrows) and effects (dashed arrows) of 1996). Studies with follicular cells from several species IGFs and IGFBPs on cells comprising the thyroid, i.e thyroid have found insulin and IGF-I receptors to coexist, and to follicular, endothelial, fibroblasts and C cells. The effects of known modulators of thyroid function and/or growth, i.e. TSH, iodide and date their actions have been found to be identical. There PKC activation, on their contribution to the pools of IGF and are no data at present on the regulation of IGF or insulin IGFBPs are shown (dotted lines) at the bottom. The effects of receptor affinity or number in human thyroid cells, TSH may not be limited to the follicular cells. TSH receptor although this may prove to be an important area for future expression is not exclusive to thyroid follicular cells, and cAMP, prostaglandins, T3 and T4, which are produced rapidly by the investigation. follicular cells after TSH receptor activation, may have paracrine While the relative expression levels of IGF peptide and effects on the other cells, influencing their secretion of IGFs and receptors may become deranged in the goitrogenic pro- IGFBPs. cess, the availability of IGF-I to component cells of the thyroid follicle may be more generally influenced and more tightly controlled by the IGF-binding proteins (Ogasawara et al. 1989), may effectively circumvent the (IGFBPs) (Minuto et al. 1989, Tode et al. 1989, Eggo IGF-I-sequestering actions of the IGFBPs. Whether a et al. 1996). IGFBPs are synthesised and secreted by protease capable of effecting such a cleavage reaction exists thyroid cells in culture, and their production is determined within the thyroid gland has yet to be determined. by several regulators of thyroid growth and function. Control of IGF-I bioavailability within the follicular Although the types of IGFBPs produced by thyroid cells microenvironment may also be effected by iodide supply. differ between species, inhibition of synthesis by TSH is In cultured porcine thyroid cells, IGF-I gene transcription common. This could increase the availability of IGF-I and and peptide release are potently suppressed by intracellular contribute to the TSH-mediated increase in growth and organified iodide (Beere et al. 1995, Hofbauer et al. 1995), function of the follicular epithelium (Bachrach et al. 1988, in parallel with a progressive reduction in the rate of 1989, 1991, Eggo et al. 1991, Wang et al. 1991). Con- cell proliferation in FRTL-5 cells (Becks et al. 1988, versely, inhibitors of thyroid function produce increases in Tramontano et al. 1989). The reactive species of iodide is thyroidal IGFBP synthesis, particularly that of IGFBP-3 postulated to be an iodolactone derivative of arachidonic (Bachrach et al. 1991, Eggo et al. 1991, 1996). IGFBPs are acid (Dugrillon et al. 1994, Pisarev et al. 1994), although not, however, always inhibitory to IGF bioactivity and further studies of the role of iodide per se, as well as thyroid some of the forms produced, for example those bound to hormones and other organified intermediates of iodide, are the extracellular matrix, may facilitate IGF-I interaction needed. Although not yet conclusively proven, derepres- with its receptor. IGFBPs may also prolong the half-life of sion of IGF-I synthesis and release after transfer of follicular IGF internalised in cells by allowing nuclear uptake and cells to iodide-deficient conditions may be the in vitro localisation (Michell et al. 1997) or they may have effects counterpart of a mechanism that contributes, at least independent of their interaction with IGFs. A final mech- partially, to the pathogenesis of iodide-deficiency goitre. anism controlling the equilibrium of IGF–IGF receptor– Even under iodide-replete conditions, loss or impairment IGFBP interaction may reside in the cell-mediated of a component critical to the correct intracellular process- processing of IGF-I. Proteolytic removal of three amino ing of iodide may similarly derepress the synthesis and acids from the N-terminal region produces a product that autocrine bioavailability of thyroidal IGF-I. is able to interact with the IGF receptor but not the Figure 1 summarises the current knowledge of the binding protein. The processing of IGF-I to a truncated production and effects of IGF-I and IGFBPs by the cells form in this way, as first detected in porcine uterus comprising the human thyroid, i.e follicular, endothelial,

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fibroblast and C cells. The contribution of these cells to the of IGFBP-3 (human) (Eggo et al. 1996) and a decrease in pool of IGFs and IGFBPs within the gland is shown in IGF-I synthesis (porcine) (Beere et al. 1991). Both actions solid arrows and, where known, the biological effects of are consistent with a decline in follicular IGF-I bioavail- IGFs and IGFBPs on the growth of the cells are shown. ability, which may serve to reinforce the direct suppression Little is known of the paracrine role of the C cells, from of cell proliferation activity afforded by TGFâ1. TGFâ1 which medullary thyroid carcinomas arise. One of their synthesis at an mRNA level has been confirmed within products, somatostatin, has been shown to have inhibitory follicular cell cultures derived from the thyroid glands of effects on both FRTL-5 cell growth and function, but numerous species. In porcine follicular cells (Cowin et al. their contribution to the IGF/IGFBP pool is not known. 1992), only TGFâ1 is present and TGFâs 2 and 3 have The figure also shows the effect of TSH, which is probably not been detected. Analysis of TGFâ1 in thyroid cell- due to its interaction with thyroid follicular cells. How- conditioned culture medium by bioassay and receptor ever, TSH binding to its receptors will result in increased assay has revealed predominantly the mature (i.e. active) cAMP which freely diffuses from the cells. Similarly form of the peptide, suggesting that normal follicular cells prostaglandin synthesis and release will be altered by TSH, in culture both synthesise TGFâ1 and retain an endogen- which undoubtedly can have paracrine effects. Another ous mechanism for TGFâ1 activation (Cowin et al. 1992, effect of TSH will be to increase thyroid hormone Cowin & Bidey 1995). Indirect evidence for the presence (tri-iodothyronine (T3) and thyroxine (T4)) synthesis and of active TGFâ1 in such cultures has also been confirmed release. All the cells in the thyroid will be responsive to T3 by the enhanced rate of follicular cell proliferation after and T4, although their effects on IGF/IGFBP synthesis immunoadsorption of autocrine TGFâ1 (Cowin & Bidey and release are not known. Iodide effects may be mediated 1995). The precise mechanism of thyroidal TGFâ1 acti- through their ability to modulate T3 and T4 synthesis vation remains to be elucidated, but support for a con- which will be stimulatory at low concentrations and tributory role of the plasminogen/plasminogen activator inhibitory at high concentrations. The direct effects of (PA) system comes from the observed release of tissue (t-) iodide per se in the thyroid follicular cells, which can and urokinase (u-)-like PAs in response to TSH (Mak et al. maintain a high intracellular iodide concentration, and the 1984), the upregulation by TSH of endogenous cathepsins effects of the organified intermediate are also possible (Phillips et al. 1989), and the increased TGFâ1 bioactivity mediators of follicular production of growth factors. Also in medium conditioned by plasmin-treated follicular cells included in the figure are the effects of epidermal growth (Cowin & Bidey 1994). factor (EGF) and protein kinase C (PKC) activation. Although exposure to iodide induces a potent down- Thyroid cells may be exposed to EGF in the bloodstream regulation of IGF-I synthesis in thyroid follicular cells from or through TGFá, secreted by endothelial cells, which at least some species (Beere et al. 1995, Hofbauer et al. interacts with the EGF receptor. Activation of the EGF 1995), changes in the expression of TGFâ1 have also been receptor or PKC stimulates IGFBP secretion (Eggo et al. observed under similar conditions. Thus the major TGFâ1 1996) from the thyroid follicular cells. mRNA species (2·5 kB) is upregulated in porcine follicular cells after exposure to iodide (Cowin et al. 1992, Yuasa et al. 1992), and this was initially thought to explain, Latent and activated forms of TGFâ and at least in part, the expansion of the thyroid follicular mass their receptors in endemic iodide deficiency (Gru¨beck-Loebenstein et al. 1989). However, in rats, thyroid hyperplasia induced While an increase in IGF-I availability is a probable factor by iodide deficiency and goitrogen is paradoxically in enlargement of the thyroid mass, the actions are likely to accompanied by an increase in TGFâ1 expression and the be integrated with those of further factors of autocrine or arrest of goitre growth after 4 weeks. This initially paracrine origin within the thyroid follicle. As observed in surprising result is thought to reflect a critical role of numerous other epithelial models, TGFâ1 is a potent TGFâ1 in stabilising goitre mass (Logan et al. 1994). inhibitor of thyroid follicular cell proliferation in vitro Evidence that this may reflect a direct action of TSH on (Morris et al. 1988, Tsushima et al. 1988, Gru¨beck- TGFâ1 synthesis is supported by the in vitro findings of a Loebenstein et al. 1989, Taton et al. 1993), reflecting direct stimulatory effect of TSH on thyroidal TGFâ1 blockade of the cell cycle through suppression of the expression in FRTL-5 cells (Pekary et al. 1995) and of a actions of the cyclins and their regulating kinases (Ewen greatly enhanced proliferative response to TSH after et al. 1993, Geng & Weinberg 1993, Koff et al. 1993). The TGFâ1 immunoadsorption from porcine follicular cell potent suppression of cell proliferation afforded by TGFâ1 cultures (Cowin et al. 1996). Presumably therefore, under thus serves to oppose both the effect of TSH and the conditions of iodide deficiency in vivo, the reduction of mitogenic action of autocrine growth factors. Interestingly, TGFâ1 expression would be masked and indeed reversed several links have been demonstrated between TGFâ1 and by the TGFâ1 synthesis response to TSH. thyroidal IGF-I bioavailability. For example, exposure of The role of TGFâ1 in MNTG, as opposed to iodine- follicular cells to TGFâ1 leads to an increase in the release insufficiency goitre, is less clear. Some forms of epithelial

Journal of Endocrinology (1999) 160, 321–332

Downloaded from Bioscientifica.com at 09/28/2021 08:55:23AM via free access Growth factors and goitrogenesis · SPBIDEYand others 325 hyperplasia are known to be associated with aberrant TGFâ1-mediated growth control (Cui et al. 1995, Roberts 1998), but clarification of the role for TGFâ1 in MNTG is clouded by conflicting reports; Perlino et al. (1996), for example, have reported a reduction in TGFâ1 in hyper- plastic, compared with normal, thyroid tissue, while a contemporary report (Morosini et al. 1996) has recorded higher levels in recurrent goitre after thyroid surgery than in patients remaining in remission. Nevertheless, the evidence from the rat model implicating a critical role of TGFâ1 in the limitation of iodide-deficiency goitre sug- gests that dysregulation of the stimulatory growth factor profile of the hyperplasia of MNTG may include a key change in TGFâ1 bioavailability, which is clearly Figure 2 Secretion (solid arrows) and effects (dashed arrows) of deserving of further study. TGFâ1 on cells comprising the thyroid. The effects of known As in cells from other forms of epithelial hyperplasia, modulators of thyroid function/growth, i.e. TSH and iodide, and studies of follicular cells from MNTG have revealed a high the putative role of the plasminogen activators (PA) are shown incidence of insensitivity to the actions of TGFâ1 (Asmis (dotted lines) at the bottom. PAs are positively regulated by TSH and may activate latent TGFâ. The effects of T3 and T4 on TGF et al. 1996) irrespective of the total TGFâ1 content of the secretion or receptor expression from any of the different cell cell-conditioned medium (Cowin & Bidey 1996). The types are not known. concept of a localised cellular resistance to TGFâ1 within the MNTG-affected thyroid gland is supported by a loss of response to TGFâ1 in follicular cells from feline goitres Figure 2 summarises our current knowledge of TGFâ (Asmis et al. 1996). Although changes in TGFâ1 receptor production and effects in the thyroid. Some of the regu- (TâR) expression or signal transduction remain to be latory data shown have not been confirmed in human confirmed as factors effecting loss of TGFâ1 responsive- thyroids. The contributions of all the cell types to the pool ness in thyroid hyperplasia, mutations in the type 2 TâR of this growth factor are shown and the effects of TGFâ on isoform are commonplace in epithelial neoplasia (Kim & these cell types are shown, where known. The role of PAs Kim 1996), while other epithelial tumours are deficient in as potential activators of latent TGFâ is also shown. TSH the ALK-5 isoform of the type I TâR (Baldwin et al. effects, which, as noted previously, may invoke the 1996). Interestingly, and as seen in other epithelia (Kim & secretion of powerful paracrine factors from the follicular Kim 1996), follicular cells from some human MNTGs also cells, and the effects of iodide and T3 and T4 are shown. demonstrate impaired TGFâ1 processing (Cowin & Bidey 1996). This suggests that a loss of a component critical to the cell membrane-mediated proteolysis of the latent FGFs and FGF receptors TGFâ1 precursor may impair local TGFâ1 bioavailability at the follicular cell level. In some hyperplastic epithelia, Follicular cells from MNTG tissue express both FGF-1 including the prostate (Timme et al. 1995) and and FGF-2, together with FGF receptor-1 (FGFR1) endometrium (Gold et al. 1994), TGFâ1 resistance is (Thompson et al. 1998), and this expression is greatly accompanied by high levels of endogenous TGFâ1, which increased compared with the normal thyroid. In rats, may induce expansion of the underlying TGFâ1- compelling evidence for the role of FGF in goitrogenesis responsive mesenchyme. Accordingly, and in contrast comes from in vivo experiments in which administration of with the growth-suppressive effect exerted on epithelia, it FGF-1 produced a 43% increase in thyroid weight by is conceivable that TGFâ1 of follicular cell origin may 6 days and a decrease in thyroid deiodinase in 2–8 h enhance the growth of perifollicular fibroblasts within the (Chanoine et al. 1992, DeVito et al. 1992). FGF has been immediate vicinity of the thyroid follicle, so that the found in pig (Emoto et al. 1991) and sheep thyroid cells in perifollicular mesenchyme may continue to expand. This culture, and TSH has been shown to increase FGF-2 may contribute to the fibrotic lesions and excessive matrix expression in the latter (Hill et al. 1994). There is indirect deposition commonly found in MNTG-affected thyroid evidence for FGF production in FRTL-5 cells, for which tissue. Whether as a consequence of cellular TGFâ1 these factors are potent mitogens (Logan et al. 1992). In resistance or diminished cellular processing therefore, the primary cultures, the effects of FGF on thyroid cells appear balance of current evidence suggests that, as in thyroid inconsistent but this may be related to regulation of tumorigenesis (Blaydes et al. 1996), a localised change in receptor expression. An initial report of studies with dog TGFâ1 bioavailability within the follicular microenviron- thyroid cells showed mitogenic (Roger & Dumont 1984) ment represents a key factor in the development of thyroid and dedifferentiating effects (Gerard et al. 1989), while hyperplasia. studies using human thyroid cells found no effect on

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growth (Taylor et al. 1993). FGFs are not normally secreted in free form, and their effectiveness as mitogens is dependent upon release from the extracellular matrix. As is the case with TGFâ therefore, the role of the FGFs in thyroid remodelling very likely depends on the presence of specific proteases to release them from sequestration within the extracellular matrix. Potential candidates for such cleavage are the PAs, which thyroid follicular cells produce in abundance (Mak et al. 1984) as well as cathepsins and matrix metalloproteinases.

Expression of growth factors and their receptors in thyroids of goitrogen-treated rats Figure 3 Secretion (solid arrows) and effects (dashed arrows) of In the goitrogen-treated rat, thyroidal FGF-2 expression is FGFs on cells comprising the thyroid. Only FGF-1 and FGF-2 effects are considered, although other FGFs may be expressed and maximally induced after 1 week of treatment, precedes may have different effects. The effects of known modulators of DNA synthesis and subsequently declines in parallel with thyroid function/growth, i.e TSH and iodide, are shown (dotted the growth velocity of the gland. An increase in FGF-2 lines) at the bottom. The putative role of the PAs, which are immunoreactivity in the goitrous thyroid glands of these positively regulated by TSH, in releasing FGF from the matrix is shown. The effects of T3 and T4 on FGF production and FGF animals has been histologically localised to the thyrocytes receptor expression in any of the different cell types are not (Logan et al. 1994, Patel et al. 1996). Large reserves of known. FGF-2 are sequestered within the perifollicular stroma of the rat thyroid gland, unlike that in human thyroid, and an earlier stage in the goitrogenic response may involve pro- FGFR1 expression and that of IGF-I and IGFBP-2 and -3, teolysis of FGF-2 and release from the stromal store. and under such conditions no goitre or histological changes FGFR-1 mRNA is also expressed in the thyrocytes and are evident, despite the fall in T3 to almost undetectable increases approximately 1 week after that of FGF-2 levels as a consequence of iodide deficiency. Subsequent mRNA. This observation provides evidence that FGF-2 treatment of goitrogen-exposed hypophysectomised rats may drive the expression of its own receptor (Becks et al. with TSH gives rise to a hyperplastic tissue response and 1994). In the same model, thyrocyte IGF-I mRNA and increased abundance of FGF-2 and FGFR1, IGF-I, peptide are induced within 1 week of goitrogen treatment, IGFBP-2 and IGFBP-3. Hypophysectomy also prevents and are increased further at 2 weeks. Like FGF-2, IGF-I an increase in TGFâ expression in response to goitrogen levels thereafter decline with growth velocity and are that is not reversed by treatment with T3. These results reduced further during goitre regression. IGFBP-2 and suggest that in iodine-deficiency goitre in the rat, TSH is IGFBP-3 mRNAs and protein levels increase progressively a direct mediator of the FGF and IGF expression which with goitrogen treatment and decline during regression drives the goitre, while TGFâ expression may also be (Phillips et al. 1994). This may represent a negative regu- linked to prevailing T3 levels. lation of the mitogenic action of IGF as goitre homoeostasis Figure 3 summarises current knowledge of FGF pro- is established. These data contrast with those from in vitro duction and actions in the thyroid and is limited to FGF-1 cell cultures where TSH inhibits follicular cell IGFBP and FGF-2. Some of the data have not been confirmed in synthesis. Whether the differences are due to the species, human thyroid cells. The role(s) of other members of the to the contributions from the other cells in the thyroid FGF family and the expression of FGFR isoforms in (either the follicular cells or the pool of IGFBPs during the MNTG compared with normal tissue await to be deter- goitrogenic response) or to differences in the aetiology of mined. The role of known modulators of thyroid function, iodide-deficiency goitre and MNTG remains to be clari- i.e. TSH and iodide, and the putative role of PAs in fied. IGFBP-5 is expressed only in the C cells and its releasing FGF from the extracellular matrix are shown. expression declines during goitrogen administration but The effects of T3 and T4 on FGF and FGF receptor is more abundant during goitre regression as C cell hyper- production by any of the cells are not known. plasia occurs. TGFâ1 is present in C cells even in normal animals and is abundant during the C cell hyperplasia that accompanies goitre regression (Logan et al. 1994). Angiogenic factors Hypophysectomised rats have been used to determine the intermediary role of TSH in triggering the changes in Expansion of the follicular epithelium during thyroid growth factor expression in experimentally induced goitre. hyperplasia is dependent upon a co-ordinated expansion Hypophysectomy prevents the rapid rise in FGF-2 and of the vascular bed supplying the thyroid gland. As a

Journal of Endocrinology (1999) 160, 321–332

Downloaded from Bioscientifica.com at 09/28/2021 08:55:23AM via free access Growth factors and goitrogenesis · SPBIDEYand others 327 consequence of their established angiogenic actions, the FGFs may participate in paracrine actions between follicu- lar and endothelial cells within the expanding tissue mass, in addition to autocrine actions between adjacent follicular cells. Goodman & Rone (1987) showed that rat thyroid cells in culture (both primary and FRTL cells) produced endotheliotropic chemoattractant activity. They did not identify the factor, but their data convincingly supported the hypothesis of Wollman et al. (1978) that the growth of perifollicular blood vessels was stimulated by angiogenic factors secreted by follicular cells. Similarly, Greil et al. (1989) showed the release of an endothelial cell growth factor from pig thyroid cells. Recent data have shown that VEGF, an angiogenic growth factor, is synthesised by thyroid follicular cells and is increased in goitres in the rat, Figure 4 Secretion (solid arrows) and effects (dashed arrows) of and in human thyroid cells in vitro, following TSH the angiogenic factors VEGF, PlGF and ANG on cells comprising stimulation (Sato et al. 1995). Although this growth factor the thyroid. The effects of known modulators of thyroid increases the permeability of the endothelium and is growth/function, i.e TSH and iodide, are shown (dotted lines) at the bottom. The role of thrombospondin, which is produced by mitogenic for endothelial cells in the thyroid as elsewhere follicular cells, is thought to be antiangiogenic and is illustrated. (Sato et al. 1997), its effects on human thyroid follicular The effects of T3 and T4 on angiogenic factor production and cell growth and function are not known. KDR and Flt, the receptor expression are not known. receptors for VEGF, were until recently thought to be exclusive to cells of the vasculature. However, they have paracrine factors may also be involved in controlling the now been located in diverse tissues, including trophoblast angiogenic response within the hyperplastic thyroid gland. cells (Ahmed et al. 1997) and smooth muscle cells from the One of the products of plasminogen cleavage by PA in the uterus (Brown et al. 1997). Significantly, recent data presence of -cysteine or reduced glutathione is angiostatin obtained using FRTL-5 cells indicate that this clonal (Stathakis et al. 1997). Since the synthesis of both uPA and thyroid cell line produces not only VEGF but also the tPA are increased by TSH (Mak et al. 1984), angiostatin Flt-1 receptor, as demonstrated by reverse transcriptase levels may also be raised. Conceivably, this factor may act PCR (Wang et al. 1998). Addition of VEGF to FRTL-5 to inhibit the angiogenic response and limit goitre size. As cultures blocks TSH-stimulated growth and function, in the control of thyroid follicular cell proliferation itself, presumably through the Flt-1 receptor because the KDR angiogenesis within the hyperplastic thyroid gland is receptor was not found (Wang et al. 1998). PlGF has also dependent upon specific factors being available in the been identified as a product of rat thyroid follicular cells. correct location at a given time. In order for angiostatin to Within the thyroid gland of goitrogen-treated rats, PlGF be effective therefore, plasminogen and glutathione, or binds only to the Flt-1 receptor and this is increased by another reducing agent, must both be available. Figure 4 chronic activation of the thyroid by TSH (Viglietto et al. illustrates our current knowledge of the secretion and 1997). Presumably therefore PlGF may exert the same effects of VEGF, PlGF and ANG in the thyroid. The effect as VEGF, at least in FRTL-5 cells. These novel effects of known modulators of thyroid function, i.e. TSH findings may implicate VEGF in the growth attenuation and iodide, are shown at the bottom of the figure. As noted found during goitrogenesis in the rat. Induction of VEGF above, TSH stimulates follicular cell secretion of paracrine synthesis by TSH, while initially promoting angiogenesis, factors. Thrombospondin, which is secreted by thyroid may also act as a brake on the mitogenic stimulation of the follicular cells (Prabakaran et al. 1993), is generally thought follicular cells by TSH, in an analogous manner to TGFâ. to have a negative effect on the angiogenic process. In the The study of Viglietto et al. (1997) did not, however, rat model of goitre, thrombospondin is reduced to negli- reveal consistent staining of VEGF receptors on the gible levels within 2 weeks of goitrogen treatment (Patel follicular cells of goitrogen-treated rats, although endo- et al. 1996) consistent with inhibition by TSH of its thelial cell VEGF receptors were increased. Whether secretion, shown in vitro (Bellon et al. 1994). The effects of human thyroid follicular cells possess VEGF receptors and T3 and T4 on the production of the angiogenic factors and can respond to VEGF remains to be determined. their receptors are not known. No data are as yet available on the goitrogenic role of the newest member of the angiogenic growth factor Endothelins, atrial natriuretic peptide (ANP) and family, angiopoietin (ANG) and its receptor Tie-1, their receptors although it is reasonable to predict its involvement in goitrogenesis. Whether its actions are restricted to the Endothelin-1 (ET-1) is secreted by rat and pig thyroid endothelium will also need to be determined. Other follicular cells (Colin et al. 1992) and human (Tseng et al.

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1993) and FRTL-5 cells (Vainio et al. 1996). ET-1 is a least at high ANP concentrations) are coupled to the 21-amino acid polypeptide formed by proteolytic cleavage release of cGMP (Tseng et al. 1990). ANP treatment from a large precursor molecule by the action of endo- inhibits thyroglobulin release from human thyroid cell thelin converting enzyme (ECE), a membrane-bound cultures, secondary to a reduction in intracellular cAMP neutral metalloprotease. ET-1 is widely distributed (Sellitti et al. 1989). In FRTL-5 cells, cGMP accumulation throughout the body and is synthesised primarily, but not is functionally coupled to the ANP receptor. TSH down- exclusively, by the vascular endothelium. ET-2 and ET-3 regulates ANP receptor number (Tseng et al. 1991) in are synthesised by vascular smooth muscle cells. Receptors human thyroid cells but there are no data on the regulation for ETs are classified as ETA, which, when expressed on of ANP expression. In endothelial cells, both ANP and smooth muscle cells, are responsible for vasoconstriction, ET-1 regulate VEGF synthesis, ANP being inhibitory and ETB, which are found in the endothelial cells and are whereas ET-1 is stimulatory (Pedram et al. 1997). In this responsible for the release of nitric oxide, prostacyclin and way, these vasoactive peptides can effect a close regulation ANP. The role of ET-1 in the thyroid itself has been of endothelial cell proliferation and invasion. Since examined using ET-1 knock-out mice. Homozygous mice VEGF, ANP and ET-1 are all products of thyroid follicular were found to have smaller thyroid glands than normal, cells, derangements in their mutual regulation and inter- which showed no midline fusion (Kurihara et al. 1995). action are likely to be important in determining the ET-2 and -3 have been found to bind to porcine thyroid balance of cell growth promotion and limitation in cells, implying redundancy in the ligands capable of the angiogenesis associated with goitrous expansion of the interacting with the receptors. ET-1 inhibits iodide uptake thyroid gland. in pig thyroid cells but not apparently by inhibiting cAMP formation (Tsushima et al. 1994). In contrast, in FRTL-5 cells, ET-1 inhibits TSH-stimulated DNA synthesis by Other growth factors and their receptors interfering with cAMP production (Miyakawa et al. 1992). In human thyroid cells in culture, Eguchi et al. (1993) HGF is secreted from normal human thyroid cells and is a found that nanomolar concentrations induced growth, and potent mitogen for both these cells (Eccles et al. 1996) and Tseng et al. (1993) found that TGFâ1 stimulated ET those from dog thyroids (Dremier et al. 1994). In the latter secretion and increased ET receptor number. This is in study, HGF, like FGF, was only found to have growth- contrast with the effects of TSH which had no effect on promoting and dedifferentiating effects if added shortly either ET synthesis or receptor number. In pig thyroid after the cells were plated, i.e. at the rapid growth stage. cells, treatment with TSH reduced the mRNA for ET-1 This would indicate a cell density-dependence of HGF whereas supraphysiological levels of iodide (0·1 mM) receptor expression. An early study of mRNA encoding increased both mRNA and protein levels (Isozaki et al. the HGF receptor, known as met, in normal human 1993). In studies of goitre induction and involution in rats, thyroid tissue demonstrated abundant mRNA expression Colin et al. (1994, 1995) showed that mature ET-1 within but an absence of protein (Di Renzo et al. 1991). the thyroid gland was increased 5-fold during the hyper- However, in thyroid carcinoma, c-met protein levels were plastic stage, and remained high in the subsequent rapid elevated over 100-fold. Expression of met and HGF has phase of involution. Both ETA and ETB receptors were recently been examined in non-neoplastic nodules using detectable, as was ECE. These workers also examined the immunostaining (Trovato et al. 1998). In 40% of the nitric oxide synthase genes (NOS I, II and II) and found samples analysed, 1–3% of the cells were positive for HGF that NOS I and III were increased in the expansive stage of and its receptor. By comparison with normal (0%) and goitrogenesis, whereas during involution their levels rap- non-papillary carcinoma (0%), this is elevated but does not idly returned to normal. Concurrent treatment with both a approach the positivity found in papillary carcinoma goitrogen and a NOS inhibitor reduced the vascular ex- (15–46% cells positive for HGF and c-met in 90% of the pansion by more than 30%. Because of the highly vascular- samples). The potency of HGF as a mitogen is significantly ised nature of thyroid tissue, it is not surprising that factors greater than that of other growth factors, therefore this capable of controlling vascular contractility are available factor clearly has the potential to influence thyroid growth locally. That these factors have effects on follicular cells is and function. In considering the actions of factors such as more surprising, but illustrates again the diversity of para- HGF, FGF and VEGF in the goitrogenic process, the crine communications between the component cell types possible existence of binding proteins that may determine within the thyroid gland, and the delicate balance that or modify the nature of the cellular responses should be exists between expression of growth factors, their receptors considered. These may be truncated forms of the receptors and other mediators such as binding proteins and proteases. as found for FGF (Hanneken et al. 1994) or, like the ANP has been identified in human and pig thyroid IGFBPs, they may have a completely different structure. follicular cells and is secreted basolaterally by pig thyroid In any event, their contribution to the regulation of the cells in culture (Sellitti & Hughes 1990). High-affinity biological activity of the corresponding growth factor, ANP receptors are found on human thyroid cells which (at which is at present unknown, may be critical.

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With the exception of IGFs, all of the autocrine/ Bachrach LK, Liu FR, Burrow GN & Eggo MC 1989 paracrine factors in the thyroid have dedifferentiating Characterization of insulin like growth factor binding proteins from sheep thyroid cells. Endocrinology 125 2831–2838. effects on thyroid follicular cells and, except for TGFâ and Bachrach LK, Eggo MC, Burrow GN, Liu F, TramT&PowellDR possibly VEGF, are mitogenic. 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