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MOLECULAR CHARACTERIZATION OF AN IODIDE TRANSPORT DEFECT (ITD) CAUSED BY A Na + /I - SYMPORTER (NIS) MUTATION (G395R), AND IMMUNOHISTOCHEMICAL ANALYSIS OF NIS PROTEIN EXPRESSION IN THYROID CANCER. By Orsolya Dohán M.D. Semmelweis Medical School Budapest Mentors: Nancy Carrasco MD, Professor, Albert Einstein College of Medicine, Bronx, New York, USA Miklós Tóth MD PhD, Doctor of the National Academy of Sciences, Hungary Doctoral School For Clinical Science in Medicine Chair: Zsolt Tulassay MD PhD, corresponding member of the National Academy of Sciences, Hungary Program 2/7: Experimental and clinical endocrinology Program leader: Rudolf de Chatel MD PhD, Doctor of the National Academy of Sciences, Hungary Budapest, 2006 September 1 TABLE OF CONTENTS SUMMARY....4 INTRODUCTION Thyroid Physiology... 6 Structural and Functional Characterization of NIS.10 Regulation of NIS Expression and Function..11 TSH modulates NIS phosphorylation..14 Regulation of NIS expression by I Effect of spatial organization of thyroid cells.16 Iodide Transport Defect (ITD) Role of NIS in Thyroid Cancer...19 RESEARCH OBJECTIVES 23 Aim 1. Analysis of the G395R NIS mutation at the molecular level.23 Aim 2. Immunohistochemical analysis of NIS protein expression and subcellular localization in differentiated thyroid cancer 24 MATERIALS AND METHODS 26 Site directed mutagenesis..26 COS-7 cells transfection..26 Iodide Uptake...26 Membrane preparation...27 Immunoblot analysis 27 Immunofluorescence analysis.28 Flow cytometry.28 Surface biotinylation 28 Immunohistochemical analysis 29 RESULTS..30 Aim 1. Analysis of the G395R NIS mutation at the molecular level.30 Characterization of activity and expression of G395R in COS cells..30 Effects of other charged amino acid substitutions at position 395 on NIS expression and activity in COS cells 31 2 Effects of neutral amino acid residues at position 395 on NIS expression and activity in COS cells.33 Kinetic analysis of I - transport mediated by NIS mutant proteins...33 Aim 2. Immunohistochemical analysis of NIS protein expression and subcellular localization in differentiated thyroid cancer...37 DISCUSSION AND CONCLUSIONS.40 Aim 1. Analysis of the G395R NIS mutation at the molecular level.40 Aim 2. Immunohistochemical analysis of NIS protein expression and subcellular localization in differentiated thyroid cancer...43 REFERENCES...46 ACKNOWLEDGMENTS..56 PUBLICATIONS OF ORSOLYA DOHÁN 57 Publications originating from this doctoral thesis..57 Other publications.57 3 SUMMARY. The Na + /I - symporter (NIS) is a plasma membrane glycoprotein that mediates active iodide uptake in the thyroid the essential first step in thyroid hormone biosynthesis and in other tissues, such as stomach, salivary and lactating mammary glands. NIS plays key roles in thyroid pathophysiology as the route by which I- reaches the gland for thyroid hormone biosynthesis, and as a means for diagnostic imaging and for radioiodide therapy in thyroid cancer. We investigated at the molecular level two conditions with absent or diminished thyroidal I - uptake: 1) a NIS mutation (G395R) causing congenital iodide transport defect resulting hypothyroidism, 2) differentiated thyroid cancer. 1) Several NIS mutations have been shown to cause I - transport defect (ITD), a condition that, if untreated, can lead to congenital hypothyroidism and ultimately cretinism. The study of ITD-causing NIS mutations provides valuable insights into the structurefunction and mechanistic properties of NIS. Here we report the thorough analysis of the G395R NIS mutation. We observed no I - uptake activity at saturating or even supersaturating external I - concentrations in COS cells transiently transfected with G395R NIS cdna, even though we demonstrated normal expression of G395R NIS and proper targeting to the plasma membrane. Several amino acid substitutions at position 395 showed that the presence of an uncharged amino acid residue with a small side-chain at position 395 is required for NIS function, suggesting that glycine 395 is located in a tightly packed region of NIS. Substitutions of large amino acid residues at position 395 resulted in lower V max without affecting K m values for I - and Na +, suggesting that these residues hamper the Na + /I - coupling reaction. 2) We analyzed the Na + /I - symporter (NIS) protein expression in 57 thyroid cancer samples by immunohistochemistry with high-affinity anti-nis Abs. As many as 70% of these samples exhibited increased NIS expression with respect to the normal surrounding thyroid tissue. Most significantly, NIS was located in these samples either in both the plasma membrane and intracellular compartments simultaneously, or exclusively in intracellular compartments. This suggests that NIS is clearly expressed or even overexpressed in most thyroid cancer cells, but malignant transformation in some of these 4 cells interferes either with the proper targeting of NIS to the plasma membrane, or with the mechanisms that retain NIS in the plasma membrane after it has been targeted. The results further indicate that, in addition to inducing NIS expression in cases where it is absent (~30%), improvements in 131 I radioablation therapy might result from promoting targeting of NIS to the plasma membrane in the majority (~70%) of thyroid cancers. 5 INTRODUCTION. Thyroid Physiology For decades, the phenomenon of iodide (I - ) concentration has been viewed as one of the most distinctive and intriguing attributes of the thyroid gland. Given that I - is an essential component of the thyroid hormones T 3 and T 4, it is advantageous for the gland to have a highly specialized system for the accumulation of most of the available I - in the blood. Indeed, with its follicular morphology, the thyroid is optimally configured not only to transport but also to store iodine to meet future needs, thus reflecting the gland's remarkable adaptation to iodine's scarcity in the environment. To make thyroid hormone biosynthesis possible, dietary ingested I - is first actively transported from the blood against a concentration gradient across the plasma membrane into the cytoplasm of the thyroid follicular cells (i.e. I - uptake). Then, I - is passively translocated from the cytoplasm of these cells into the follicular lumen (i.e. I - efflux). The lumen, surrounded by the epithelial cells, is an extracellular compartment filled with colloid, the main component of which is the large protein thyroglobulin (Tg). I - reaches the cell/colloid interface, the site where hormone biosynthesis primarily occurs (1). The Na + /I - symporter (NIS) is the plasma membrane glycoprotein that mediates active I - uptake into the thyroid follicular cells. This process is the crucial first step in thyroid hormone biosynthesis. NIS couples the inward translocation of Na + down its electrochemical gradient to the simultaneous inward uphill translocation of I - against its electrochemical gradient. Two Na + are transported per each I -. The Na + gradient that provides the driving force for I - uptake is maintained by the Na + /K + ATPase (2). Both NIS and the Na + /K + ATPase are located on the basolateral side of the follicular cells, facing the blood supply (Fig. 1). On the other hand, I - efflux to the follicular lumen has been suggested to be mediated by a different plasma membrane transporter putatively located on the apical side of the follicular cells, facing the colloid. The identity of this transporter remains unsettled. Most probably, I - efflux is mediated by Cl - channels. Once I - is at the cell/colloid interface, the anion is organified by the enzyme thyroid peroxidase (TPO). 6 Figure 1. Iodide transport and biosynthetic pathway of thyroid hormones T 3 and T 4 in the thyroid follicular cell. Thyroid follicles are comprised of a layer of epithelial cells surrounding the colloid. The basolateral surface of the cell is shown on the left side of the figure, and the apical surface on the right. Active accumulation of I -, mediated by the Na + /I - symporter (NIS) [top circle], driven by the Na + gradient generated by the Na + /K + ATPase [bottom circle]; once I - effluxes towards the colloid [cylinder], (TPO) [triangle] catalyzes the organification of I - on the thyroglobulin (Tg) molecule. Dotted line pointing from the apical to the basolateral surface indicates endocytosis of iodinated Tg, followed by its phagolysosomal hydrolysis and secretion of thyroid hormones. Organification involves I - oxidation and its incorporation into some tyrosyl residues within the Tg molecule, leading to the subsequent coupling of iodotyrosine residues. Iodinated Tg is stored extracellularly in the colloid. In response to demand for thyroid hormones, phagolysosomal hydrolysis of endocytosed iodinated Tg occurs. T 3 and T 4 are secreted into the bloodstream, and non-secreted iodotyrosines are metabolized to tyrosine and I -, a 7 reaction catalyzed by the microsomal enzyme iodotyrosine dehalogenase. This process facilitates reutilization of the remaining I -. All of these steps are stimulated by TSH. (Fig.1) The environmental scarcity of I -, underscores the importance of NIS activity for thyroid physiology and for overall health and development. It has been estimated that 30% of the world s population is at risk of iodine deficiency disorders (IDDs), 750 million people suffer from goiter, and more than 5 millions suffer from cretinism ( This illustrates how difficult it would be for people to stay euthyroid and healthy in the absence of a specialized I - transport mechanism in the thyroid (that is, in the absence of NIS), even in areas where iodine is not particularly scarce. Iodine deprivation early in life hampers not only overall development, but most dramatically the development of the nervous system. As the mediator of the first step of the thyroid hormone biosynthetic pathway, NIS plays a key role in thyroid pathophysiology. For over 60 years, the ability of the thyroid to accumulate I - via NIS has been the basis for the notably successful use of radioiodide in the diagnosis and treatment of thyroid diseases, including hyperthyroidism and thyroid cancer and its metastases (3). However, no molecular information on NIS was available until 1996, when the cdna encoding rat NIS was isolated by expression cloning in Xenopus laevis oocytes (4). This development marked the beginning of the molecular characterization of NIS, and spurred numerous new studies on NIS using a wide variety of techniques and approaches. On the basis of the rnis cdna sequence, the human (5), mouse (6), and pig (7) NIS cdnas were subsequently cloned. Interestingly, NIS has been shown to be the mediator of I - transport not only in the thyroid but also in several extrathyroidal tissues, such as salivary glands (8-10), gastric mucosa (10), and lactating mammary gland (10), thus disproving the previously held view of NIS as a thyroid-specific protein (Fig. 2). Moreover, NIS has been found to be differently regulated and subjected to distinct post-translational modifications in each tissue where it is expressed (10-12). 8 Figure 2. Immunohistochemical analyses of NIS expression in human iodideconcentrating tissues. From top to bottom: Normal thyroid (original magnification: 400 x. Salivary gland, basolateral NIS staining in the salivary ductal cells (original magnification: 400 x), Gastric mucosa, basolateral NIS staining of the gastric mucinsecreting cells (original magnification: 1,000 x). One of the most promising recent developments in NIS research, that NIS is endogenously expressed in human breast cancer (10). Another high-impact trend is a series of studies by several investigators who have successfully obtained ectopic NIS expression by gene transfer techniques in prostate cancer (13, 14), myeloma (15-17), glioma cells (18) 9 melanoma cells (19) and in liver (20), among others. All these findings have given NIS its current unique position as the molecule with the tantalizing potential, whether by endogenous or ectopic expression, to render a wide variety of cancers amenable to effective treatment with radioiodide. Figure 3. Secondary structure model of NIS. Transmembrane segments are numbered with Roman numerals I-XIII. The N-terminus faces the extracellular milieu and the C- terminus the cytosol. N-glycosylation sites are indicated by arrows and the leucine zipper motif in the VI transmembrane segment is shaded gray. Serines on the C-terminus are indicated. Structural and Functional Characterization of NIS Both the availability of the NIS cdna (4, 5) and the generation of specific anti-nis Abs (21-23) have made it possible to carry out a thorough structure/function characterization of NIS (11, 12, 21, 24-28). Rat NIS is a protein of 618 amino acids (relative molecular mass 65,196); both human and pig NIS, which contain 643 amino acids, are highly homologous (75.9% and 74.2%, respectively) to rat NIS. Based on extensive experimental testing, a NIS 10 secondary structure model with 13 transmembrane segments has been proposed (Fig. 3). The predicted length of these segments ranges from 20 to 28 amino acid residues, except for transmembrane segment V, which contains 18 residues. The amino and carboxy termini face extra and intracellularly, respectively (21). NIS is a glycoprotein; three of its Asp residues (225, 485, 497) are glycosylated in the endoplasmic reticulum (11). Glycosylation is not essential for proper NIS function, as indicated by the observation that a nonglycosylated NIS mutant is properly targeted to the plasma membrane and displays I - transport activity with an identical Km value (~20-30 µm) to that of wild type NIS (33). The ca 70 amino acid-long hydrophilic carboxy terminus is the main phosphorylated region of the protein (12). Freeze-fracture electron microscopy studies of NIS-expressing Xenopus laevis oocytes revealed the appearance of 9 nm intramembrane particles corresponding to NIS (2). The size of these particles suggested that NIS may function as a multimeric protein. A putative leucine zipper motif constituted by leucines at positions 199, 206, 213 and 220 may be the structural basis for NIS oligomerization. Regulation of NIS Expression and Function TSH and I - are the two main factors that regulate NIS expression: TSH stimulates and I - decreases it. Hence, TSH stimulation and I - depletion of residual thyroid carcinoma tissue are the two most important modulators routinely used to optimize radioiodide treatment. To achieve maximum therapeutic effect, thyroidectomized patients must have TSH levels above 30 mu/l and must have been on a low I - diet for two weeks prior to initiation of radioiodide treatment (29). TSH has long been known to be a key regulator not only of NIS expression but also of thyroidal I - uptake (i.e., NIS activity). No thyroidal NIS expression is observed in hypophysectomized rats (because of the lack of TSH), but thyroidal NIS expression is restored as early as 24 h after treatment with TSH. In intact (i.e., non-hypophysectomized) rats, treatment with the I - organification inhibitor propylthiouracil causes elevated TSH levels, which in turn lead to higher NIS expression than in control animals (21). TSH 11 regulates NIS expression at both the transcriptional and posttranscriptional levels. Several groups have demonstrated that TSH upregulates I - transport by a camp-mediated increase in NIS transcription, while withdrawing TSH causes decreased camp levels and diminished NIS transcription (30). The detailed analysis of the rat and human NIS promoters has confirmed the significant role of Pax8 in NIS expression (31-33). In rat, the proximal NIS promoter was found to contain a TTF1 binding site and a TSH-responsive element where a putative transcription factor NTF-1 (NIS TSH-responsive factor 1) interacts (31). NIS upstream enhancer (NUE 2495 to -2260) contains two Pax-8 binding sites and a degenerate CRE (camp-responsive element sequence), which are essential for full TSH camp-dependent transcription of NIS (31). Interestingly, during chronic TSH stimulation when the catalytic subunit of PKA is downregulated, camp is still able to stimulate NIS transcription, indicating the existence of both PKA-dependent and independent mechanisms (31). Recently, a thyroid-specific farupstream (-9847 to 8968) enhancer in the human NIS gene highly homologous to the rat NUE has been reported. It contains putative Pax-8 and TTF-1 binding sites and a CRElike sequence. The TTF-1 binding site is not required for full activity (32, 33). FRTL-5 cells are rat-thyroid-derived, well-differentiated normal thyroid epithelial cells that grow in media supplemented with TSH. These cells are frequently used as an in vitro model system to study TSH regulation. In FRTL-5 cells, NIS expression is TSH dependent. Kaminsky et al (34) observed that, in the absence of TSH in the medium, intact FRTL-5 cells did not transport I -, whereas membrane vesicles prepared from the same TSHdeprived cells surprisingly maintained their I - transporting ability. This suggested that mechanisms other than transcriptional might also operate in regulating NIS activity in response to TSH. Riedel et al (12) investigated this phenomenon in detail. They observed that in the absence of TSH, there was no de novo NIS synthesis in FRTL-5 cells, while previously synthesized NIS was redistributed from the plasma membrane to intracellular membrane compartments. (Fig. 4) These authors also demonstrated that NIS has a long half-life: 5 days in the presence and 3 days in the absence of TSH. Considering the TSH regulation of NIS expression and the long half-life of NIS, it is clear that NIS mrna levels 12 Figure 4. A: Indirect immunofluorescence analysis of NIS localized in the plasma membrane of FRTL-5 cells kept in the presence of TSH. B: intracellulare NIS localization in TSH deprived FRTL- 5 cells. C: schematic representation of NIS plasma membrane localization and iodide transport in FRTL-5 cells kept in the presence of TSH D: schematic representation of NIS localized in the intracellulare membrane compartments in TSH deprived FRTL-5 cells, resulting in lack of iodide transport. alone are not a good indicator of actual NIS protein levels. Instead, NIS protein levels must be assessed directly with anti-nis Abs. In addition, it is also essential to keep in mind that NIS protein expression, in turn, does not necessarily correlate with NIS activity, because such factors as subcellular distribution of NIS to the plasma membrane play a key role in NIS function; hence, it is crucial to quantitate NIS activity (Fig. 4 and 5). Figure 5. Multiple levels of NIS regulation 13 TSH modulates NIS phosphorylation The mechanism by which TSH regulates the subcellular distribution of NIS is unknown. Phosphorylation has been shown to be implicated in the activation and subcellular distribution of several transporters (35-37). NIS has several consensus sites for kinases, including those for camp-dependent protein kinase, protein kinase C, and casein kinase-2 (12, 38). Furthermore, TSH actions in the thyroid are mainly mediated by camp. All these points raised the possibility that phosphorylation might be involved in the regulation of NIS subcellular distribution. When FRTL-5 cells were labeled with 32 Pi, lysed, and immunoprecipitated with anti-nis Ab, it was observed in the autoradiogram that NIS was phosphorylated, independently of the presence of TSH in the culture medium (12). The phosphopeptide map obtained after NIS digestion with trypsin was markedly different when TSH was present from that when TSH was absent (12). The predominant phosphorylated region of NIS was determined by treatment of the immunoprecipitated symporter with CNBr. CNBr cleaves polypeptides at methionine residues. The anti-nis Ab generated against the last 16 amino acids of NIS recognized an 11-kDa polypeptide observed also by autoradiog
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