TSH-activated signaling pathways in thyroid tumorigenesis

Molecular and Cellular Endocrinology 213 (2003) 31–45

Review

TSH-activated signaling pathways in thyroid tumorigenesis Marcos Rivas, Pilar Santisteban∗ Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Cient´ıficas and Universidad Autónoma de Madrid, Arturo Duperier # 4, E-28029 Madrid, Spain

Abstract Thyrotropin (TSH) is considered the main regulator of thyrocyte differentiation and proliferation. Thus, the characterization of the different signaling pathways triggered by TSH on these cells is of major interest in order to understand the mechanisms implicated in thyroid pathology. In this review we focus on the different signaling pathways involved in TSH-mediated proliferation and their role in thyroid transformation and tumorigenesis. TSH mitogenic activities are mediated largely by cAMP, which in turn may activate protein kinase (PKA)-dependent and independent processes. We analyze the effects of increased cAMP levels and PKA activity during cell cycle progression and the role of this signaling pathway in thyroid tumor initiation. Alternative pathways to PKA in the cAMP-mediated proliferation appear to involve the small GTPases Rap1 and Ras. We analyze the Ras effectors (PI3K, RalGDS and Raf) that are thought to mediate its oncogenic activity, as well as the ability of Ras to induce apoptosis in thyrocytes. Finally, we discuss the activation of the PLC/PKC cascade by TSH in thyroid cells and the role of this signaling pathway in the TSH-mediated proliferation and tumorigenesis. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Thyrotropin; Thyrocyte; Tumorigenesis

1. Introduction Thyrotropin (TSH), a heterodimeric glycoprotein hormone synthesized by pituitary thyrotrophs, is considered the main regulator of thyroid function and growth acting through its G protein-coupled receptor (TSHR) in the cell surface of thyrocytes (Vassart and Dumont, 1992; Szkudlinski et al., 2002). TSH activities are largely mediated by an increase in cAMP intracellular levels (via Gs proteins), which activate multiple signaling pathways to regulate thyroid differentiation, proliferation and function (Medina and Santisteban, 2000; Kimura et al., 2001). In addition, some activities are mediated by the activation of the PLC–Ca2+ cascade (via Gq proteins), which has been shown to promote proliferation while inducing dedifferentiation of thyrocytes (Bachrach et al., 1985; Roger et al., 1986). Therefore, inappropriate activation of the signals triggered by TSH may contribute to a sub-set of thyroid pathologies, including thyroid cancer. In order to understand thyroid proliferation, and eventually thyroid pathology, several cell systems from different

∗

Corresponding author. E-mail address: [email protected] (P. Santisteban).

species have been studied. For still unknown reasons, bovine and pig thyrocytes (Gerard et al., 1989; Dumont et al., 1991) do not respond to TSH as a mitogenic stimulus. Therefore, we have compiled the information obtained from the more commonly analyzed cell systems concerning the effects of TSH on thyroid proliferation, which are the stable cell lines derived from rat thyrocytes FRTL-5, WRT, and PCCL3 cells (Ambesi-Impiombato et al., 1980; Brandi et al., 1987; Fusco et al., 1987) and primary cultures from dog (Rapoport, 1976; Roger et al., 1987) and human thyrocytes (Roger et al., 1988; Williams et al., 1988). Such diversity of cell models used implicates that sometimes different, if not opposite, mechanisms that lead to thyroid proliferation have been described (Kimura et al., 2001). Data obtained from both cell cultures and animal models demonstrated that TSH has a mitogenic effect on thyroid follicular cells. However, TSH-induced proliferation seems to be dependent on the presence of additional growth factors, such as insulin/IGF-1 or those present in serum (Medina and Santisteban, 2000; Kimura et al., 2001). Moreover, TSH can indirectly stimulate proliferation by inducing the expression of autocrine growth factors (Takahashi et al., 1990; Becks et al., 1994) or growth factor receptors (Burikhanov et al., 1996; Cocks et al., 2000) that may act as permissive factors for the proliferative effects of TSH. The interaction between

0303-7207/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2003.10.029

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TSH and other growth factors (mainly insulin/IGF-1), and its effects on thyroid proliferation has extensively been analyzed (Ariga et al., 2000; Medina and Santisteban, 2000; Kimura et al., 2001; Dremier et al., 2002) and will not be discussed in this review. Similarly, detailed information about the role of cyclins/CDKs and immediate early genes in the TSH-mediated cell cycle progression has been compiled elsewhere (Deleu et al., 1999; Medina and Santisteban, 2000; Wang et al., 2000; Kimura et al., 2001; Dremier et al., 2002).

2. The TSH-receptor TSH controls thyroid function by binding to the TSH-receptor (TSHR), a seven-transmembrane receptor (Fig. 1) located at the cell surface of thyrocytes that regulates thyroid cell proliferation as well as thyroid hormone synthesis and release (Postiglione et al., 2002; Szkudlinski et al., 2002). Studies from transgenic mice indicate that the TSHR is important in both maintaining the follicular organization and controlling the thyroid gland growth (Marians et al., 2002; Postiglione et al., 2002). Activation of TSHR in thyroid membranes induces the coupling of different G proteins (Laugwitz et al., 1996; Allgeier et al., 1997). However, most of the activities of TSHR are mediated through a heterotrimeric Gs protein, which activates the adenylate cyclase/cAMP cascade (Vassart and Dumont, 1992). In human and rat thyrocytes TSH can also stimulate the Gq/PLC cascade (discussed below) (Medina and Santisteban, 2000; Kimura et al., 2001), and in dog and human thyrocytes TSH also activates Gi, which partially opposes the stimulation through Gs and is not related to TSH-mediated proliferation (Allgeier et al., 1997). TSHR induces the dissociation of Gs in its ␣ and ␤␥ subunits. In turn, G␣s subunits stimulate the adenylate cyclase with the subsequent increase in cAMP (Fig. 1) (Vassart and Dumont, 1992). The G␣s plays an essential role in TSH-mediated proliferation and activated G␣s mutant reproduces the mitogenic effects of TSH in rat FRTL-5 cells (Muca and Vallar, 1994). Moreover, microinjection of a G␣s-specific antibody abolishes TSH-stimulated DNA synthesis in rat WRT cells (Meinkoth et al., 1992). Noteworthy, ␤␥ subunits have been shown to modulate intracellular signals involved in cell growth in different cell types (Camps et al., 1992; Coso et al., 1996; Katz et al., 1992; Yamauchi et al., 1997), although the precise role of these subunits in thyroid cell proliferation remains essentially unknown. Constitutive activation of the cAMP cascade has been experimentally shown to cause increased proliferation of thyroid cells in vitro and in vivo, however, its role in thyrocyte transformation is not well understood. Thus, transgenic mice with constitutive activation of the cAMP signaling pathway in thyroid by the specific expression of a G␣s mutant (Michiels et al., 1994), the A2 adenosine receptor (Ledent

Fig. 1. Hypothetical model of the signaling pathways involved in the TSH proliferation of rat thyrocytes. Most of the activities of TSHR are mediated through a heterotrimeric Gs protein, which activates the adenylate cyclase (AC)/cAMP cascade. PKA-dependent and -independent pathways activated by cAMP are depicted. Although increased cAMP levels are required for thyrocytes to enter the cell cycle, in rat (but not in dog) thyrocytes, cAMP induces its own degradation by phosphodiesterases (PDEs) to allow cell cycle progression. In rat and human thyrocytes TSH can also activate the PLC–Ca2+ pathway via members of the Gq/11 family. Cross-talk between these two main signaling pathways is depicted. Symbols: arrows, activation; blocked bars, inhibition; black circle, phosphorylation of still unknown significance.

et al., 1992), or the cholera toxin A1 subunit (Zeiger et al., 1997), develop thyroid hyperplasia and hyperthyroidism but not carcinomas. Therefore, constitutive activation of the cAMP signal transduction cascade does not appear to be sufficient to induce thyroid cell transformation. Similarly, somatic activating mutations of the TSHR or G␣s (oncogene Gsp) inappropriately induce adenylate cyclase activity and promote a relatively unrestrained growth drive in thyroid cells. In fact, defects in either the TSHR or G␣s genes account for most autonomously functioning thyroid adenomas (Russo et al., 1995b; Parma et al., 1997), but few thyroid carcinomas with activating point mutations of the TSHR have been reported (Russo et al., 1995a; Esapa et al., 1997; Fagin, 2002). These observations fit with the notion that mutations in TSHR could be an initiating event in thyroid

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tumorigenesis, but the further tumor development must be dependent on mutations of additional growth-regulatory genes. Activating mutations are more commonly found, and are thought to be more oncogenic, in the TSHR than in its downstream transducer G␣s (Ludgate et al., 1999). Thus, in rat FRTL-5 cells the expression of both TSHR and G␣s mutant proteins that induce cAMP accumulation lead to a TSH-independent proliferation. However, only the TSHR mutant is able to induce neoplastic transformation (Fournes et al., 1998). This data suggest that not all the proliferative signals of the TSHR go through G␣s, and maybe other pathways (such as the PLC/PKC, or that induced by the ␤␥ subunits) should be taken into account.

3. cAMP cAMP can have either mitogenic or anti-mitogenic effects depending on the cell type (Iyengar, 1996). In thyroid cells, TSH acting through cAMP stimulates proliferation, although the participation of other factors such as insulin/IGF-1, bFGF, EGF or those present in serum, are required for TSH to display a full mitogenic activity (Medina and Santisteban, 2000; Kimura et al., 2001). Recent data indicate that although increased intracellular cAMP levels are required for thyrocytes to proliferate, the response of rat thyrocytes to cAMP is dependent on the time of delivery of the cAMP signal relative to the phase of the cell cycle. Thus, in rat thyrocytes cAMP effects on cell cycle are biphasic. Increased intracellular cAMP levels are required to enter G1 from G0 but after its initial effects, cAMP is no longer required for G1 phase progression, and even inhibits it when maintained at high levels (Villone et al., 1997). In keeping with these observations, the group of Conti proposes that cAMP induces its own degradation, with the subsequent progression through the cell cycle, by activation of phosphodiesterase activity in rat FRTL-5 cells (Oki et al., 2000; Takahashi et al., 2001). In contrast, different mechanism/s must be operating in dog primary cultures, since cAMP must be continuously elevated by TSH to directly control progression through G1 phase (Roger et al., 1999; Van Keymeulen et al., 2001). The role of cAMP in thyroid tumor progression is not well understood. Thyrocytes require cAMP to proliferate and constitutive activation of the cAMP signaling pathway induces thyroid hyperplasia. However, increased cAMP levels does not appear to be sufficient to generate toxic thyroid adenomas (Derwahl et al., 1998) and far more complex mechanisms may be required in the pathogenesis of these tumors. Moreover, some data suggest that cAMP may be acting as a protective factor in the thyroid tumor progression. Thus, cAMP acts as a growth inhibitor in some human thyroid tumoral cell lines (Derwahl et al., 1993; Ohta et al., 1997) and activation of oncogenic Ras has been suggested to induce apoptosis when intracellular cAMP levels are increased (Shirokawa et al., 2000).

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4. PKA In rat, dog, and human thyrocytes the activity of cAMP-dependent protein kinase (PKA) is required for the mitogenic stimulation by TSH (Kimura et al., 2001). Classically it has been considered that PKA mediates the specific intracellular signaling events occurring after cAMP stimulation (Fig. 1). However, in the last years, different observations have indicated that PKA does not account for all the intracellular targets of cAMP, and the same occurs in cAMP-mediated proliferation of thyroid cells. Thus, microinjection of heat-stable PKA inhibitor (PKI) in dog thyrocytes and rat WRT cells partially inhibited TSH/cAMP mediated proliferation (Kupperman et al., 1993; Dremier et al., 1997). Paradoxically, microinjection of the PKA catalytic subunit failed to stimulate proliferation. Therefore, these results indicated that activation of PKA is required but is not sufficient to mimic cAMP-dependent proliferation, and suggested that there must be another PKA-independent mechanisms that contribute to cAMP-stimulated cell cycle progression. There are at least two types of PKA holoenzymes, PKAI and II, which are formed by the assembly of two regulatory (PKA-R) and two catalytic (PKA-C) subunits (Doskeland et al., 1993). The distinctive characteristics of the PKA holoenzymes are largely determined by the structure and properties of their PKA-R subunits. Unlike PKAI, which is typically cytosolic, PKAII (␣ and ␤) are often targeted to certain subcellular location (Golgi-centrosome area and in the cytoskeleton) by specific anchor proteins (AKAPs) (Edwards and Scott, 2000; Feliciello et al., 2001; Michel and Scott, 2002). Activation of the PKA is achieved when cytoplasmic cAMP binds the PKA-R subunits, which then releases free PKA-C subunits allowing their nuclear localization and the subsequent activation of PKA-dependent mechanisms (Taylor et al., 1990). Each isozyme displays different affinity for cAMP and has a different turnover (Weber and Hilz, 1986), suggesting that both holoenzymes may decode cAMP signals with different duration and targets. In rat thyrocytes, the cAMP-PKA pathway is initially upregulated early in G1 and then downregulated later in this phase (Villone et al., 1997). Feliciello and collaborators propose that regulation of the cAMP-PKA pathway after entry into cell cycle dose not primarily reflect changes in cAMP levels (as discussed above). Instead, it may result from changes in: (i) the subcellular location of PKAII isozymes (which would involve AKAPs), (ii) the concentrations of the PKA-RII subunits and (iii) the capacity of these subunits to bind PKA-C subunits (Feliciello et al., 2000). An inactivating mutation of the PKA-RIA subunit (PRKARIA), that leads to stimulation of the PKA pathway, has been related to benign endocrine neoplasms, functioning thyroid nodules and even follicular carcinomas observed in patients with the Carney complex (Kirschner et al., 2000). Therefore, PRKARIA has been proposed as a tumor suppressor gene. In contrast, as far as we are aware,

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no activating mutations in the PKA-C subunits have been described in endocrine tumors (Esapa and Harris, 1999). 5. CREB One of the best-characterized PKA-substrates is the ubiquitous transcription factor CREB (cAMP-response element (CRE) binding protein) (Fig. 1). PKA phosphorylates CREB at serine 133 and activates it to stimulate the transcription of many cAMP-responsive genes by binding to CRE sequences in their promoters (Della Fazia et al., 1997; Richards, 2001). CREB activity has been shown to be important in the TSH/cAMP/PKA pathway that leads to thyrocyte proliferation either in vitro or in vivo (Woloshin et al., 1992; Uyttersprot et al., 1999; Nguyen et al., 2000), however, CREB activity does not appear to be sufficient to fully mimic the TSH-dependent DNA synthesis. Changes in PKA-C subunits directly affect CREB activity and, in turn, the expression of CRE-containing genes. Arsmstrong and collaborators suggested that stimulation of rat FRTL-5 cells with cAMP reduces the levels in PKA-C protein leading to a refractory period with a lack of transcription of CRE-containing genes in response to TSH stimulation (Armstrong et al., 1995). Nuclear exclusion of the PKA-C subunit has been proposed to occur after overexpession of v-Ras or PKC activation in rat FRTL-5 cells (Gallo et al., 1992, 1995). This effect on PKA-C location is correlated with a reduction in the transcriptionally active form of CREB and the subsequent loss in the thyroid differentiated state. However, contradictory results are found in rat WRT cells, where injection of oncogenic Ras into quiescent cells did not restrict endogenous PKA-C subunit from entering the nucleus after stimulation with TSH, and no alteration in CREB phosphorylation or CRE-regulated gene expression was found (Kupperman et al., 1996). Various alterations of the CREB family of transcription factors can be observed in endocrine tumors (Rosenberg et al., 2002). However, the role of the PKA/CREB system in the development of thyroid tumors is not yet clarified since no alterations in the levels of CREB protein (total and Ser(133)-phosphorylated) (Peri et al., 2002) or even a marked reduction in activated phospho133-CREB (Brunetti et al., 2000) can be found in hyperfunctioning thyroid adenomas. The functional relevance of the phosphorylation of the transcription factor CREB by other kinases such as PDK1, AKT or MAPKs (Pierrat et al., 1998; Shaywitz and Greenberg, 1999; Hansen et al., 1999) has not yet been explored in regard to thyrocyte proliferation, although cAMP does not appear to phosphorylate CREB in other sites besides Ser-133 in thyrocytes (Armstrong et al., 1995). 6. PI3K Phosphoinositide-3-kinases (PI3Ks) generate specific inositol lipids that have been implicated in a wide range of cel-

lular activities including control of proliferation and apoptosis (Rameh and Cantley, 1999). Among the three different classes in which the multiple isoforms of PI3Ks are divided, class I PI3Ks is the most widely studied. Class I PI3Ks are heterodimers composed of a 85 kDa regulatory subunit (p85) and a 110 kDa catalytic subunit (p110) (Kapeller and Cantley, 1994; Domin and Waterfield, 1997). In thyrocytes, PI3K is activated by many growth factors such as insulin/IGF-1, hepatocyte growth factor (HGF), or epidermal growth factor (EGF) (Medina and Santisteban, 2000; Kimura et al., 2001). Treatment with PI3K inhibitors (wortmannin and LY2944002) or the expression of a dominant negative form of PI3K causes a G1 arrest of rat thyroid cells stimulated to proliferate with TSH (Cass et al., 1999; Medina et al., 2000). These observations suggested that PI3K could be involved in the TSH/cAMP-dependent growth of rat thyrocytes (Fig. 1). However, no direct activation of PI3K by cAMP or PKA has been demonstrated either in rat or dog thyrocytes (Coulonval et al., 2000; Ciullo et al., 2001). Nevertheless, PKA appears to stabilize the p85–p110 complex by phosphorylation of the p85 subunit, and this has been proposed to be the initial step leading to the interaction between Ras and PI3K upon cAMP stimulation in FRTL-5 cells (Ciullo et al., 2001). Moreover, the activation of the PI3K by other growth factors (such as insulin/IGF-1) may be required for thyrocytes to proliferate upon TSH stimulation (Coulonval et al., 2000). The biological effects of PI3K are mediated through downstream kinases such as PDK1, Akt/PKB, and p70s6k (Pullen and Thomas, 1997; Downward, 1998; Vanhaesebroeck and Alessi, 2000). In general, PI3K activates PDK1, which then phosphorylates Akt/PKB and p70s6k (Fig. 1), among other kinases, which are crucial effectors in the oncogenic signaling mediated by PI3K (Blume-Jensen and Hunter, 2001). The small GTPase Rac-1 (a member of the Rho family of GTPases) is also activated downstream from PI3K and contributes to p70s6k activation in WRT cells (Cass et al., 1999).

7. p70s6k p70 ribosomal S6 kinase (p70s6k) has been suggested to play an essential role in the proliferation of various cell types and is activated in response to virtually all mitogenic stimuli as well as oncogenes (Chou and Blenis, 1995). In dog and rat WRT thyroid cells TSH, acting through cAMP/PKA, activates p70s6k (Cass and Meinkoth, 1998; Coulonval et al., 2000) and, at least in WRT cells, this activity is required for TSH-stimulated DNA synthesis. However, the role of the PI3K in the activation of p70s6k by cAMP has not been clarified. In dog and WRT cells the PI3K inhibitor LY294002 (15 ␮M) inhibited cAMP-dependent stimulation of p70s6k (Cass and Meinkoth, 1998; Coulonval et al., 2000). However, wortmannin (100 nM) (another PI3K inhibitor) seemed to be less efficient than LY294002 and higher doses (200 nM)

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were needed to observe little effect in the cAMP-dependent stimulation of p70s6k in WRT cells (Cass et al., 1999). These results point to the notion that PI3K is involved in the cAMP pathway that leads to the activation of p70s6k. However, the role of PI3K in this pathway is not clear for several reasons. (i) The different abilities of LY294002 and wortmannin to inhibit p70s6k phosphorylation. (ii) The lack of specificity in the action of these PI3K inhibitors (Brunn et al., 1996). (iii) In dog thyrocytes, TSH and cAMP are not coupled to the formation of 3-phosphoinositides (Coulonval et al., 2000). Thus, the role of PI3K in the activation of p70s6k by cAMP is controversial, although it cannot be totally excluded. In dog thyroid cells, insulin/IGF-1 do not have a mitogenic effect by themselves but are able to stimulate p70s6k (Coulonval et al., 2000). Since insulin and IGF-1 are considered as permissive factors for the TSH-induced proliferation, this data suggest that the activation of p70s6k might be necessary, but not sufficient, for the induction of DNA synthesis in thyrocytes.

8. Akt/PKB Akt, also known as PKB, is the cellular homologue of the AKT8 retrovirus transforming oncogene (Bellacosa et al., 1991). Akt/PKB is a serine/threonine kinase with high homology to PKA and PKC which function has been linked to diverse processes such as diabetes, cell proliferation, differentiation, transformation and apoptosis (Brazil and Hemmings, 2001; Testa and Bellacosa, 2001). cAMP modulates Akt/PKB activity either positively or negatively in a cell type-specific manner. However, there is not always a direct relationship between the effects of cAMP on proliferation (as activator or as inhibitor) and its effects on Akt/PKB activity in the different cell types (Filippa et al., 1999; Kim et al., 2001; Wang et al., 2001). In thyroid cells, Akt/PKB has been implicated in the stimulation of cell proliferation by TSH and cAMP (Fig. 1). However, the mechanism by which cAMP regulates Akt/PKB activity and the role of this event in cell proliferation remains unclear. In rat WRT cells, cAMP signaling agents stimulated Akt/PKB activity and, contrary to the initial findings (Cass et al., 1999), this stimulation is dependent on both PKA and PI3K activities (Tsygankova et al., 2001). In contrast, more recently, Lou and collaborators have shown that TSH/cAMP rapidly inhibits both basal and insulin-stimulated Akt/PKB activity in rat PCCL3 cells in a mechanism that require the phosphorylation of the small GTPase Rap1b by PKA (Lou et al., 2002). Therefore, these authors propose that cAMP inhibits Akt/PKB activity despite of the proliferative effects of cAMP on thyroid cells. Finally, in primary dog thyrocytes TSH does not stimulate Akt/PKB activity, which is in agreement with the absence of PI3K stimulation by TSH in this cell system (Coulonval et al., 2000). Therefore, in dog thyroid cells, Akt/PKB is not involved in the cAMP-dependent pathways leading to thyrocyte proliferation, although the

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stimulation of Akt/PKB by insulin/IGF-1 might account for the permissive action of these factors in the TSH-mediated proliferation of these cells (Coulonval et al., 2000). It appears that modulation of Akt/PKB activity is not sufficient to explain cAMP-mediated mitogenesis in thyrocytes, moreover insulin (or serum) maximally stimulates Akt/PKB but these agents by themselves have little or no effect on G1 /S entry in thyrocytes (Kimura et al., 2001). However, the expression of a constitutively active Akt/PKB induces both survival and a hormone-independent growth insensitive to contact inhibition in rat thyroid cells (De Vita et al., 2000; Saito et al., 2001). Therefore, a possible involvement of Akt/PKB in thyroid cancer can be postulated.

9. Rap1 Guanine nucleotide binding proteins are biochemical transducers that switch between an active GTP-bound and an inactive GDP-bound state (Bourne et al., 1990) in a tightly regulated manner. Changes in the active or inactive conformation are controlled by several regulatory proteins: guanine exchange factors (GEFs); GTPase-activating proteins; and guanine nucleotide dissociation inhibitors. The identification of novel cAMP-binding proteins that exhibit GEF activity, called Epacs (for exchange protein activated by cAMP) or cAMP-GEFs (de Rooij et al., 1998; Kawasaki et al., 1998a), opened new doors for the cAMP actions that are independent of PKA. These actions include the activation of small GTPases such as Rap1, Rap2 and possibly Ras (Nancy et al., 1999; Pham et al., 2000). Epac expression is highly enriched in thyrocytes (Kawasaki et al., 1998b) and thus it is likely to have an important function in the activation of the proliferative and differentiation programs that are mediated by cAMP but are independent of PKA. Currently much attention has been focused on Rap1 (Fig. 1), a small GTPase highly related to Ras that, although originally identified as an antagonist of Ras action, exhibits many Ras-independent effects including a role in signaling pathways initiated by cAMP (Bos et al., 2001). It has been recently shown that TSH/cAMP induce the GTP-bound form of Rap1 in a PKA independent manner (Fig. 1) in rat and dog thyrocytes (Dremier et al., 2000; Tsygankova et al., 2001). The mechanistic aspects of this activation have not been elucidated, however, it is thought to be mediated by Epac. It is important to differentiate between Rap1a and Rap1b activities. In rat WRT thyrocytes, Rap1a has been related to the differentiation rather than to the proliferation process (Tsygankova et al., 2001), while in rat PCCL3 cells Rap1b appears to be involved in the cAMP-mediated proliferation. Moreover, in PCCL3 cells, TSH/cAMP stimulation of DNA synthesis requires the action of Rap1b in a manner dependent on its phosphorylation by PKA (Ribeiro-Neto et al., 2002). In contrast, in primary dog thyrocytes, although TSH (as well as other mi-

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togenic and non-mitogenic stimuli) activated Rap1 proteins, microinjection of Rap1b failed to induce proliferation (Dremier et al., 2000). GTPase Rap1b is endowed with both mitogenic and tumorigenic properties (Altschuler and Ribeiro-Neto, 1998), but the role of the cAMP-Epac-Rap1 signaling pathway in thyroid tumorigenesis is yet to be known. It has been suggested that this pathway does not play a major role in the generation of cold thyroid follicular adenomas since no mutation in either Epac, Rap1a or Rap1b could be observed in ten nodules studied by Vanvooren and collaborators (Vanvooren et al., 2001).

10. RAS The Ras proteins, H-Ras, N-Ras, and K-Ras, are membrane-bound guanine nucleotide-binding proteins which transduce signals from cell membrane to nucleus displaying a central role in the control of cell growth and differentiation in mammalian cells (Barbacid, 1987; KhosraviFar and Der, 1994). In rat WRT thyrocytes, overexpression of a constitutive inactive Ras form (Ras N17) significantly reduces TSH-mediated proliferation. Moreover, concomitant overexpression of Ras N17 with a constitutive inactive PKA fully suppresses TSH-mediated proliferation, suggesting that both PKA and Ras are required for the full mitogenic action of TSH (Kupperman et al., 1993). In these WRT cells, TSH activation of ectopically expressed Ras is mediated by cAMP but is not dependent on PKA activity (Tsygankova et al., 2000), however, the role of guanine nucleotide exchange factors (GEFs) in this activation as well as the effect of cAMP on endogneous Ras remain to be elucidated. Overexpression of oncogenic Ras in rat thyrocytes resulted in growth factor-independent proliferation, loss of differentiation and tumor formation in nude mice (Fusco et al., 1982, 1987; Avvedimento et al., 1991; Francis-Lang et al., 1992; Kupperman et al., 1996), and sustained proliferation, although without loss of differentiation in primary human thyrocytes (Gire and Wynford-Thomas, 2000; Jones et al., 2000). However, in dog thyrocytes the activation of endogenous Ras by extracellular stimuli is not sufficient to trigger mitogenesis (Van Keymeulen et al., 2000). Ras, in its active GTP-bound form, can interact with multiple downstream effectors including Raf-1 (Vojtek et al., 1993; Warne et al., 1993), RalGDS (Albright et al., 1993), PI3K (Rodriguez-Viciana et al., 1994), and other Ras-binding proteins (Marshall, 1995b), as well as members of the Rho family (Khosravi-Far et al., 1995; Prendergast et al., 1995; Qiu et al., 1995; Bar-Sagi and Hall, 2000) (see Fig. 1). In the last few years it has become progressively clear that integration of multiple functions is required to mimic Ras effects and ultimately lead to full transformation (White et al., 1995; Khosravi-Far et al., 1996; Marshall, 1996; Joneson and Bar-Sagi, 1997; Katz and McCormick, 1997).

One of the downstream effectors of Ras is PI3K, which catalytic subunit (p110) is recruited by Ras in a GTP-dependent manner (Rodriguez-Viciana et al., 1994). Expression of a Ras mutant that binds selectively to PI3K conferred TSH-independent proliferation in rat WRT cells (Cass and Meinkoth, 2000) and activation of PI3K is necessary (although not sufficient) for the Ras-induced proliferation in human thyrocytes (Gire et al., 2000). Since both Ras and PI3K are required for TSH-induced cell cycle progression of rat thyrocytes, it appears that both may be components of the same signaling cascade. Another effector for Ras is Ral GDS (Ral GDP dissociation stimulator) (Fig. 1), a GEF for the Ras related protein Ral (Kikuchi and Williams, 1996). Interestingly, several results suggested a role for Ral GDS/Ral in Ras-mediated proliferation and transformation in fibroblasts (Urano et al., 1996; White et al., 1996). In rat WRT cells, overexpression of an activated Ras mutant that selectively binds Ral GDS (RasV12, G37) stimulated DNA synthesis in quiescent and TSH-treated cells, and microinjection of a dominant negative RalA (RalA N28) reduced DNA synthesis stimulated by Ras and TSH. These data support the idea that RalGDS may be an effector of Ras in the cAMP-mediated growth stimulation (Miller et al., 1997). Ral A has been shown to potentiate the oncogenic effects of Ras in NIH 3T3 fibroblast (Urano et al., 1996), however, the overexpression of RasV12, G37 in WRT cells did not stimulate the morphological transformation and anchorage-independent growth observed with the expression of a different Ras mutant that selectively binds Raf (Miller et al., 1998). Therefore, although RalGDS is involved in the effects of TSH and Ras on thyrocytes proliferation, its role in thyroid transformation remains largely unknown. One of the best-characterized Ras effectors is the Raf family of serine/threonine kinases (Raf-1, Raf-A and Raf-B) (Morrison and Cutler, 1997; Yuryev and Wennogle, 1998). Ras interaction with Raf leads to activation of a kinase cascade consisting of mitogen activated protein kinase (MAPK) kinase (MEK-1 and MEK-2), which in turn activate p42 and p44 MAPK/extracellular signal regulated kinases (ERKs) (Marshall, 1994). Activated MAPK translocate to the nucleus where they phosphorylate many substrates, including transcription factors, resulting in immediate-early gene induction (Davis, 1995; Marshall, 1995a; Whitmarsh and Davis, 1996). The Raf/MEK/MAPK cascade is thought to mediate many of the proliferative and transforming effects of activated Ras (Cowley et al., 1994; Mansour et al., 1994; Pages et al., 1994). In rat WRT cells, where cAMP has been suggested to activate Ras in a PKA independent manner (Tsygankova et al., 2000), TSH does not stimulate MAPK activity and even reduces growth factors-stimulation of the MAPK pathway (Miller et al., 1997). Moreover, in rat PCCL3 and WRT thyrocytes, TSH, acting through cAMP/PKA, was proposed to impair Ras signaling to Raf-1/MAPK while enhancing the signaling to other effectors such as PI3K and Ral GDS (al-Alawi et al., 1995; Miller et al., 1998; Ciullo et al., 2001). These results

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would indicate that the effects of Ras after TSH stimulation are mediated via PI3K and RalGDS rather than by the MAPKs signaling pathway. In sharp contrast, Iacovelli and collaborators observed that in rat FRTL-5 cells, TSH activates the MEK/ERK pathway in a cAMP-dependent PKA-independent mechanism (Iacovelli et al., 2001). It is worth pointing out that previous effects of TSH over MAPK activation in FRTL-5 had been reported, but these effects were cAMP-independent and did not involve the TSHR so they were eventually ascribed to contaminants of TSH preparations (Correze et al., 2000). Similarly, a cAMP-independent activation of p42/p44 MAPK has been reported in human thyrocytes (Saunier et al., 1995), although Kimura and collaborators claim that this effect is not inhibited by antibodies neutralizing TSH or blocking TSH receptors (Kimura et al., 2001) and thus may also reflect the contamination of TSH with growth factors. Interestingly, the TSH-mediated activation of the MAPKs observed by Iacovelly is reproduced by cAMP-elevating agents. Moreover, RasN17 inhibited the effects of TSH over MAPK activation, and the MEK inhibitors UO126 and PD85059 (as well as RasN17) reduced TSH-stimulated proliferation (Iacovelli et al., 2001). These results would suggest that, in FRTL-5 cells, TSH promotes proliferation at least in part by activation of the Ras/Raf/MAPK pathway. However, contradictory results were found in our laboratory, where PD85059 did not affect TSH-mediated proliferation of FRTL-5 cells (Medina et al., 2000). Therefore, the activation of the MAPK pathway in response to TSH, and its role in cell proliferation is controversial. Whether the different results concerning the stimulation of the MAPK pathway in response to TSH/cAMP in these closely related cell lines of rat thyrocytes is due to different experimental conditions is not yet clarified. In primary dog thyrocytes, TSH and cAMP do not stimulate the phosphorylation and activity of p42/p44 MAPKs (Lamy et al., 1993). Moreover, TSH and cAMP reduce the basal levels of GTP-Ras (Van Keymeulen et al., 2000). These data supports the lack of MAPK activation in the cAMP dependent pathway, but due to a reduction in the activated form of Ras rather than by the uncoupling of the Ras-Raf signaling pathway. Nevertheless, in dog thyrocytes MAPK inhibitors reduced DNA synthesis triggered by TSH in the presence of insulin, even though TSH does not activate MAPKs. Therefore, Ras and MAPKs are not activated by TSH, but contribute to the TSH/cAMP-mediated mitogenesis in dog thyrocytes (Kimura et al., 2001). Finally, in primary human thyrocytes activation of the MAPK pathway is necessary, but not sufficient, for the effects of Ras on cell proliferation (Gire et al., 1999). However, the MAPK cascade synergises with the PI3K pathway to mimic, at least in part, Ras-induced proliferation (Gire et al., 2000). Several reports on the high frequency of Ras mutations in both benign and malignant follicular neoplasia suggest that Ras activation is an early event in thyroid tumorigenesis (Lemoine et al., 1989; Namba et al., 1990; Karga et al., 1991;

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Farid et al., 1994; Esapa et al., 1999; Suarez, 1998). Thyroid specific expression of mutant K-Ras in transgenic mice is associated with development of follicular neoplasms (Santelli et al., 1993a,b). In addition, several members of the Rho family of small GTPases have been implicated in Ras-mediated transformation of different cell types (Prendergast et al., 1995; Pruitt and Der, 2001), including thyrocytes (Medina et al., 2002), although the effectors by which Ras feeds into these proteins have not been firmly established. There are considerable data demonstrating that oncogenic Ras enhances cell proliferation. Alternatively, there is also evidence that acute expression of Ras may trigger an antitumorigenic apoptotic response in thyroid cells (Gire and Wynford-Thomas, 2000; Shirokawa et al., 2000; Cheng and Meinkoth, 2001; Fagin, 2002; Cheng et al., 2003). Activated (Val 12 mutation) H-Ras accelerates cell proliferation in PCCL-3 thyrocytes, however, in the presence of TSH, H-RasV12 induced a transient increase in cell proliferation during the first couple of cell cycles, but later inhibited TSH-mediated cell growth via initiation of programmed cell death. These effects are in part mediated via the ERK signal transduction pathway and are only observed with concomitant stimulation with TSH in a PKA-independent manner (Shirokawa et al., 2000; Fagin, 2002). These data suggested that cAMP maybe acting as a protective factor avoiding the oncogenic mechanisms triggered by Ras and redirecting cells to apoptosis. In contrast, Cheng and coworkers claim that acute expression of Ras induces apoptosis of rat thyrocytes (including PCCL3 cells) either in the absence or presence of TSH (Cheng et al., 2003). To this point, we do not know whether such contradictory results may be due to the difference in the culture medium conditions. Thus, Shirokawa and collaborators used higher doses of TSH (10 mU/ml) than Cheng and collaborators (1 mU/ml), and include additional components in the culture medium that, as in the case of somatostatin, could affect TSH activity (Medina et al., 2000). In human thyrocytes the activation of PI3K is necessary but not sufficient to reproduce Ras proliferative effects (Gire et al., 2000). Interestingly, the PI3K inhibitor LY294002 induced apoptosis in colonies expressing RasV12 although no effect was observed in normal thyrocytes (Gire et al., 2000). These results suggest that PI3K activity can suppress the apoptotic effects of Ras, as has been previously observed in fibroblast (Kauffmann-Zeh et al., 1997), and allow tumor progression. Therefore, it is tempting to speculate that the activity of Ras as a tumor initiator may be dependent on the primacy of the cAMP or PI3K signaling pathways, which in turn may be conditioned by the alterations in other regulatory cascades. Thus, cAMP may serve as a protective factor while PI3K would promote tumor progression. Moreover, activation of the PKC by phorbol esters in human thyrocytes appears to tip the balance of pro-apoptotic and anti-apoptotic signals generated by Ras in favor of apoptosis (Fig. 2) (Hall-Jackson et al., 1998).

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RAS

MAPKs

CHROMOSOME INSTABILITY cAMP PI3K PKC

APOPTOSIS

TUMOR INITIATION

Fig. 2. Contribution of different signaling pathways to the final oncogenic Ras effect in thyrocytes. Overexpression of oncogenic Ras has been shown to produce genomic destabilization in some tumors and thyroid cells. This effect appears to involve the MAPKs signaling pathway. Some of the effects of Ras on apoptosis may be secundary to DNA damage. Alternatively, the genomic instability evoked by Ras may allow some cells with additional defects to initiate the tumor clone. cAMP, in a PKA-independent manner, has been suggested to act as a protective factor avoiding the oncogenic mechanisms triggered by Ras and leading cells to apoptosis. PI3K activity appears to suppress the apoptotic effects of Ras and allow tumor progression. Activation of PKC by phorbol esters has been suggested to contribute to the apoptotic effects of Ras. Symbols: arrows, positive contribution; blocked bar, negative contribution.

Overexpression of oncogenic Ras has been shown to produce genomic destabilization and chromosome aberrations in some tumors (Ichikawa et al., 1990, 1991; de Vries et al., 1993) and PCCL3 cells (Saavedra et al., 2000). Therefore, the effects of Ras on apoptosis may be, at least in part, secondary to DNA damage. Alternatively, the genomic instability evoked by Ras may allow some cells with additional genetic defects to develop a resistance to the apoptotic stimulus and thus to initiate the tumor clone (Fig. 2). The effects of Ras on chromosome stability appears to be mediated in part by activation of MEK1 (Saavedra et al., 2000; Shirokawa et al., 2000; Fagin, 2002) suggesting that the MAPK signaling pathway might contribute to the tumorigenic effects of Ras. However, overexpression of a constitutively active form of MEK-1 in FRTL-5 cells is not sufficient to mimic the oncogenic effects of Ras, and concomitant activation of MEK-1 and Rac-1 can reproduce only a subset of the Ras-induced effects (Cobellis et al., 1998), thus supporting the idea that the integration of multiple signals activated by oncogenic Ras are required to obtain full malignant transformation. 11. PKC It is well established, that most effects of TSH on the thyroid gland, including stimulation of proliferation, thy-

roid hormone synthesis, and expression of thyroid-specific genes, are transmitted mainly by the adenylate cyclase pathway (Dumont et al., 1992; Medina and Santisteban, 2000; Kimura et al., 2001). However, in human (Laurent et al., 1987; Yanagita et al., 1996) and rat FRTL-5 cells (Bone et al., 1986; Field et al., 1987), but not in dog thyrocytes (Mockel et al., 1991; Raspe et al., 1992), TSH can also stimulate the ␤-isoforms of PLC (PLC␤), via coupling of the receptor to members of the Gq/11 family (Jhon et al., 1993; Allgeier et al., 1994) (Fig. 1). PLC catalyzes the hydrolysis of PtdIns (4,5)P2 yielding the second messengers 1,2-diacylglycerol (DAG) and Ins (1,4,5)P3. DAG activates PKC while Ins (1,4,5)P3 facilitates an increase in intracellular Ca2+ (Newton, 1997). In rat FRTL-5 cells TSH has been suggested to increase DAG via phospholipase D (PLD), which produces DAG from phosphatidylcholine hydrolysis, suggesting an alternative mechanism for TSH-dependent activation of PKC (reviewed in Medina and Santisteban, 2000). In primary human thyrocytes, activation of the PLC–Ca2+ cascade has been proposed to require 10-fold higher concentrations of hormone than those required to activate adenylate cyclase (Yanagita et al., 1996; Laurent et al., 1987). However, D’Arcangelo and collaborators described that physiological concentrations of TSH are sufficient to increase cytosolic Ca2+ levels in a mechanism that involves the activation of PLC in these cells (D’Arcangelo et al., 1995). In rat FRTL-5 thyrocytes, activation of the PLC–Ca2+ cascade by TSH has been controversial because 100–1000-fold higher concentrations of hormone than those required to activate adenylate cyclase are needed (Bone et al., 1986; Field et al., 1987; Wang et al., 1994). Nevertheless, it has been shown that stimulation with P1-purinergic agonists (such as adenosine) not only enhances the TSH-induced activation of the PLC cascade in rat FRTL-5 cells, but also inhibits the TSH-induced accumulation of cAMP (Sho et al., 1991) and a similar phenomenon has been shown in human thyrocytes (Yanagita et al., 1996). Moreover, repetitive stimulation with TSH increases the capability of the TSHR to induce Ca2+ mobilization (Metcalfe et al., 1998). Therefore, these results suggest that along with TSH, additional physiological stimuli (such as adenosine) or repetitive stimulation with the hormone may be required to activate the PLC cascade. Activation of this pathway may contribute to thyroid proliferation since TSH, through Ca2+ mobilization, has been proposed to regulate various aspects of thyroid physiology (Weiss et al., 1984; Raspe et al., 1991; Tahara et al., 1991) including cell growth (Isozaki et al., 1992), and activation of PKC in thyroid cells has been related to an increase in cell proliferation. Thus, in rat thyrocytes, activation of PKC by phorbol esters have a mitogenic effect (Bachrach et al., 1985; Lombardi et al., 1988; Fujimoto and Brenner-Gati, 1992; Portella et al., 1998). This effect is cAMP independent. Moreover PKC interferes with cAMP production (Lombardi et al., 1988; Sho et al., 1991), which could be related to the lack of differentiation observed after activation of PKC by phorbol ester treatment of thyrocytes.

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Similarly, increased cAMP levels appear to interfere the activation of the PLC cascade by TSH (Laglia et al., 1996). Therefore, the cross-talk between the two main signaling pathways (cAMP and PLC) activated by TSH add another element in the complexity of the analysis of the TSH-mediated effects on thyroid proliferation and differentiation. The PKC family is divided in three subgroups of isozymes based on sequence homology and cofactor requirements (Ohno et al., 1991; Newton, 1997). PKC isozymes are involved in signal transduction pathways controlling growth, differentiation and apoptosis. In addition, PKCs are the major cellular receptors for the tumor promoter phorbol esters and related compounds (Castagna et al., 1982). In FRTL-5 and PCCL-3 cells, activation of PKC stimulates proliferation while inducing a less differentiated phenotype (Gallo et al., 1992; Portella et al., 1998) mimicking the effects of Ras transformation, which indicates a possible role as an effector downstream from Ras. Moreover, PKC activities are increased in thyrocytes expressing v-Ras genes (Spina et al., 1988) and PKC activation increases the transforming activity of v-K-Ras and H-Ras oncogenes (Portella et al., 1998). It has been proposed that v-Ras via PKC inhibits the transmission of cAMP-PKA signal reducing the levels of the PKA-C subunit in the nucleus of rat FRTL-5 cells (Gallo et al., 1992, 1995; Feliciello et al., 1996). This data would account for the effects of Ras and PKC on thyroid differentiation, but still have to be reconcile with their effects on thyroid proliferation. However, contradictory results were obtained in WRT cells, where Ras did not interfere with the accumulation of PKA-C in the nucleus (Kupperman et al., 1996). Therefore, these authors propose that the effects of Ras and PKC on thyroid proliferation and differentiation are mediated through mechanisms distal to the PKA-C trafficking. Recent data suggest that the effects of PKC in the proliferation of FRTL-5 cells may be mediated by an activation of the Janus Kinases (JAK)/signal transducer activator of transcription, STAT3 pathway (Park et al., 2002) (Fig. 1). Treatment with TSH activated STAT3 phosphorylation and PKC inhibitors blocked STAT3 activation induced by either TSH or by stimulators of cAMP, while PKA inhibitors did not affect STAT-3 activation (Park et al., 2002). Therefore, these authors suggest that PKC may be involved in the pathway downstream from cAMP (but not from PKA). STAT3 mediates diverse cytokine responses, such as cellular proliferation and transformation, by activating nuclear gene transcription (Ihle, 1995; Ivashkiv, 1995; Darnell, 1997). However, the functional significance of STAT3 activation in FRTL-5 cells remains to be clarified. Increased enzymatic activity of PLC and PKC in thyroid carcinomas has been reported (Hatada et al., 1992; Kobayashi et al., 1993). Moreover, PKC activation by TSH in human thyrocytes has been suggested to stimulate invasion and growth (Hoelting et al., 1993). However, in thyroid tumors the TSHR-Gq protein-PLC␤ signaling pathway is not likely to be activated. Firstly, because the reduced expression of TSHR in neoplastic thyroid tissues (Brabant

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et al., 1991; Ohta et al., 1991; Ros et al., 1999) and, secondly because, by feedback regulation, PKC appears to prevent activation of PLC␤ by the TSHR in thyroid carcinoma cells (Broecker et al., 1997). Thyroid-specific expression of a constitutively active mutation of the G␣q in mice has been shown to induce thyroid hyperplasia (Ringel et al., 1998), although no such activating mutations in either ␣q or ␣11 subunits were found in thyroid neoplasms and are unlikely to be responsible for the elevated PLC activities in thyroid cancer (Ringel et al., 1998). Thus, increased PLC and PKC activities described in thyroid tumors may be due to stimulation by other growth factor receptors (Lemoine et al., 1991; Berridge, 1993). In addition, in a subset of thyroid neoplasms, high PKC activities may be due to a mutation of the PKC␣ gene that leads to an increased expression of PKC isozymes (Prevostel et al., 1995). Interestingly, activating mutations in the TSHR that constitutively activate both the adenylate cyclase and the PLC cascades have been reported in non-autoimmune hyperthyrodism (Biebermann et al., 2001) and thyroid nodules (Parma et al., 1995; Van Sande et al., 1995; Corvilain et al., 2001). The contribution of these mutations to the tumorigenic process is not known and no clear differences in the phenotype of these nodules were observed when compared with those with constitutive activation of the adenylate cyclase pathway exclusively. However, the study of transgenic mice characterized by the chronic stimulation of both adenylate cyclase and PLC due to the expression of a constitutively active mutant of the ␣1B adrenergic receptor in thyroid, demonstrated that the cAMP and the PLC cascades can cooperate in vivo toward the development of thyroid follicular malignancies (Ledent et al., 1997).

12. Concluding remarks In this work, we have given an overview of the pathways involved in TSH-mediated thyrocyte proliferation and their role in cellular transformation and tumorigenesis. TSH promotes cell growth by elevating intracellular cAMP levels. However, constitutive activation of the cAMP pathway does not appear to be sufficient to induce thyroid transformation and additional alterations in cell regulatory mechanisms may be required. Moreover, some data suggest that cAMP might be acting as a protective factor avoiding the tumor progression of thyroid cells. Apart from the classical cAMP/PKA pathway, cAMP stimulates cell cycle progression through a PKA-independent mechanism that involves the activation of Rap1 and Ras. PI3K and RalGDS appear to contribute to the oncogenic properties of Ras. However, the activation of the Raf/MAPK cascade by Ras upon TSH stimulation is controversial. Interestingly, although Ras mutations are thought to be an early event in thyroid tumorigenesis, recent data indicate that Ras activation may also induce an apoptotic effect in

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thyroid cells. Thus, it appears that only when the thyrocyte is able to overcome (by still unknown reasons) the apoptotic signal, the cell is committed to initiate the tumor clone. In human thyrocytes, TSH can activate the PLC/PKC signaling pathway, although such activation in other species is a matter of controversy. Activating mutations of the TSHR that constitutively activate both the cAMP and the PLC/PKC signaling pathway have been described in thyroid nodules. However, the contribution of the activation of the PLC/PKC cascade to the development of such nodules is not known. In sum, inappropriate activation of the different signaling pathways involved in TSH-mediated proliferation may lead to tumor initiation of thyroid cells, although additional alterations in other growth-regulatory systems appears to be needed to display full tumoral transformation.

Acknowledgements The works from the authors referred in this review has been supported by Grants BMC 2001-2087 (MCyT), CAM 08.1/0037/2001-1, and FIS, RCGC (C03/10) (Spain).

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