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Molecular Endocrinology Laboratory, VA Greater Los Angeles Healthcare System, Departments of Medicine and Physiology, David Geffen School of Medicine at UCLA, Los Angeles, California 90073, USA
¹ Third Department of Medicine, Yamanashi University, Yamanashi 409-3898, Japan

(Requests for offprints should be addressed to G A Brent; Email: gbrent{at}ucla.edu)

	Abstract

Top Abstract Introduction TSH regulation of NIS... Transcriptional regulation of... Signal transduction of the... Post-translational regulation of... NIS expression in thyroid... Transcriptional regulation of... Promotion of NIS expression... NIS expression in normal... NIS expression and iodide... Iodide organification and iodide... Promotion of the NIS... Conclusions References

 
The sodium/iodide symporter (NIS) mediates iodide uptake in the thyroid gland and lactating breast. NIS mRNA and protein expression are detected in most thyroid cancer specimens, although functional iodide uptake is usually reduced resulting in the characteristic finding of a ‘cold’ or non-functioning lesion on a radioiodine image. Iodide uptake after thyroid stimulating hormone (TSH) stimulation, however, is sufficient in most differentiated thyroid cancer to utilize ß-emitting radioactive iodide for the treatment of residual and metastatic disease. Elevated serum TSH, achieved by thyroid hormone withdrawal in athyreotic patients or after recombinant human thyrotropin administration, directly stimulates NIS gene expression and/or NIS trafficking to the plasma membrane, increasing radioiodide uptake. Approximately 10–20% differentiated thyroid cancers, however, do not express the NIS gene despite TSH stimulation. These tumors are generally associated with a poor prognosis. Reduced NIS gene expression in thyroid cancer is likely due in part, to impaired trans-activation at the proximal promoter and/or the upstream enhancer. Basal NIS gene expression is detected in about 80% breast cancer specimens, but the fraction with functional iodide transport is relatively low. Lactogenic hormones and various nuclear hormone receptor ligands increase iodide uptake in breast cancer cells in vitro, but TSH has no effect. A wide range of ‘differentiation’ agents have been utilized to stimulate NIS expression in thyroid and breast cancer using in vitro and in vivo models, and a few have been used in clinical studies. Retinoic acid has been used to stimulate NIS expression in both thyroid and breast cancer. There are similarities and differences in NIS gene regulation and expression in thyroid and breast cancer. The various agents used to enhance NIS expression in thyroid and breast cancer will be reviewed with a focus on the mechanism of action. Agents that promote tumor differentiation, or directly stimulate NIS gene expression, may result in iodine concentration in ‘scan-negative’ thyroid cancer and some breast cancer.

	Introduction

Top Abstract Introduction TSH regulation of NIS... Transcriptional regulation of... Signal transduction of the... Post-translational regulation of... NIS expression in thyroid... Transcriptional regulation of... Promotion of NIS expression... NIS expression in normal... NIS expression and iodide... Iodide organification and iodide... Promotion of the NIS... Conclusions References

 
The thyroid contains 70–90% total iodine in the body (9–10 mg; Riggs 1952). The thyroid gland must trap about 60 µg iodine/day from the circulation to maintain adequate thyroid hormone production. The sodium/iodide symporter (NIS, or SLC5A5 on the NCBI database; http://www.ncbi.nlm.nih.gov/) is expressed on the basolateral membrane of thyroid follicular cells and mediates the accumulation of iodide from the bloodstream to thyroid follicles (Dai et al. 1996). NIS is a membrane-bound glycoprotein with 13 trans-membrane domains and belongs to the sodium/solute symporter family (Dohan et al. 2003). In normal thyroid tissue, NIS transports two Na⁺ and one I^– down the Na⁺ion gradient generated from the activity of Na⁺/K⁺ ATPase (Fig. 1). NIS actively transports iodide producing an iodine concentration gradient from the thyroid cell to extracellular fluid greater than 30:1. Ouabain, which inhibits the Na⁺/K⁺ ATPase, blocks thyroidal iodide uptake (Eggo et al. 1986, Carrasco 1993). Trapped iodide in the follicular cells is released into the lumen through the apical iodide transporter (AIT or SLC5A8; Rodriguez et al. 2002) and pendrin (SLC26A4, the product of Pendred’s syndrome gene; Royaux et al. 2000, Yoshida et al. 2004, Fig. 1). Thyroglobulin (Tg) is localized at the apical membrane outside the follicular cells, where internal tyrosyl residues are iodinated by thyroid peroxidase (TPO). The thyroid oxidases (ThOX1 and ThOX2; De Deken et al. 2000) produce oxidative conditions by generation of H₂O₂, and are required for the normal function of TPO. Since iodide is bound to an organic compound, this process is known as ‘organification’ of iodide. Thyrotropin (TSH) increases the expression of genes involved in thyroid iodide metabolism and thyroid hormone synthesis, including NIS, Tg, and TPO (Dunn & Dunn 2001).

View larger version (17K): [in this window] [in a new window]   Figure 1 Schematic representation of iodide transport in the thyroid gland.

Most differentiated thyroid cancer has an excellent prognosis; survival rates are 93–98% in papillary cancer, and 85–92% in follicular cancer (Gilliland et al. 1997, Dean & Hay 2000). Many metastatic-differentiated thyroid carcinomas, as well as undifferentiated anaplastic cancer, however, do not concentrate sufficient ¹³¹I for therapy (Maxon & Smith 1990). In these cases, other therapeutic options, such as surgical removal of metastases (Niederle et al. 1986), external radiation (Kim & Leeper 1983, Tubiana et al. 1985), or chemotherapy (Kim & Leeper 1983, Shimaoka et al. 1985, Ain et al. 1996), are utilized, but are largely unsuccessful (Tyler et al. 2000). The correlation between NIS expression in thyroid tumors and their ability to concentrate radioiodine has been confirmed (Caillou et al. 1998, Castro et al. 2001). NIS mRNA expression in papillary thyroid cancer with a poor prognosis is markedly decreased (Ward et al. 2003). The regulation of NIS expression in normal and malignant thyroid cells has been investigated extensively and the various agents that have been recognized to influence expression are summarized (Table 1).

	TSH regulation of NIS and other iodide transporters in normal thyroid tissue

Top Abstract Introduction TSH regulation of NIS... Transcriptional regulation of... Signal transduction of the... Post-translational regulation of... NIS expression in thyroid... Transcriptional regulation of... Promotion of NIS expression... NIS expression in normal... NIS expression and iodide... Iodide organification and iodide... Promotion of the NIS... Conclusions References

 
The thyroid follicle has a two-step process of iodide transport to accumulate iodide into the lumen, and three transporters, NIS, AIT (SLC5A8; Rodriguez et al. 2002), and pendrin (SLC26A4; Royaux et al. 2000), mediate the process. NIS on the basolateral membrane of thyroid follicular cells mediates iodide accumulation into the cells from the bloodstream. AIT and pendrin are expressed on the apical membrane (Bidart et al. 2000, Royaux et al. 2000, Rodriguez et al. 2002), and transport the trapped iodide to the lumen (Yoshida et al. 2002, 2004). NIS has a very high affinity for iodide (K_m 20–40 µM; Weiss et al. 1984b, Mandell et al. 1999, Kogai et al. 2000b), so that thyroid cells can concentrate iodide from the bloodstream up to 2 mM (Yoshida et al. 2002). Pendrin requires a relatively high concentration of iodide (more than 1 mM) in the cytoplasm to function as an iodide transporter (Yoshida et al. 2004). Iodide efflux by pendrin to the lumen, therefore, likely depends on functional NIS expression.

TSH stimulation of NIS mRNA and protein expression are mediated by the cAMP pathway in rodent cell lines, Fisher rat thyroid cell line (FRTL)-5 cells (Weiss et al. 1984a, Kogai et al. 1997), PC Cl3 immortalized rat thyroid cells (Trapasso et al. 1999), and human primary thyroid cells (Saito et al. 1997, Kogai et al. 2000a). The upregulation of NIS in response to TSH is at both the transcriptional and the post-translational levels. TSH stimulates the NIS promoter and NIS upstream enhancer (NUE; Endo et al. 1997, Ohmori et al. 1998, Ohno et al. 1999, Taki et al. 2002), increases the half-life of the NIS protein, and stimulates the trafficking of the NIS to the plasma membrane (Riedel et al. 2001). Direct stimulation of the TSH receptor in Graves’ disease by antibody (Saito et al. 1997, Caillou et al. 1998, Jhiang et al. 1998, Castro et al. 1999, Lazar et al. 1999), constitutive activating mutations of the TSH receptor in a hyperfunctioning thyroid adenoma (Mian et al. 2001), or the weak agonist human chorionic gonadotropin (hCG; Hershman 1999, Arturi et al. 2002), activate the cAMP pathway and result in marked NIS expression. In contrast, the expression of AIT is not increased in hyperfunctioning thyroid adenomas or Graves’ disease (Lacroix et al. 2004, Porra et al. 2005). The expression of pendrin protein is increased in hyperfunctioning thyroid tissues, possibly due to post-transcriptional upregulation by TSHR signaling (Bidart et al. 2000, Mian et al. 2001). Iodide transport in the thyroid gland is regulated by TSH, primarily through the stimulation of NIS expression.

	Transcriptional regulation of NIS by TSH in normal thyroid cells

Top Abstract Introduction TSH regulation of NIS... Transcriptional regulation of... Signal transduction of the... Post-translational regulation of... NIS expression in thyroid... Transcriptional regulation of... Promotion of NIS expression... NIS expression in normal... NIS expression and iodide... Iodide organification and iodide... Promotion of the NIS... Conclusions References

 
Recent progress from sequencing of human and rat genomes has allowed comparison of the NIS coding and regulatory gene sequence from various species. Since TSH stimulates NIS expression in both human and rodent thyroid cells, the regulatory region(s) for NIS induction by TSH was expected to be in sequences common to human and rat NIS genes. The human NIS gene maps to 19p13.2-p12 (Smanik et al. 1997) and contains 14 introns in 22 116 bases as measured from the first to the last exon. The rat NIS gene is markedly smaller in size compared with human (9260 bases), although the number of exons is the same and the size of mRNA is similar. The next gene upstream of NIS on the human genomic sequence, ribosomal protein L18a (RPL18a), is located 8657 bases upstream from the first exon of NIS, while the distance on the rat genome is only 2130 bases (Fig. 2A). The similarity of the 5'-flanking region (from the NIS-coding sequence to the RPL18a-coding sequence) between human and rat is only 11.8%.

View larger version (35K): [in this window] [in a new window]   Figure 2 Comparison of the human and rat sodium/iodide symporter (NIS) gene. (A) Three homologous regions (more than 60% homology) in the 5'-flanking region are shown. (B) Comparison of the NIS upstream enhancer (NUE) sequences of human and rodents. A Pax-8 element (PB) is missing on the human NUE.

These regulatory regions contain putative cis-elements for thyroid-selective transcription factors, Pax-8 (a paired domain containing transcription factor) and thyroid transcription factor-1 (TTF-1 or Nkx-2.1, a homeo-domain containing transcription factor), both of which are required for thyroid development and differentiation (De Felice & Di Lauro 2004). The NIS gene promoter/enhancer is regulated by these transacting factors although with some variation among species (Endo et al. 1997, Ohmori et al. 1998, Ohno et al. 1999, Taki et al. 2002). The NUE requires Pax-8 and cAMP-responsive element binding protein (CREB) for its full activity (Ohno et al. 1999, Taki et al. 2002). CREB is one of the basic-leucine zipper (B-ZIP) transcription factor, containing a leucine zipper domain, which mediates DNA binding and dimerization to form homodimers or heterodimers with other B-ZIP proteins (Vinson et al. 2002). A participation of other B-ZIP proteins, such as c-Fos, c-Jun, and the activating transcription factor-2 (ATF-2), may play a role in NUE activation (Chun et al. 2004).

Regulation of the NIS proximal promoter

The core promoter region of the NIS gene contains a TATA-like motif (AATAAAT) and a GC box (CCCGCCCC). Binding of Sp-1 and an ‘Sp-1-like’ protein to the GC-box has been demonstrated, and is required for full activity of the NIS basal promoter (Xu et al. 2002). The rat NIS proximal promoter contains two cis-elements for thyroid-specific transcription factors, TTF-1 between –245 and –230 (Endo et al. 1997) and NIS TSH-responsive factor-1 (NTF-1) around –405 (Ohmori et al. 1998). TSH/cAMP-induced upregulation of the rat NIS gene expression requires NTF-1, which also contributes to TTF-1-mediated thyroid-specific NIS gene expression (Ohmori et al. 1998). The binding of NTF-1 to the cis-element is diminished by an oxidizing agent, diamide, and restored by the reducing agent dithiothreitol, suggesting oxidation/reduction (redox) state regulation of NTF-1 (Ohmori et al. 1998). The human NIS 5'-flanking region contains two putative NTF-1 sites with a consensus sequence, GNNCGGANG, located –558 to –550 (one base mismatch) and –439 to –430 (two base mismatch; Kogai et al. 2001).

Characterization of the NUE

The NUE responds strongly to TSH and cAMP stimulation in thyroid cells (Ohno et al. 1999, Schmitt et al. 2002, Taki et al. 2002, Lin et al. 2004). In the rodent NIS gene, the NUE contains two Pax-8 elements (PA and PB, see Fig. 2B). A cAMP-responsive element (CRE)-like sequence (TGACGCA) is located between the two Pax-8 elements (Ohno et al. 1999, Lin et al. 2004). In the human NUE, one of the Pax-8 elements downstream of the CRE is missing (Schmitt et al. 2002, Taki et al. 2002), reducing sequence similarity between human and rodent to about 70% (Fig. 2B). Our mutagenesis study of these elements indicated that both the Pax-8 element and the CRE-like sequence, but not the TTF-1 element, are required for NUE activity (Taki et al. 2002). TSH and cAMP agonists significantly activate the NUE through the Pax-8 element and the CRE-like sequence in human and rodent cells (Ohno et al. 1999, Taki et al. 2002).

Redox state regulation of the NIS promoter

The redox state regulates a number of cellular responses by modifying the status of redox-sensitive cysteine residues on signal transduction molecules and transcription factors. Some NIS gene regulatory factors, such as Pax-8 (Puppin et al. 2004), rat TSH-responsive factor NTF-1 (Ohmori et al. 1998), and p38-mitogen-activated protein kinase (MAPK; Pomerance et al. 2000), are regulated by redox state. TSH stimulates the reduction of Pax-8 and binding to the cis-element in thyroid cells (Kambe et al. 1996). TSH increases the expression of redox factor-1 (Ref-1, also called apurinic apyrimidinic endonuclease, APE; Asai et al. 1997), a nuclear enzyme mediating reduction of transcription factors (Nakamura et al. 1997, Rothwell et al. 1997), as well as the translocation of Ref-1 into the nucleus (Tell et al. 2000). Ref-1 stimulates Pax-8 DNA binding in thyroid cells (Tell et al. 1998a,b).

	Signal transduction of the NUE in thyroid

Top Abstract Introduction TSH regulation of NIS... Transcriptional regulation of... Signal transduction of the... Post-translational regulation of... NIS expression in thyroid... Transcriptional regulation of... Promotion of NIS expression... NIS expression in normal... NIS expression and iodide... Iodide organification and iodide... Promotion of the NIS... Conclusions References

 
TSH stimulates the NUE in thyroid cells through the cAMP pathway (Ohno et al. 1999, Schmitt et al. 2002, Taki et al. 2002, Lin et al. 2004). TSHR is a guanine nucleotide-binding protein (G-protein) coupled receptor and activates adenylyl cyclase through stimulatory G protein (Gs protein) to generate cAMP. An adenylyl cyclase agonist, forskolin, increases cAMP accumulation and activates the NUE in thyroid cells, mimicking the TSHR stimulation (Ohno et al. 1999, Taki et al. 2002). cAMP activates both protein kinase-A (PKA)-dependent pathways and PKA-independent pathways in thyroid cells. These pathways include PKA-CREB, APE/Ref-1-Pax-8, and two of three major MAPK pathways, the extracellular signal-regulated kinase (ERK) MAPK pathway and the p38 MAPK pathway (Fig. 3).

View larger version (43K): [in this window] [in a new window]   Figure 3 TSH signal transduction to the NIS gene in thyroid cells. CHOP, CCAAT/enhancer binding protein (C/EBP) homologous protein.

Members of the Ras superfamily (Rho family) of small guanosine triphosphate (GTP)-binding proteins are involved in the regulation of cell growth, differentiation, cytoskeletal reorganization, and protein kinase activation. Rap1 (Tsygankova et al. 2001) and Rac1 (Pomerance et al. 2000), Ras family members, have been reported mediators of TSH-stimulated NIS expression in thyroid cells.

In mammalian cells, guanine-nucleotide-exchange factors (GEFs) positively regulate small GTP-binding proteins in response to a variety of signals. GEFs catalyze the dissociation of GDP from the inactive GTP-binding proteins. GTP can then bind and induce structural changes that allow interaction with effectors. Some GEFs bind to cAMP and are directly activated by cAMP (cAMP-GEFs; Kawasaki et al. 1998, de Rooij et al. 2000). Some investigators have reported that TSH upregulates Rap1 in a PKA-independent manner in thyroid cells (Dremier et al. 2000, Iacovelli et al. 2001, Tsygankova et al. 2001). Rap1 is one of the effectors of cAMP-GEFs, suggesting the possibility that TSH/cAMP regulates Rap1 through cAMP-GEFs. On the other hand, PKA activates Rap1 by phosphorylation in mammalian cells (Hata et al. 1991, Altschuler et al. 1995). The Rap1, therefore, is regulated by both cAMP-GEFs and PKA, and this dual regulation likely brings about the PKA-dependent and -independent pathways in thyroid cells.

Rap1 may contribute to TSH induction of NIS expression in FRTL-5 rat thyroid cells (Tsygankova et al. 2001), likely through the v-raf murine sarcoma viral oncogene homolog B1 (BRAF) MAP kinase (MEK)1/2–ERK1/2 MAPK pathway (Iacovelli et al. 2001, Taki et al. 2002, Fig. 3). Dominant-negative mutant of Rap1A partially (50%) inhibits the cAMP-induced NUE activity (Chun et al. 2004). TSH stimulates the MEK1/2–ERK1/2 MAPK pathway in FRTL-5 cells through Rap1 and BRAF without PKA activation (Iacovelli et al. 2001). An inhibitor of MEK1/2, PD98059, partially (43%) inhibits human NUE activation by forskolin (Taki et al. 2002). In contrast, the activation of the MEK–ERK pathway in response to TSH is not observed in primary dog thyrocytes (Vandeput et al. 2003). The regulation of NUE by MEK–ERK may vary among different species or cell lines.

Phosphatidylinositol 3-kinase (PI3K) phosphorylates the inositol ring of phosphatidylinositol and related compounds at the 3'-position. These products serve as second messenger-signaling molecules for regulating cell growth and differentiation. The TSH/cAMP stimulation of cell proliferation with insulin-like growth factor-I (IGF-I) is dependent on a small GTP-binding protein Ras and the PI3K in thyroid cells (Cass & Meinkoth 2000, Coulonval et al. 2000, Saito et al. 2001). Although TSH activates PI3K signaling, PI3K downregulates NIS expression in thyroid cells (Cass & Meinkoth 2000, Garcia & Santisteban 2002). Expression of a Ras mutant that selectively stimulates PI3K markedly decreases the TSH-induced NIS expression in Wistar rat thyroid (WRT) cells (Cass & Meinkoth 2000). IGF-I inhibits the cAMP-induced NIS expression in FRTL-5 cells through PI3K activation (Garcia & Santisteban 2002). An evaluation of the rat NIS gene regulatory sequence has indicated that the region between –1947 and –1152 is responsible for the inhibitory effect of IGF-I (Garcia & Santisteban 2002).

TSH and cAMP activate another MAPK pathway, MKK3/6–p38 MAPK, through a small GTP-binding protein Rac1 (Pomerance et al. 2000). An inhibitor of the p38 MAPK, SB203580, significantly decreases the cAMP-induced NIS mRNA expression in FRTL-5 cells (Pomerance et al. 2000). The TSH/cAMP stimulates the p38-MAPK phosphorylation PKA-dependently, but not PKC or PI3K (Pomerance et al. 2000). TSH stimulates the phosphorylation of MKK3/6, p38-MAPK, and ATF-2, a substrate of the p38 (Pomerance et al. 2000). The ATF-2 has been reported to bind the CRE-like sequence in the rat NUE (Chun et al. 2004). Reactive oxygen species is involved in the activation of p38-MAPK by cAMP (Pomerance et al. 2000), indicating redox regulation of the p38-MAPK pathway in thyroid cells. Another substrate of p38-MAPK, CCAAT/enhancer-binding homologous protein (CHOP), is also involved in the TSH stimulation of the rat NUE (Pomerance et al. 2003), although binding of CHOP to the NUE has not been confirmed.

Some B-ZIP transcription factors, CREB1, ATF1, and/or the cAMP-response element modulator (CREM) , bind to the CRE-like regulatory sequence in the human NUE. These B-ZIP factors form homodimers or heterodimers with other B-ZIP proteins using a leucine zipper (B-ZIP) domain and then bind to these cis-elements on the target gene (Vinson et al. 2002). The cAMP-activated PKA phosphorylates the CREB to regulate cAMP-responsive genes, such as Tg, TPO, TSHR (Nguyen et al. 2000), and NIS (Chun et al. 2004). Our study has indicated the requirement of the CRE-like element in the human NUE for the cAMP-induced NUE activity, and the binding of CREB1, ATF1, and/or CREM to the CRE-like element (Taki et al. 2002).

Thyroid-specific genes Tg (Civitareale et al. 1989, Sinclair et al. 1990, Donda et al. 1991, Zannini et al. 1992, Berg et al. 1996, Espinoza et al. 2001) and TPO (Mizuno et al. 1991, Francis-Lang et al. 1992, Zannini et al. 1992, Miccadei et al. 2002) require both Pax-8 and TTF-1 for full gene expression. Recent studies have demonstrated the critical role of Pax-8 in TSH regulation of NIS through the NUE (Ohno et al. 1999, Taki et al. 2002). The role of TTF-1, however, in human NIS gene expression, is likely less important. Overexpression of exogenous TTF-1 significantly increases the expression of Tg and TPO, but not NIS, in human thyroid cancer cells (Furuya et al. 2004b). TTF-1 is not recruited to the human NIS proximal promoter (Kogai et al. 2001) and does not stimulate promoter activity (Schmitt et al. 2001).

	Post-translational regulation of NIS by TSH

Top Abstract Introduction TSH regulation of NIS... Transcriptional regulation of... Signal transduction of the... Post-translational regulation of... NIS expression in thyroid... Transcriptional regulation of... Promotion of NIS expression... NIS expression in normal... NIS expression and iodide... Iodide organification and iodide... Promotion of the NIS... Conclusions References

 
TSH stimulates NIS expression at the post-translational level in FRTL-5 rat thyroid cells (Riedel et al. 2001). TSH stimulates NIS trafficking to the membrane and prolongs the half-life of NIS protein from 3 to 5 days (Riedel et al. 2001). Although TSH in the medium is required to maintain FRTL-5 cells (Ambesi-Impiombato et al. 1980), they can survive without TSH for up to 10 days (Kogai et al. 1997). The removal of TSH markedly reduces iodide uptake, NIS mRNA, and NIS protein expression in these cells (Kogai et al. 1997, Paire et al. 1997, Riedel et al. 2001). The addition of TSH restores NIS expression with a time lag between the induction of iodide uptake and NIS protein expression (Kogai et al. 1997). TSH induces NIS protein in 36 h to ~80% maximum, while the iodide uptake reaches only ~25% maximum at 36 h (Kogai et al. 1997). Immunocytochemistry in FRTL-5 cells shows an intense staining of NIS on plasma membrane with continuous TSH treatment, while intracellular staining is observed 3 days after withdrawal of TSH (Riedel et al. 2001).

The NIS protein in FRTL-5 cells is randomly distributed on the plasma membrane and does not exhibit cell polarity (Paire et al. 1997). In contrast, in normal thyroid gland, NIS protein is expressed on the basolateral membrane, but not on the apical side facing the lumen (Paire et al. 1997, Caillou et al. 1998, Jhiang et al. 1998, Castro et al. 1999). We developed a culture system for primary human thyroid cells, which survives for up to 3 months (Curcio et al. 1994, Kogai et al. 2000a). The primary cells in monolayer constitutively expressed the thyroid-specific genes, TTF-1, Pax-8, Tg, TPO, and NIS (Curcio et al. 1994, Perrella et al. 1997). TSH treatment stimulates cAMP production (Curcio et al. 1994) as well as the expression of NIS mRNA and protein (Kogai et al. 2000a). No significant induction of iodide uptake, however, was observed in the monolayer cells, even after TSH stimulation (Kogai et al. 2000a). Specific culture conditions promote the formation of three-dimensional ‘follicle-like’ structures with a periodic acid schiff (PAS)-positive colloid filled lumen. In contrast to monolayers, TSH stimulation of the follicles results in significant induction of iodide uptake (Kogai et al. 2000a). These results indicate that cell polarity is required for the expression of the functional NIS in the human primary thyroid cells, which likely promotes NIS translocation to the basolateral membrane. The importance of thyroid follicle structure for NIS induction has been replicated in primary porcine thyroid preparations (Bernier-Valentin et al. 2006).

A role for NIS glycosylation and phosphorylation in the regulation of NIS trafficking to the membrane has been proposed (Dohan et al. 2003). The cytoplasmic carboxyl-terminal domain of NIS contains a PDZ target motif and a dileucine motif tail that are important for protein–protein interactions. A recent study reported that the deletion of these motifs prevented the transport and insertion of NIS protein into the plasma membrane (Dohan et al. 2005). Those motifs are likely to be involved in the post-translational regulation of NIS by TSH.

	NIS expression in thyroid cancer

Top Abstract Introduction TSH regulation of NIS... Transcriptional regulation of... Signal transduction of the... Post-translational regulation of... NIS expression in thyroid... Transcriptional regulation of... Promotion of NIS expression... NIS expression in normal... NIS expression and iodide... Iodide organification and iodide... Promotion of the NIS... Conclusions References

 
The NIS expression in the primary tumor of differentiated thyroid cancer has been studied extensively, although findings have differed. Several investigators reported reduced expressions of NIS mRNA and protein in papillary and follicular thyroid cancer (Smanik et al. 1997, Arturi et al. 1998, Caillou et al. 1998, Ryu et al. 1998, Lazar et al. 1999, Ringel et al. 2001). Increased NIS expression, however, has been reported in papillary cancer (Saito et al. 1998, Dohan et al. 2001, Wapnir et al. 2003). An immunohistochemical analysis of NIS with an affinity-purified high-sensitivity anti-human NIS polyclonal antibody has shown strong positive staining in ~68% of the 72 cases of papillary cancer using conventional whole tissue sections (Wapnir et al. 2003).

NIS expression in the primary tumor of differentiated thyroid cancer and iodide uptake in recurrent or metastatic cancer, are correlated (Castro et al. 2001, Min et al. 2001). Positive NIS protein staining in primary intrathyroidal tumor was reported in 86% case of papillary cancer (Castro et al. 2001). The subsequent whole body scan with ¹³¹I, following the endogenous TSH stimulation achieved by thyroxine withdrawal, shows positive iodide uptake in metastatic tumor in 90% patients with NIS-positive primary tumors (Castro et al. 2001). The expression of TSHR is usually retained in differentiated thyroid cancers (Brabant et al. 1991), except for insular carcinoma (Gerard et al. 2003), although significant cytoplasmic distribution has been observed (Mizukami et al. 1994, Gerard et al. 2003). A study with primary culture of papillary thyroid cancer cells has shown increased iodide uptake after TSH treatment (Saito et al. 1998), consistent with the data from clinical specimens.

In normal thyroid tissue, NIS protein is expressed on the basolateral membrane, even when the serum TSH level is in the normal range, while the NIS protein in differentiated thyroid cancer is expressed predominantly in the cytosol (Saito et al. 1998, Dohan et al. 2001, Wapnir et al. 2003). The loss of tissue polarity is a characteristic change seen in epithelial tumors (Fish & Molitoris 1994). NIS-trafficking and correct polarity, therefore, are likely impaired in thyroid cancer. Our study with the three-dimensional culture of primary thyroid cells indicates the importance of cell polarity in the full expression of functional NIS (Kogai et al. 2000a). Restoration and/or stimulation of the trafficking of NIS in thyroid cancer are likely to increase the efficacy of ¹³¹I therapy.

	Transcriptional regulation of NIS in thyroid cancer

Top Abstract Introduction TSH regulation of NIS... Transcriptional regulation of... Signal transduction of the... Post-translational regulation of... NIS expression in thyroid... Transcriptional regulation of... Promotion of NIS expression... NIS expression in normal... NIS expression and iodide... Iodide organification and iodide... Promotion of the NIS... Conclusions References

 
Some thyroid papillary cancer cells express reduced NIS mRNA, likely due to the reduced activity of the NIS promoter (Kogai et al. 2001, Puppin et al. 2004). The human NIS gene region –596 to –415 has reduced promoter expression in thyroid cancer cells compared with FRTL-5 cells (Kogai et al. 2001). Binding of unknown nuclear factor(s) to the region –596 to –415 is decreased or absent in the BHP 2–7 cells compared with FRTL-5 cells (Kogai et al. 2001).

An anti-oxidative stress nuclear factor, Ref-1, is related to the upregulation of Pax-8 by TSH in thyroid cells, stimulating the human NIS regulatory sequence (up to –2.4 kb of 5'-flanking region) activity with Pax-8 or an ubiquitous transcription factor early growth response (Egr)-1 in HeLa cells (Puppin et al. 2004). Impaired translocation of Ref-1 to the nuclei has been reported in papillary and anaplastic thyroid cancer cell lines as well as thyroid cancer tissues (Russo et al. 2001). The reduced NIS promoter activity in some thyroid cancer cells, therefore, may be due to the reduced Ref-1 localization in the nuclei (Puppin et al. 2004).

The potent enhancer NUE requires Pax-8 binding for the full activity (Ohno et al. 1999, Taki et al. 2002). Endogenous Pax-8 expression is markedly reduced in 70% differentiated thyroid cancer, especially in aggressive disease (Fabbro et al. 1994, Puglisi et al. 2000).

Papillary thyroid cancer (20–85% with geographic variation) has frequent somatic rearrangements of the RET receptor (RET/PTC; Santoro et al. 1992, 2002, Chua et al. 2000, Nikiforov 2002), leading to a constitutive activation of the RET tyrosine kinase. The auto-activated RET receptor stimulates cell proliferation and motility through the ERK–MAPK pathway (Melillo et al. 2005). One of the most common RET/PTC rearrangement, RET/PTC1, consists of the intracellular portion of RET with the tyrosine kinase domain fused to H4, a ubiquitous gene of unknown function. The RET/PCT1 expression impairs the activity of Pax-8, which is required for the full activation of the NUE (Ohno et al. 1999, Taki et al. 2002), in PC Cl3 rat thyroid cells (De Vita et al. 1998). Reduced expression of NIS has been reported in a PC Cl3 constitutively expressing exogenous RET/PTC1 (Trapasso et al. 1999, Venkateswaran et al. 2004). In transgenic mice of thyroid cancer model with thyroid-targeted RET/PTC1 expression, iodide uptake is decreased in thyroid glands (Jhiang et al. 1996). In RET/PTC1-expressing PC Cl3 rat thyroid cells, there is reduced localization of PKA to the nucleus (Venkateswaran et al. 2004). Forskolin and substitution of exogenous PKA restore the NIS expression in the RET/PTC1-expressing cells, suggesting interference of RET/PTC1 with PKA-dependent signaling. On the other hand, our study has indicated a partial interruption of the NUE by the blocking of MEK–ERK pathway in FRTL-5 rat thyroid cells (Taki et al. 2002). Activating mutations of BRAF (Kimura et al. 2003) and an activating rearrangement of the BRAF with A-kinase anchor protein 9 (AKAP9-BRAF; Ciampi et al. 2005) have been reported recently in some papillary thyroid cancer. Crosstalk between the ERK pathway and signaling to NIS gene expression remains to be further investigated.

	Promotion of NIS expression in thyroid cancer

Top Abstract Introduction TSH regulation of NIS... Transcriptional regulation of... Signal transduction of the... Post-translational regulation of... NIS expression in thyroid... Transcriptional regulation of... Promotion of NIS expression... NIS expression in normal... NIS expression and iodide... Iodide organification and iodide... Promotion of the NIS... Conclusions References

 
To achieve the maximum iodide uptake in metastatic-differentiated thyroid cancer, TSH stimulation is required. This is achieved either by withdrawal of thyroid hormone replacement after a total thyroid-ectomy or by administration of rhTSH (Dow et al. 1997). The efficacy of rhTSH is generally equivalent to thyroid hormone withdrawal in the detection of thyroid cancer (Jarzab et al. 2003).

Some differentiated thyroid cancers (approximately 10–20%), however, do not concentrate radioiodide, even after TSH stimulation (Robbins et al. 1991, Schmutzler & Koehrle 2000). TSH unresponsiveness of NIS induction is unlikely to be due to the absence of TSHR. Almost all differentiated thyroid cancer expresses the TSHR protein (Brabant et al. 1991, Mizukami et al. 1994, Gerard et al. 2003). Reduced expression of TSHR, however, is associated with a poor prognosis in papillary thyroid cancer (Tanaka et al. 1997). Failure of signal transduction and/or transcription factors required for NIS gene expression is likely to be responsible for the lack of iodide accumulation in aggressive differentiated thyroid cancer. Defects in NIS protein trafficking and membrane insertion may also play a role. Recent studies have demonstrated the potential for NIS induction in aggressive thyroid cancer by re-differentiation agents, such as nuclear receptor ligands and inhibitors of epigenetic modifications.

NIS induction by nuclear hormone receptor ligands

Retinoic acid (RA), a vitamin A derivative, plays a pivotal role in development, differentiation, and cell growth. RA action is mediated through two families of nuclear receptors, retinoic acid receptors (RARs), and retinoid X receptors (RXRs). RA induces re-differentiation and apoptosis in cancer cells (Hong & Itri 1994). In thyroid cancer cells, RA induces type-I 5'-deiodinase (Schreck et al. 1994) and NIS (Schmutzler et al. 1997). Treatment for 24 h with all-trans RA (tRA; 10^–6 M) markedly increased NIS mRNA expression in two follicular thyroid cancer cell lines, FTC-133 and FTC-238 (Schmutzler et al. 1997). Treatment of FRTL-5 rat thyroid cells with tRA, however, downregulates NIS mRNA (Schmutzler et al. 1997). These findings suggest differential regulation of NIS expression by RA in normal and malignant thyroid tissues.

Based on the findings of the RA induction of ‘re-differentiation’ in thyroid cancer cell lines, clinical trials have been conducted to evaluate the efficacy of RA for improving radioiodide uptake in recurrent/metastatic thyroid cancer (Simon et al. 1996, 1998, 2002a, Grunwald et al. 1998, Koerber et al. 1999, Gruning et al. 2003). In most of these studies, treatment with 13-cis RA has been used. 13-cis RA is isomerized to tRA and/or 9-cis RA in tissues, and activates RAR and/or RXR (Blaner 2001), with less toxicity (Hixson et al. 1979) and a longer half-life (Brazzell et al. 1983), compared with tRA. These clinical studies have shown that 20–42% aggressive differentiated thyroid cancer responds to RA treatment by an increase in radioiodide uptake (Simon et al. 1996, 1998, 2002a, Grunwald et al. 1998, Koerber et al. 1999, Gruning et al. 2003, Coelho et al. 2004). In a study of 50 patients with advanced invasive or metastatic thyroid cancer and negative iodide scans, an oral dose of 13-cis RA (1.5 mg/kg) was given for 5 weeks (Simon et al. 2002a). After 13-cis RA treatment, 13 patients had a marked increase in radioiodide uptake in the invasive or metastatic tumor, and eight patients had a modest increase in radioiodide uptake (Simon et al. 2002a). Reduced tumor volume was observed in seven of the 21 cases with functional NIS expression after the treatment with 80–270 mCi ¹³¹I following the 13-cis RA treatment (Simon et al. 2002a). In some published reports with a small number (5–25) of cases, a marked increase in iodide uptake has been shown in follicular cancer, but not in papillary cancer (Grunwald et al. 1998, Gruning et al. 2003, Coelho et al. 2004). The studies, however, have not been randomized prospective studies of matched groups that would be necessary to confirm an effect of RA treatment.

The first demonstration of in vitro NIS induction was in follicular thyroid cancer cell lines (Schmutzler et al. 1997). Differential response of some thyroid cancer cell lines to RA has been described; cell lines expressing both RARß and RXR demonstrate significant growth suppression with RA, whereas cell lines lacking these isoforms do not respond to RA (Haugen et al. 2004). Differential expression of RAR isoform may be important to predict the NIS induction in thyroid cancer with RA treatment, as well as histological difference. NIS expression and iodide uptake are increased in some breast cancer cells (Kogai et al. 2000b, 2004). The duration of the NIS expression with maximum function, however, is only a few days during the in vivo RA treatment (Kogai et al. 2004). In addition, a large systemic dose (160 mg/kg) is required to maximize the uptake in the mouse models (Kogai et al. 2004). Further evaluations are likely to be required in aggressive thyroid cancer to adjust the dose and duration of RA, as well as relationship among the histology, the tumor RAR isoform expression profile, and the response to RA.

Stimulation of another nuclear receptor, PPAR-, increases NIS expression in some thyroid cancer cell lines in vitro. Troglitazone, a PPAR- ligand, has been reported to increase the NIS mRNA significantly in the FTC-133 follicular thyroid cancer cell line and the TPC-1 papillary thyroid cancer cell line, but not in a Hurthle-cell cancer cell line (Park et al. 2005). Since troglitazone inhibits cell proliferation and induces apoptosis in some papillary thyroid cancer cell lines in vitro and in vivo (Ohta et al. 2001), a combination of troglitazone and radioiodide therapy might provide a synergistic inhibitory effect on some thyroid cancers.

Alteration of chromatin structure with histone deacetylase (HDAC) inhibitor

Epigenetic modifications, such as histone deacetylation and DNA hypermethylation, are commonly detected in human cancer cells, relevant to de-differentiation and proliferation. Alteration of these epigenetic changes has been a target for re-differentiation in cancer cells.

Histone acetyltransferases and HDACs affect the acetylation status of histones, influencing gene expression (Marks et al. 2001). Inhibitors of HDACs induce growth arrest, differentiation, and/or apoptosis in many cancer cells (Marks et al. 2001). Some HDAC inhibitors, such as depsipeptide (FR901228) and valproic acid, have been reported to increase NIS expression in thyroid cancer cell lines (Kitazono et al. 2001, Zarnegar et al. 2002, Fortunati et al. 2004, Furuya et al. 2004b).

Depsipeptide significantly induces NIS mRNA and iodide uptake in follicular thyroid cancer cell lines (FTC-133 and FTC-236) and two anaplastic cancer cell lines (SW-1736 and KAT-4) at a low concentration (1 ng/ml) in vitro (Kitazono et al. 2001). Pharmacokinetics of the depsipeptide in patients have indicated that levels of more than 500 ng/ml are achieved without significant toxicity, promising to obtain the NIS-inducible concentration in patients (Kitazono et al. 2001). Another group has tried depsipeptide in a papillary thyroid cancer cells (BHP 18–21v) and an anaplastic cancer cell line (ARO), and found that 3–10 ng/ml of depsipeptide induces the NIS mRNA, protein, and iodide uptake, as well as Tg and TPO in association with iodide organification (Furuya et al. 2004b). The expression of TTF-1, but not Pax-8, is increased by depsipeptide in both cell lines (Furuya et al. 2004b). Since the overexpression of exogenous TTF-1 induces Tg and TPO (Furuya et al. 2004a), TTF-1 is likely to be responsible for the induction of iodide organification and decreased iodide efflux by the treatment of depsipeptide. The in vivo effect of depsipeptide on the iodide uptake has been confirmed in a BHP 18–21v xenograft model (Furuya et al. 2004b).

Another HDAC inhibitor, trichostatin A (TSA), also induces NIS mRNA in some papillary cancer cell lines (Zarnegar et al. 2002, Furuya et al. 2004b), a follicular cancer cell line (Zarnegar et al. 2002), an anaplastic cancer cell line (Furuya et al. 2004b), and a Hurthle-cell cancer cell line (Zarnegar et al. 2002). The induction of iodide uptake, however, has not yet been confirmed in thyroid cancer cells treated by TSA (Kogai et al. 2001).

Recently, the anticonvulsant valproic acid, acting as a HDAC inhibitor (Marks et al. 2001), has been shown to induce NIS expression in a papillary cancer cell line, NPA, and the anaplastic cancer cell line, ARO (Fortunati et al. 2004), although the induction is relatively modest. HDAC inhibitors, especially depsipeptide, have a potential to increase radioiodide uptake in some aggressive thyroid cancer tumors.

Effects of hypermethylation on NIS promoter activity

Expression of some tissue-specific genes is regulated by cytidine methylation in a CpG dinucleotide sequence on regulatory sequences near the transcription start site (Antequera et al. 1990). The prevalence of abnormal methylation pattern of selected genes in thyroid tumors is high (Matsuo et al. 1993). The human NIS gene has three CpG-rich regions around the translation start site, the core promoter region (about 100 bp from the transcription start site), the 5'-untranslated region, and the coding region of the first exon (Venkataraman et al. 1999). The demethylation agent, 5-azacytidine, restores NIS mRNA expression and iodide uptake in three papillary cancer cell lines, NPA, KAT-5, and KAT-10, but not in two follicular cancer cell lines, MRO and WRO (Venkataraman et al. 1999). A correlation has been observed between the successful restoration of NIS expression by 5-azacytidine and demethylation of the 5'-untranslated region (Venkataraman et al. 1999). On the other hand, their evaluation of methylation status in thyroid cancer tumor specimen has revealed no significant correlation between the methylation status of these CpG-rich regions and NIS mRNA expression in thyroid cancer tumor samples (Venkataraman et al. 1999). The hypermethylation of the NIS 5'-untranslated region could be one of the factors contributing to reduced NIS expression in some thyroid cancers. The demethylation agent, 5-azacytidine, has the potential to restore radioiodide uptake in some thyroid cancer tumors.

Thyroid cancer models

Concentration of radioiodine in response to TSH stimulation is observed in 70% of metastatic thyroid cancers (Robbins et al. 1991). However, only a few normal thyroid cell lines (Weiss et al. 1984b, Berlingieri et al. 1993, Venkataraman et al. 1998) and thyroid cancer cell lines (Ohta et al. 1996, 1997, Kogai et al. 2001) express endogenous NIS in response to TSH stimulation. Most of these thyroid cell lines have de-differentiated and lost expression of TSH-R and TTF-1, which are expressed in most differentiated thyroid cancers. Several transgenic mouse models of thyroid cancers have been developed with thyroid-targeted expression of oncogenes, including SV40-large T antigen (Ledent et al. 1991), human papilloma virus (HPV)-E7 oncogene (Ledent et al. 1995, Coppee et al. 1996), RET/PTC1 (Jhiang et al. 1996, Santoro et al. 1996), RET/PTC3 (Powell et al. 1998), and TRK-T1 (Russell et al. 2000). These transgenic mice may provide better models to evaluate the regulation of NIS in thyroid cancer.

	NIS expression in normal breast tissue

Top Abstract Introduction TSH regulation of NIS... Transcriptional regulation of... Signal transduction of the... Post-translational regulation of... NIS expression in thyroid... Transcriptional regulation of... Promotion of NIS expression... NIS expression in normal... NIS expression and iodide... Iodide organification and iodide... Promotion of the NIS... Conclusions References

 
Iodide accumulation in the lactating breast has been recognized for more than 50 years (Honour et al. 1952, Brown-Grant 1957, Grosvenor 1960, Eskin et al. 1974, Thorpe 1976, Strum 1978, Bakheet et al. 1998, Perros et al. 2003). Once the NIS gene was cloned and available for study, a correlation between NIS expression and iodide uptake in lactating mammary glands was demonstrated (Cho et al. 2000, Tazebay et al. 2000). NIS is expressed on the basolateral membrane of alveolar cells in mammary glands (Spitzweg et al. 1998) and is markedly induced during lactation (Cho et al. 2000, Tazebay et al. 2000). The alveolar cells concentrate iodide in milk, 6 to 15-fold relative to the plasma iodide concentration (Thorpe 1976). Part of the trapped iodide (~20%) is organified by peroxidases in the alveolar cells and lumens adjacent to the alveolar cells (Strum 1978, Etling & Gehin-Fouque 1984, Shah et al. 1986).

Radioiodide therapy and imaging for thyroid diseases are contraindicated in breast-feeding patients. After thyroid imaging with ¹³¹I or ¹²³I, cessation of breast feeding is recommended until breast milk radioactivity levels are at a safe level (Stabin & Breitz 2000). Since the therapeutic administration of ¹³¹I for thyroid cancer (150 mCi) delivers approximately 2 Gy (200 rad) to the mammary glands, it is recommended that breast feeding should be discontinued (Stabin & Breitz 2000).

No correlation between thyroid uptake and breast uptake has been reported, suggesting differential regulation of iodide uptake in the thyroid and mammary glands (Eskin et al. 1974). Recent reports have demonstrated that fetoplacental estrogen and two pituitary hormones, oxytocin and prolactin, play an important role in the induction of NIS in the lactating mammary glands (Cho et al. 2000, Tazebay et al. 2000). Estradiol produces a modest induction of NIS in mammary glands from ovariectomized mice (Tazebay et al. 2000). In contrast, estradiol decreases the NIS expression in FRTL-5 rat thyroid cells (Furlanetto et al. 1999). The treatment of mice with the combination of oxytocin, prolactin, and estradiol markedly induces the NIS in mammary glands, while each hormone alone is not sufficient for NIS induction (Tazebay et al. 2000). Basal levels of these three hormones are significantly increased in late pregnancy and the lactogenic hormones, prolactin and oxytocin, are still elevated during the first few months of the post-partum period. The surge of oxytocin during lactation is likely to be important for the maximum induction of NIS in mammary glands.

There is a particular concern regarding the impact of ¹³¹I treatment for thyroid cancer in the post-partum period. Bromocriptine, which inhibits the secretion of prolactin, partially inhibits the iodide uptake in the lactating mammary glands in rats (Cho et al. 2000). Breast uptake of ¹³¹I during the treatment of thyroid cancer in the post-partum period may increase the risk of breast cancer (Preston et al. 2002, Zheng et al. 2002). Cessation of breast feeding and the administration of bromocriptine have been reported to reduce ¹³¹I uptake in breast tissues (Hsiao et al. 2004).

NIS activity is the primary regulator of iodide accumulation in the lactating breast, although other transporters may make a small contribution (Shennan 2001). A sulfate/iodide exchanger that is inhibited by 4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid has been identified in rat mammary gland explants (Shennan 2001). Increased pendrin expression has also been reported in the lactating mammary gland (Rillema & Hill 2003). Since NIS is expressed on the basolateral membrane in the lactating mammary glands, other transporters, like pendrin, may mediate release of the trapped iodide into the lumen.

	NIS expression and iodide uptake in breast cancer

Top Abstract Introduction TSH regulation of NIS... Transcriptional regulation of... Signal transduction of the... Post-translational regulation of... NIS expression in thyroid... Transcriptional regulation of... Promotion of NIS expression... NIS expression in normal... NIS expression and iodide... Iodide organification and iodide... Promotion of the NIS... Conclusions References

 
It has been