RET/PTC1-MEDIATED PHOSPHOTYROSINE SIGNALING PATHWAYS INVOLVED IN THYROID CELL TRANSFORMATION DISSERTATION

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1 RET/PTC1-MEDIATED PHOSPHOTYROSINE SIGNALING PATHWAYS INVOLVED IN THYROID CELL TRANSFORMATION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University by Anjli Venkateswaran, B. Tech * * * * * The Ohio State University 2004 Dissertation Committee: Dr. Sissy M. Jhiang, Adviser Dr. Michael C. Ostrowski Dr. Gustavo W. Leone Dr. Ing-Ming Chiu Approved by Adviser The Ohio State Biochemistry Program

2 ABSTRACT The RET/PTC family of oncogenes are detected in human papillary thyroid carcinomas. RET/PTC1 is the most common form of RET/PTC that is detected in the general population. The overall aim of this study is to investigate the phosphotyrosine signaling pathways downstream of RET/PTC1 oncoprotein that mediate dedifferentiation in PC Cl 3 immortalized rat thyroid cells. Previously, site directed mutagenesis was performed to generate single tyrosine 404 or 451 to phenylalanine (F) mutants, thus abolishing phosphorylation at Y404 or Y451, and consequent downstream signaling. In this study, PC Cl 3 cell lines stably expressing either RET/PTC1, RET/PTC1 Y294F, RET/PTC1 Y404F or RET/PTC1 Y451F were generated to 1) investigate the mechanism of RET/PTC1 mediated NIS reduction, 2) investigate the role of py404 or py451 on NIS reduction and TSH-independent proliferation and 3) compare global gene expression profiles expression by microarray analysis and identify novel genes that are dysregulated by RET/PTC1 or RET/PTC1 Y/F expression. RET/PTC1 expression was demonstrated to interfere with the thyroid stimulating hormone (TSH) mediated cyclic AMP (camp)-protein kinase A (PKA) signaling pathway in PC Cl 3 rat thyroid cells by inhibiting cpka nuclear accumulation. Stimulation of the camp-pka pathway by forskolin, 8-Br-cAMP or catalytic PKA expression in the nucleus were able to reverse the effect of RET/PTC1 on NIS expression and function. ii

3 In chapter 3, the effect of single Y/F mutagenesis on RET/PTC1 subcellular localization, catalytic PKA nuclear localization, reduction of NIS protein expression and function and TSH-independent proliferation was investigated. Y/F mutagenesis of py451 reduced RET/PTC1 plasma membrane localization while py Y404 mediated PKC-Raf-MEK signaling was found to be important for RET/PTC1 mediated NIS reduction. Interestingly, further stimulation of PKC activation in RET/PTC1 expressing cells was sufficient to reduce forskolin mediated cpka nuclear accumulation, NIS protein expression and function. Microarray analysis was performed to compare gene expression profiles between PC Cl 3 parental, RET/PTC1, RET/PTC1 Y/F expressing cell lines and to identify novel genes that are dysregulated by RET/PTC1 or RET/PTC1 expression. Osteonectin, HIF-1α, aquaporin 5 and alpha crystallin B were identified as novel genes upregulated by RET/PTC1. Interestingly, RET/PTC1 Y294F which has reduced tyrosine kinase activity showed reduced expression of genes involved in tumor progression. In order to investigate the role of onset and expression level of RET/PTC1 on thyroid tumorigenesis, I generated and characterized PC Cl 3 CMV-Tet-On TRE- RET/PTC1 cell lines which have doxycycline (dox) inducible RET/PTC1 expression. TRE-RET/PTC1 transgenic mice were also generated and characterization of the mice for dox-inducible RET/PTC1 expression are currently underway. A bi-directional TRE construct expressing RET/PTC1 and luciferase was generated as a first step towards iii

4 generating bi-transgenic mice with thyroid targeted expression and dox-inducible RET/PTC1 and luciferase expression. Luciferase expression can be detected noninvasively via Xenogen imaging and therefore will serve as a reporter gene for RET/PTC1 expression. iv

5 Dedicated to my parents, Revathi and K.R. Venkateswaran, and to my husband, Manaswi Sharma v

6 ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Sissy Jhiang, for her support, and my committee members, Dr. Mike Ostrowski, Dr. Gustavo Leone and Dr. Ing-Ming Chiu for their helpful comments. I would like to thank Dr. Andrew Fischer (University of Massachusetts, Worcester), Dr. Steven Green (University of Iowa) and Drs. Jeffrey Knauf and James Fagin (University of Cincinnati) for providing several molecular reagents. Dr. Karl Kornacker (The Ohio State University) performed the statistical analysis on the microarray data. Thanks also to the OSUCCC microarray facility Herbert Auer, Jeff Palatini and David Newsom who performed the chip hybridization and to the OSU Transgenic Animal Facility for generating and maintaining the TRE- RET/PTC1 transgenic mice. I would like to acknowledge the help and support from former and current members of Dr. Jhiang s laboratory: Dr. Tara Buckwalter, Dr. Krista LaPerle, Dr. Je- Yoel Cho, Dr Kwon-Yul Ryu, Dr. Daniel Shen, Dr. Xiaoqin Lin, Dr. Derek Marsee, Dr. Katie Knostman, Zhaoxia Zhang, Douangsone Vadysirisack, Marc Lavender and Danielle Westfall. Most of all, I would like to thank my parents who have always encouraged and supported me, and my husband, Manaswi, who never stopped believing in me. vi

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8 VITA birthday...born December 27, B. Tech Industrial Biotechnology Anna University, Chennai, India present...graduate Research Associate The Ohio State University Research Publications PUBLICATIONS 1) TLF Buckwalter, A Venkateswaran, M Lavender, KMD La Perle, J-Y Cho, ML Robinson, and S M Jhiang The Roles of Phosphotyrosine-294, -404, and -451 in RET/PTC1-Induced Thyroid Tumor Formation, Oncogene, 2002 Nov 21;21(53): ) DK Marsee, A Venkateswaran, H Tao, D Vadysirisack, Z Zhang, DD Vandre and SM Jhiang Inhibition of Heat Shock Protein 90, a Novel RET/PTC1-associated Protein Increases Radioiodide Accumulation in Thyroid Cells J Biol Chem Epub ahead of print August ) A. Venkateswaran, DK Marsee, SH Green and SM Jhiang Forskolin, 8-Br-cAMP, and catalytic PKA expression in the nucleus increase radioiodide uptake and NIS protein levels in RET/PTC1 expressing cells in press J Clin Endocrinol Metab September viii

9 FIELDS OF STUDY Major Field: Biochemistry ix

10 TABLE OF CONTENTS Page Abstract...ii Dedication... v Acknowledgments...vi Vita...vii List of Tables...xi List of Figures...xii Chapters: 1. Introduction Forskolin, 8-Br-cAMP and catalytic PKA expression in the nucleus increase radioiodide uptake and NIS protein levels in RET/PTC1 expressing cells. Introduction Materials and Methods Results Discussion Signaling pathways underlying RET/PTC1 mediated NIS reduction in PC Cl 3 thyroid cells. Introduction Materials and Methods Results Discussion Comparison of gene expression profiles of PC Cl 3 parental, RET/PTC1 and RET/PTC1 Y/F expressing cells by microarray analysis x

11 Introduction Materials and Methods Results Discussion Generation and characterization of inducible RET/PTC1 expression in cultured rat thyroid cells and transgenic mice... Introduction Materials and Methods Results and Discussion Summary and Future Directions Bibliography xi

12 LIST OF TABLES Table Page 1.1 Tyrosine conversion chart showing the conversion of tyrosine residues numbering between RET, RET/PTC1, RET/PTC2 and RET/PTC Chromosomal location and expression of GDNF ligands and GFR-α family of receptors Proteins that bind to py1062 of various forms of RET Forskolin increased cpka nuclear accumulation in RET/PTC1 expressing cells Fold increase/decrease and known function of genes dysregulated in PC Cl 3 RET/PTC1 cells compared to PC Cl 3 parental cells Fold increase/decrease and known function of genes dysregulated in PC Cl 3 RET/PTC1 Y404F cells compared to PC Cl 3 RET/PTC1 cells Fold increase/decrease and known function of genes dysregulated in PC Cl 3 RET/PTC1 Y451F cells compared to PC Cl 3 RET/PTC1 cells Fold increase/decrease and known function of genes dysregulated in PC Cl 3 RET/PTC1 Y294F cells compared to PC Cl 3 RET/PTC1 cells xii

13 LIST OF FIGURES Figure Page 1.1 Key steps in thyroid hormone biosynthesis The hypothalamic-pituitary-thyroid axis GDNF-GFRα-RET signaling RET is rearranged to form the RET/PTC family of oncogenes TSH receptor mrna levels are decreased in PC Cl 3 cells with stable and acute RET/PTC1 expression Forskolin increases RAIU by 12 hours of treatment and in a dosedependent manner Forskolin increases NIS and Tg protein levels in RET/PTC1 expressing cells Br-cAMP increases radioiodide uptake in RET/PTC1 expressing cells in a temporal and dose-dependent manner Nuclear localization of GFP-tagged cpka is reduced in RET/PTC1 expressing cells compared to parental cells Nuclear localization of cpka is increased by forskolin in RET/PTC1 expressing cells xiii

14 2.7 Transient expression of GFP-cPKAnls increases RAIU and NIS protein levels Selective loss of tyrosine phosphorylation of RET/PTC1 Y/F mutants Adapter protein binding to RET/PTC1 and RET/PTC1 Y/F mutants PLCγ and STAT3 phosphorylation in PC Cl 3 parental, RET/PTC1 and RET/PTC1 Y/F cell lines RET/PTC1 py451 is important for targeting RET/PTC1 to the membrane Effect of PMA and GFX on cpka nuclear accumulation and NIS protein levels in PC Cl 3 parental, RET/PTC1 and RET/PTC1 Y/F cells Raf and MEK inhibition increase cpka nuclear accumulation and NIS protein levels in RET/PTC1 and RET/PTC1 Y451F cells Effect of PMA and BAY or PMA and PD98059 combination treatments on cpka nuclear accumulation in PC Cl 3 parental, RET/PTC1 and RET/PTC1 Y/F cells RET/PTC1 Y404F partially reversed the effect of RET/PTC1 on NISmediated radioiodide uptake Radioiodide uptake of PC Cl 3 RET/PTC1 and RET/PTC1 Y/F cells treated with DMSO control, PMA, GFX, BAY or PD Constitutively active MEK expression decreases cpka nuclear accumulation, NIS protein levels and RAIU xiv

15 3.10 PMA treatment abolished forskolin mediated increase in cpka nuclear accumulation, NIS protein levels and RAIU in PC Cl 3 RET/PTC1 and RET/PTC1 Y/F cells Representative real-time RT-PCR analysis of thyroid differentiation markers (NIS, TPO) and TTF-1 transcription factor mrna levels in PC Cl 3 parental, RET/PTC1 and RET/PTC1 Y/F cells Representative real-time RT-PCR analysis of osteonectin and α crystallin B mrna levels in PC Cl 3 parental, RET/PTC1 and RET/PTC1 Y/F cells Representative real-time RT-PCR analysis of gadd153 transcription factor mrna levels in PC Cl 3 parental, RET/PTC1 and RET/PTC1 Y/F cells Representative PC Cl 3 Tet-On clonal cell populations with doxinducible luciferase activity Transient expression of prevtre-ret/ptc1 is induced by dox Representative Western blots of dox-inducible RET/PTC1 expression in PC Cl 3 Tet-On TRE-RET/PTC1 stable clonal cell populations RET/PTC1 expression is regulated by dox dosage Temporal profile of RET/PTC1 expression RAIU of PC Cl 3 Tet-On RET/PTC1 cells treated with dox for different time periods xv

16 5.5 Dox-inducible RET/PTC1 expression is reversible RAIU and NIS expression of PC Cl 3 Tet-On cells is not reversible upon dox withdrawal Cloning strategy of prevtre-ret/ptc Cloning strategy of pbi-luc-ret/ptc Characterization of dox-inducible luciferase activity of pbi-luc- RET/PTC Dox treatment significantly increased RET/PTC1 expression in PC Cl 3 Tet-On cells transiently transfected with pbi-luc-ret/ptc xvi

17 CHAPTER 1 INTRODUCTION The normal thyroid gland The thyroid gland is an endocrine gland that has two functions: to produce thyroid hormones (triiodothyrononine or T3 and tetraiodothyronine or T4) and calcitonin. Thyroid hormones are essential for development and metabolism while calcitonin is involved in calcium homeostasis. The thyroid gland is composed mainly of follicular thyroid cells with interspersed clusters of C-cells. Thyroid hormone biosynthesis occurs in the follicular cells while calcitonin is produced in the C-cells. The tissue architecture of the thyroid gland consists of follicles which are in turn comprised of colloid surrounded by follicular cells. The rate limiting step in thyroid hormone biosynthesis is the uptake of iodine from the blood via the sodium/iodide symporter (NIS) which is expressed at the basolateral membrane of follicular cells. The iodide transported into the cell is transported to the colloid or follicular lumen by the apical transporter pendrin where it is coupled to tyrosine residues on thyroglobulin (Tg) by a reaction catalyzed by thyroid 1

18 peroxidase (TPO). Iodinated thyroglobulin is stored in the lumen as colloid and when the body requires thyroid hormone, it is endocytosed into the follicular cells where it is hydrolyzed to produce T4 and T3 that are released into the bloodstream for systemic circulation. Thyroid hormone biosynthesis is regulated by the hypothalamic-pituitarythyroid axis. When thyroid hormone levels are low, the hypothalamus releases thyrotropin releasing hormone (TRH) which stimulates the anterior pituitary to produce thyroid stimulating hormone or thyrotropin (TSH). TSH binds to the TSH receptor on the surface of follicular thyroid cells and stimulates the cyclic AMP (camp)-protein kinase A (PKA) signaling pathway to increase expression of NIS and Tg which results in increased thyroid hormone synthesis. Thyroid hormones are part of a negative feedback mechanism where excess thyroid hormone levels inhibit TSH release from the anterior pituitary. TSH signal transduction Thyrotrophin or TSH is considered to be the main regulator of proliferation and differentiation of thyroid follicular cells. TSH is a heterodimeric glycoprotein hormone that binds to the TSH receptor (TSHR). TSHR is located at the cell surface of thyrocytes and is a 7 transmembrane G-protein coupled receptor. Activation of TSHR leads to the coupling of different G proteins. Most of TSHR activity is mediated by the heterotrimeric G s protein that is composed of α, β and γ subunits. In human and rat thyrocytes, TSH can also stimulate the G q protein that activates the PLC/PKC signaling 2

19 pathway. TSH can also stimulate the G i protein which partially counteracts G s protein stimulation (Rivas and Santisteban 2003). Activated TSHR induces the dissociation of G s into α and βγ subunits. The G s α subunit stimulates the enzyme adenylate cyclase with subsequent increase of intracellular camp levels. Cyclic AMP can have either mitogenic or anti-mitogenic effects depending on cell type. Thyrocytes require camp to proliferate and constitutive activation of the camp signaling pathways results in thyroid hyperplasia. In rat, dog and human thyrocytes the activity of camp-dependent protein kinase A (PKA) is required for TSH-mediated mitogenesis (Kimura et al. 2001). PKA however, does not account for all the intracellular camp signaling effects. There are at least 2 PKA holoenzymes PKA I and PKA II, which are composed of 2 regulatory subunits (PKA-R) and 2 catalytic subunits (PKA-C). PKA I is typically cytosolic while PKA II (α and β) are often targeted to certain subcellular locations like the Golgi and the cytoskeleton by anchor proteins called AKAPs. Cyclic AMP activates PKA by binding to PKA-R subunits, which then releases the catalytic PKA subunits that enter the nucleus and phosphorylate specific targets (Taylor et al. 1990). One of the best characterized nuclear substrates of PKA is the camp-response element binding protein or CREB. PKA phosphorylates CREB at serine 133 and activates it to stimulate the transcription of several camp-responsive genes. CREB has been reported to be involved in TSH mediated proliferation (Woloshin et al. 1992). 3

20 The sodium-iodide symporter (NIS) The sodium-iodide symporter is the 13 trans-membrane glycoprotein that mediates active iodide uptake into thyroid follicular cells. This process is the first and rate-limiting step in thyroid hormone biosynthesis. NIS is localized in the basolateral plasma membrane and it couples the inward translocation of 2 sodium ions down their electrochemical gradient with one iodide ion against its electrochemical gradient. The sodium gradient that provides the driving force for iodide uptake is maintained by the sodium/potassium ATPase. Rat NIS was cloned in 1996 by the functional screening of a cdna library derived from FRTL-5 rat thyroid cells in Xenopus laevis oocytes (Dai et al. 1996) and subsequently human NIS (Smanik et al. 1996), mouse NIS (Perron et al. 2001; Pinke et al. 2001) and porcine NIS were cloned (Selmi-Ruby et al. 2003). Rat NIS is a 618 amino acid protein with a relative molecular mass of 65.2 kda while human NIS is a 643 amino acid protein with a relative molecular mass of 68 kda. NIS is detected mainly as a mature glycosylated 87 kda form. Interestingly, rat NIS has been reported to concentrate radioiodine up to 5 fold more than human NIS (Heltemes et al. 2003). The regulation of rat NIS expression has been extensively studied and the rat NIS promoter and upstream enhancer have been well characterized. Endo et al. localized a TTF-1 binding site in the proximal 2kb rat NIS promoter that confers thyroid-specific transcription but to a modest extent (Endo et al. 1997). Ohno et al. identified the rat NIS upstream enhancer (rnue) that mediates thyroid-specific transcription via the interaction of the paired box domain transcription 4

21 factor Pax8 with camp dependent pathway (Ohno et al. 1999). The rnue contains two Pax8 binding sites, two TTF-1 binding sites (one of which does not correspond to any relevant region) and one degenerate CRE-like sequence. Currently, it is believed that Pax8 and the CRE-binding factor act synergistically to maximize TSH-cAMP regulation of NIS expression. Thyroid Carcinomas Thyroid cancer accounts for about 1% of all neoplasms diagnosed in the United States and are the most prevalent endocrine malignancies. There are several forms of thyroid cancer. Carcinomas of the follicular thyroid cells include papillary thyroid carcinoma (PTC), follicular thyroid carcinomas (FTC) and anaplastic thyroid carcinomas (ATC). Another type of thyroid carcinoma is Hurthle cell carcinoma (HCC). PTCs are the most common form of thyroid cancer with an occurrence rate of about 60% while FTCs are the second most common form (15%). Higher rates of PTCs are observed in areas with radiation exposure while higher rates of FTCs are seen in iodine deficient regions (Gleich, 1999). ATCs are rarest form of thyroid follicular neoplasms but are the most aggressive form with high metastatic potential and poor prognosis. Medullary thyroid carcinomas (MTCs) are neoplasms of the C-cells and either sporadic or familial (multiple endocrine neoplasia type 2 or MEN2). MEN2 syndrome can be further divided into MEN2A and MEN2B (Gleich, 1999). 5

22 The RET proto-oncogene The RET proto-oncogene was identified as the susceptibility gene for MEN2 syndrome. Takahashi et al. reported that when human T cell lymphoma genomic DNA was transfected into NIH/3T3 cells, the cells were transformed (Takahashi et al. 1985). A novel recombinant gene was identified which was composed of ret (rearranged during transfection) and rfp (RET finger protein). After the ret gene was cloned, sequence homology revealed it to be a receptor tyrosine kinase (RTK) (Takahashi and Cooper 1987). The gene was localized to chromosome 10q11.2 and encodes an open reading frame spanning 20 exons (Ceccherini et al. 1993). The last exon (exon 21) does not contain any coding sequence (Kwok et al. 1993). The RET protein is comprised of three domains, an extracellular domain containing a signal peptide, cadherin-like motif and a cysteine-rich region, a single transmembrane domain and an intracellular region containing the tyrosine kinase (TK) domain that is split in half by a 27 amino acid insertion (Takahashi, 1988). The TK domain shares a 40-50% homology with other receptor tyrosine kinases with a conserved ATP binding site (Takahashi and Cooper 1987). Three different isoforms of RET have been identified p9, p43 and p51 and are a result of alternative splicing (Lorenzo et al. 1995). The isoforms are named based on the length of the amino acid sequence after a C-terminal splice site. All three RET isoforms are glycosylated and are detected as a kda immature form and kda mature form (Takahashi et al. 1991). The mature form is mainly detected at the plasma membrane while the immature form is mainly intracellular. 6

23 RET Ligands RET is the co-receptor for the glial cell line derived neurotrophic factor (GDNF) family. This family is comprised of GDNF, Neurturin (NTN), Persephin (PSP) and Artemin (ART). These ligands interact with multimeric receptors composed of glycosyl-phosphatidylinositol (GPI)-linked receptors and RET. Four forms of GPIlinked receptors have been identified GFRα1, 2, 3 and 4. GDNF, NTN, PSP and ARTN preferentially bind to GFRα1, 2, 3 and 4 respectively although alternative ligand-receptor interactions have been observed (reviewed in (Airaksinen et al. 1999)) and see figure 1.3. GDNF can bind to GFRα2 and GFRα3 in the presence of RET while NTN and ARTN can bind to GFRα1. The ligands bind to the GFR receptor and in turn the ligand-receptor complex interacts with RET to induce dimerization and activation of the tyrosine kinase activity. Role of RET in development Functional RET protein has been shown to be essential for kidney and enteric nervous system development. In rodent embryonic and adult tissues, RET is expressed in peripheral enteric, sympathetic and sensory neurons as well as in central motor, dopaminergic and noradrenergic neurons suggesting an important role of RET in differentiation and survival of these neurons (Tsuzuki et al. 1995). Furthermore, RET expression has also been shown in the excretory system such as the mesonephric duct and branching ureteric bud during embryogenesis although it almost disappears after 7

24 birth (Schuchardt et al. 1994). Consistent with its expression pattern, RET knockout mice lacked enteric neurons and superior cervical ganglia and had renal agenesis or dysgenesis. GDNF and GFRα1 knockout mice showed similar phenotypes as RET knockout mice except that the superior cervical ganglia were normal or moderately affected. Studies investigating the levels of RET expression during development show that RET is normally expressed during development in neural and excretory tissues in zebrafish, Drosophila, mice, rats and humans (Sugaya et al. 1994; Schuchardt et al. 1995; Tsuzuki et al. 1995; Marcos-Gutierrez et al. 1997; Widenfalk et al. 1999). Mutations that inhibit RET tyrosine kinase activity have been reported in Hirschsprung s disease in which the enteric nervous system innervation is affected, resulting in aganglionic megacolon (Edery et al. 1994; Romeo et al. 1994). RET point mutations in disease Point mutations in RET can have either a loss-of-function effect as in Hirschsprung s disease or a gain-of-function effect as in sporadic medullary thyroid carcinoma (MTC), familial MTC (FMTC) or multiple endocrine neoplasia type 2 (MEN2A and MEN2B). Mutations that cause Hirschsprung s disease have been found in locations spanning the entire RET sequence. The consequences of these mutations are usually TK inactivation or a failure to transport the mutant protein to the cell surface. In contrast, RET mutations found in sporadic MTC, FMTC and MEN2 lead to constitutive tyrosine kinase activity of RET. Patients with sporadic MTC and FMTC present with only 8

25 medullary thyroid carcinoma. MEN2A patients present with MTC (100% of cases) in conjunction with pheochromocytomas (50% of cases) and parathyroid hyperplasia (15-30% of cases). MEN2B patients develop MTC (100%) and pheochromocytomas (50% of cases) with an earlier age of tumor onset along with developmental abnormalities such as ganglioneuromatosis, medullated corneal nerves and marfanoid habitus. MEN2A mutations have been identified in one of six cysteine residues (codons 609, 611, 618 and 620 in exon 10 and codons 630 and 634 in exons 11) in the extracellular domain of RET (Eng et al. 1996). The cysteines that are mutated in MEN2A are involved in intra-molecular disulfide bond formation, so the consequence of the cysteine being mutated to a non-cysteine residue is that an unpaired cysteine residue in a RET monomer forms an inter-molecular disulfide bond with another RET monomer resulting in dimerization and activation. Approximately 90% of MEN2A mutations affect codon 634 where the most common mutation is C634R. In FMTC, the point mutations are detected in cysteine residues like MEN2A or at codons 768, 804 or 891. The most common mutation in MEN2B is a M918T mutation which has been reported in 98% of patients. Another mutation that has been reported in MEN2B is a missense point mutation at codon 883. The RET/MEN2B receptor remains a monomer in the absence of ligand binding but the M918T mutation stimulates the tyrosine kinase activity and alters substrate specificity. Consequently, RET/MEN2B remains responsive to ligands and therefore temporal and tissue-specific expression of the GDNF family of ligands may account for the clinical phenotype of MEN2B (see review (Jhiang 2000)). 9

26 RET signal transduction The RET receptor tyrosine kinase is activated by the binding of the GDNF family of ligands that first bind to the GFR receptors and then the ligand-receptor complex binds to RET which forms dimers. RET dimerization leads to autophosphorylation of specific tyrosine residues, which recruit several adapter proteins that complex with specific tyrosine residues and in turn activate various signal transduction pathways ( see review Airaksinen et al. 1999). Several adapter proteins that complex with RET have been identified. Grb10 is a member of the Grb7 family of SH2 domain adapter proteins. It has a central Pleckstrin Homology (PH) domain which may play a role in membrane targeting. Grb10 was found to bind to phospho-tyrosine (py) 905 of RET (Pandey et al. 1995). Phosphotyrosine 905 is located in the tyrosine kinase domain and tyrosine to phenylalanine mutagenesis results in decreased tyrosine kinase activity (Iwashita et al. 1996). Grb2 is an adapter protein that consists of 1 SH2 and 2 SH3 domains. Grb2 binds to phosphotyrosines via the SH2 domain and also interacts with the guanine nucleotide exchange factor Sos. It has been reported to bind directly to py1096 of the long form of RET and indirectly to py1062 (Alberti et al. 1998). Shc proteins have been reported to bind to py1062 of RET via the phosphotyrosine binding domain (PTB) of Shc (Asai et al. 1996). Shc recruits the Grb2/Sos complex and is involved in Ras/MAPK signaling. Three forms of Shc have been identified 66 kda, 52 kda and 46 kda. Enigma is a LIM domain containing proteins that recognizes tyrosine containing tight turn structures but do not require phosphorylation of the tyrosine residue for complex formation. 10

27 Enigma has been implicated in the cell periphery docking of RET/PTC2 and therefore may play a role in subcellular localization of rearranged forms of RET. Durick et al. reported that RET/PTC2 binds to Enigma and localizes near the plasma membrane (Durick et al. 1998). Phospholipase C gamma (PLCγ) has been reported to bind to Y1015 of full-length RET (Borrello et al. 1996; Durick et al. 1996). PLCγ converts the membrane phospholipids phosphoinositol to diacylglycerol and inositol tri-phosphate (IP 3 ). Diacylglycerol activates protein kinase C isoforms while IP 3 increases intracellular Ca 2+ by causing the release of Ca 2+ from intracellular stores. Different methods have been used to investigate the downstream signaling pathways of RET (reviewed in (van Weering and Bos 1998)). Prior to the identification of the GDNF family members as RET ligands, RET/MEN2A and RET/MEN2B constitutively active forms of RET were used to study signal transduction. RET/MEN2A and RET/MEN2B were reported to transform NIH/3T3 fibroblasts by inducing foci formation, tumor formation in nude mice and anchorage-independent proliferation in soft agar (Iwashita et al. 1996). Tyrosine to phenylalanine mutagenesis is one approach to dissect the role of individual phospho-tyrosine residues in RET. The rationale of this strategy is to replace the hydroxyl group containing aromatic side chain of tyrosine with the aromatic side chain of phenylalanine that lacks the hydroxyl group. Therefore, the tyrosine to phenylalanine mutagenesis would not alter the protein structure but would abolish phosphorylation, adapter protein binding and further downstream signaling. This approach has been used to identify several adapter proteins that complexed with specific RET phosphotyrosines. 11

28 Another approach is to use a chimeric EGF receptor fused to the intracellular portion of RET which was activated by EGF. Using this approach, Santoro et al. reported that the chimeric receptor weakly activated PLCγ and stimulated Ras (Santoro et al. 1994). Several reports investigating the signaling pathways downstream of phosphotyrosines 1062 and 1096 of RET have been published. Phosphotyrosine 1062 was reported to be important for the transforming potential of MEN2 forms of RET via the MAPK pathway (Asai et al. 1996). However, another group reported that py1062 also activated the PI-3K pathway in RET/MEN2A (Segouffin-Cariou and Billaud 2000). Using the Y/F mutagenesis approach, De Vita et al. showed that py1062 is important for mediating PI-3K and MAPK signal transduction in PC 12 pheochromocytoma cells and provided evidence that the p85 regulatory subunit of PI- 3K complexes directly with py1062 to mediate PI-3K/Akt signaling (De Vita et al. 2000). Besset et al. investigated the signaling pathways mediated by RET py1062 and py1096 in fibroblast cell lines stably expressing GFRα1 and either wild type RET, Y1062F, Y1096F RET or the double mutant Y1062F/Y1096F RET. Y1062 was found to be the major activator of ERK signaling with little or no contribution from Y1096 (Besset et al. 2000). In contrast, Y1096 was found to be involved in the activation of PI- 3K/Akt since the Y1062/Y1096 double mutant showed a complete loss of GDNFdependent Akt phosphorylation (Besset et al. 2000). Since py1062 can activate both MAPK and PI-3K signaling, Hayashi et al. investigated the differential adapter protein complex formation that determines whether MAPK or PI-3K is activated. They found that in GDNF stimulated cells expressing full length RET, Shc bound to py1062 and also complexed with Gab1 and Grb2. Tyrosine-phosphorylated Gab1 associated with 12

29 the p85 regulatory subunit of PI-3K whereas Shc-Grb2-Sos complex was responsible for the activation of the Ras/MAPK pathway (Hayashi et al. 2000). More recently, Melillo et al. reported that the lipid anchored FRS2 docking protein was tyrosine phosphorylated by and complexed with RET at py1062 in NIH/3T3 cells stably expressing an EGFR/RET chimeric receptor (Melillo et al. 2001). FRS-2 binding to RET increased MAPK phosphorylation and interestingly, increased membrane localization of RET/PTC3 (Melillo et al. 2001). Furthermore, the tyrosine phosphatase SHP-2 co-precipitated with FRS-2 and may be involved in regulating py1062 signaling (Kurokawa et al. 2001). Recently, the p62dok family of docking proteins were reported to complex with RET and mediate neuronal differentiation of PC12 cells. Dok family members bind directly to py1062 via their phosphotyrosine binding (PTB) domains and become phosphorylated themselves. Dok-4 and dok-5 were found to stimulate MAPK signaling and trigger neuronal outgrowths in PC12 cells (Grimm et al. 2001). Taken together, py1062 of RET is the most extensively characterized phosphotyrosine of RET and appears to regulate several downstream signaling pathways. Chromosomal rearrangements of RET The oncogenes that results from RET chromosomal rearrangements are found exclusively in human papillary thyroid carcinomas (PTCs) and are referred to as RET/PTC oncogenes. To date, ten different RET/PTC oncogenes have been identified 13

30 RET/PTC 1-7, RET/PCM1, RET/ELKS and Delta rfp/ret (see figure 1.4). The different oncogenic forms of RET/PTC vary, based on the N-terminal genes that fuse with the intracellular portion of RET to generate chimeric oncogenes. The RET/PTC oncogenes are the result of either chromosomal inversions (RET/PTC 1, 3, 4 and 5) or chromosomal translocations (RET/PTC 2, 6, 7, 8, RET/PCM1, RET/ELKS and Deltarfp/RET). Each of the RET/PTC fusion proteins contain a dimerization domain (coiled-coil motif) from the N-terminal fusion partner and therefore RET/PTC proteins are capable of ligand-independent constitutive dimerization and activation. Furthermore, the RET promoter is substituted with the N-terminal fusion partners promoters, all of which are expressed in thyroid follicular cells thus resulting in RET/PTC expression in thyroid follicular cells. The leucine zipper motif in the H4 part of RET/PTC1 is essential for RET/PTC1 oncogenic activity since deletion of the motif results in the loss of tyrosine phosphorylation and transforming activity of RET/PTC1 (Tong et al. 1997). The incidence of RET/PTC activation in spontaneous papillary thyroid carcinomas (PTCs) range from 5-30% but the incidence of RET/PTC activation in radiation-induced PTCs has been reported to be as high as 60-70%. In areas contaminated by the Chernobyl nuclear fallout, RET/PTC3 has been associated with childhood PTCs of short latency while RET/PTC1 has been associated with childhood PTCs of long latency. 14

31 Transgenic mouse models of RET/PTC thyroid tumorigenesis\ Jhiang et al. generated transgenic mice with thyroid targeted expression of RET/PTC1 and reported that the mice formed bilateral papillary thyroid carcinomas that were characterized by slow growth rate, TSH-dependent tumor progression and reduced radioiodide concentrating ability (Jhiang et al. 1996). The tumors had the histological characteristics of humans PTCs. Thyroid-specific expression of RET/PTC1 was conferred by the use of the bovine thyroglobulin promoter. The time of tumor onset was found to be dependent on the expression level of the transgene since high-copy mice developed cellular abnormalities as early as embryological day 18 (Cho et al. 1999). Furthermore, Sagartz et al. reported that the PTCs of RET/PTC1 transgenic mice were responsive to TSH and chronic TSH stimulation resulted in the development of spindle and giant cells that are characteristic of anaplasia (Sagartz et al. 1997). However, p53 overexpression was not detected in any of the tumors (Sagartz et al. 1997). Interestingly, La Perle et al. reported that RET/PTC1 transgenic mice crossed with p53 knockout mice developed anaplastic thyroid tumors that were invasive and had a high mitotic index (La Perle et al. 2000). This indicates that loss of p53 may play a role in the development of dedifferentiated anaplastic carcinoma from well differentiated papillary thyroid carcinomas. Santoro et al. generated transgenic mice with the rat thyroglobulin promoter driving thyroid-specific expression of the RET/PTC1 transgene. These mice also developed papillary thyroid carcinomas with the histological characteristics of human PTCs (Santoro et al. 1996). 15

32 Recently, Buckwalter et al. generated transgenic mice with thyroid targeted expression of RET/PTC1, RET/PTC1 Y294F, RET/PTC1 Y404F or RET/PTC1 Y451F. The aim of the study was to investigate the contribution of each phosphotyrosine signaling pathway to thyroid tumorigenesis. However, no one signal transduction pathway was found to be solely essential for RET/PTC1 tumor formation. Interestingly, RET/PTC1 Y294F mice showed the least rate of tumor formation (6%) which may be due to reduced tyrosine kinase activity. RET/PTC1 Y404F mice had a 41% tumor incidence rate while RET/PTC1 Y451F mice had a 30% tumor incidence rate. Overall, the thyroid tumor formation in all three mutants was lower than that of RET/PTC1 indicating that signaling pathways mediated by all three phosphotyrosines contribute to tumor development (Buckwalter et al. 2002). RET/PTC1 mediated thyroid cell transformation RET/PTC mediates thyroid cell transformation by decreasing expression of differentiation markers such as NIS and Tg and conferring TSH-independent proliferation. The most extensively studied cell lines are immortalized rat thyroid cell lines FRTL-5, PC Cl 3 and WRT. All these cell lines express thyroid differentiation markers such as NIS, Tg, TPO and TSHR and depend on TSH for proliferation. However, they are fairly simple to culture and can be transfected and genetically modified. Since they are untransformed cell lines, they are used extensively to investigate different aspects of thyroid biology. PC Cl 3 cells were isolated from 18 month old Fisher rats while FRTL-5 cells were isolated from 5-6 week old Fisher rats 16

33 and are derived from clonal isolation of FRTL cells (see review (Kimura et al. 2001)). We used PC Cl 3 cells for our studies since we could transfect or transduce them with a greater degree of efficiency compared to FRTL-5 cells (unpublished results). Furthermore, FRTL-5 cells have been reported to be genetically unstable and undergo clonal variability (Huber et al. 1990) while no reports have been published regarding the stability of PC Cl 3 cells. Santoro et al. generated PC Cl 3 cells stably expressing RET/PTC1 via retroviral transduction and showed that PC Cl 3 RET/PTC1 cells were able to proliferate in the absence of the six hormones normally present in culture media and that there was a loss of mrna levels of differentiation specific markers such as NIS, Tg and TPO. The morphology of PC Cl 3 RET/PTC1 cells did not significantly vary from PC Cl 3 normal cells. Interestingly, PC Cl 3 RET/PTC1 cells did not grow in soft agar or form tumors in nude mice. However, PC Cl 3 RET/PTC1 cells that were infected with Ha-Ras or Ki- Ras virus were fully transformed and formed aggressive tumors in nude mice (Santoro et al. 1993). RET/PTC1 has been reported to downregulate NIS mrna levels in PC Cl 3 rat thyroid cells (Trapasso et al. 1999) as well as decrease radioiodide accumulation in the thyroids of transgenic mice with thyroid-targeted expression of RET/PTC1 (Cho et al. 1999). The mechanisms by which RET/PTC1 downregulates NIS is not fully understood. Recently, Knauf et al. reported that py1062 of full-length RET (corresponding to py451 of RET/PTC1) mediated the reduction of NIS and Tg mrna levels via the Ras-MAPK pathway in PC Cl 3 cells acutely expressing RET/PTC3 (Knauf et al. 2003). 17

34 Pax8 and thyroid transcription factor-1 (TTF-1) are the best characterized transcription factors that are bind to and activate thyroid-specific promoters (Endo et al. 1997). TTF-1 is a homeodomain containing protein that is expressed in the thyroid, lung and diencephalon. The Tg and TPO promoters both have Pax8 and TTF-1 binding sites and De Vita et al. showed that RET/PTC downregulated Tg and TPO promoter activity in PC Cl 3 cells. Furthermore, RET/PTC decreased Pax8 transcription which reduced Pax8 mrna and protein levels but did not reduce TTF-1 expression (De Vita et al. 1998). TTF-1 function was impaired at the post translational level but the mechanism by which RET/PTC1 affects TTF-1 function has not been elucidated. Though the rat NIS promoter has been extensively characterized, the effect of RET/PTC1 on NIS promoter activity in PC Cl 3 cells has not been described. It is possible that RET/PTC1 may downregulate NIS expression by reducing NIS promoter activity. RET/PTC subcellular localization The RET/PTC oncoproteins lack a transmembrane domain and therefore cannot insert into the plasma membrane like full-length RET. However, there are reports that RET/PTC forms may localize near the cell periphery via interactions with specific docking proteins or the presence of specific domains in the N-terminal portion. RET/PTC3 is the product of the fusion of the N-terminal RFG gene and the C- terminal portion of RET. The RFG gene encodes a protein that regulates steroid receptors including the androgen receptor and peroxisome proliferators-activated receptors (PPAR-α and PPAR-γ). RFG proteins are membrane localized with a portion 18

35 at the plasma membrane. RET/PTC3 was shown to interact with RFG proteins via the RFG domain. This results in the phosphorylation of RFG and relocalization of RET/PTC3 to the cell periphery (Monaco et al. 2001). Enigma is a 455 amino acid protein that contains a N-terminal PDZ domain and three C-terminal LIM domains. Enigma interacts with RET and RET/PTC2 via the second LIM domain in a phosphorylation-independent manner. Enigma was reported to anchor RET/PTC2 at the plasma membrane (Durick et al. 1998). Though the dok family of docking proteins have been reported to interact with full-length RET, there are no reports that the RET/PTC forms interact with dok proteins. It would be interesting to investigate whether RET/PTC proteins interact with dok proteins to localize near the cell periphery. The functional consequence of RET/PTC localization to the membrane fractions or cell periphery is that specific adapter proteins are localized to specific subcellular locations. For example, PI-3K holoenzyme is localized near cellular membranes so it is possible that RET/PTCs have to localize near the membrane to activate PI-3K. Phospholipase C gamma (PLCγ) is a cytosolic enzyme whose substrate (phosphatidyl-inositol) is a component of cellular membranes. Therefore, to be catalytically active PLCγ1 has to be membrane associated to mediate downstream signaling. 19

36 Blood + 2Na 1 I - NIS NIS Iodide uptake Thyroid follicular cell Pendrin AIT TPO + H 2 2O 2 oxidation I - I - Lumen Thyroglobulin Deiodination iodination Hormone secretion MIT DIT Tg TPO + H 2 O 2 T3 T4 DIT MIT MIT Colloid DIT Thyroglobulin resorption T3 proteolysis T4 Figure 1.1: Key steps in thyroid hormone biosynthesis include basolateral membrane iodide uptake (NIS), apical iodide transport (pendrin, apical iodide transporter), iodination of thyroglobulin (Tg) by thyroid peroxidase (TPO), endocytosis, proteolysis and hormone secretion. (Na: sodium, I: iodine, H 2 O 2 : hydrogen peroxide, MIT: monoiodotyrosine, DIT: diiodotyrosine, T3: triiodothyronine and T4: tetraiodothyronine.) Modified from TLF Buckwalter Phosphotyrosine-mediated signal transduction pathways essential for RET/PTC1-induced tumor formation PhD dissertation

37 Hypothalamus TRH Pituitary T3 T4 TSH Thyroid Figure 1.2: The hypothalamic-pituitary-thyroid axis. The release of thyrotropin releasing hormone (TRH) from the hypothalamus, and subsequently, thyroid stimulating hormone (TSH) from the pituitary leads to thyroid hormone secretion (T3, T4) from the thyroid. Acting by negative feedback regulation, T4 and T3 inhibit further production of TSH. 21

38 GDNF NTN ART PSP RET LBD GFRα1 GFRα2 TM GFRα3 GFRα4 TK Grb7/10-(p)Y 905 Y(p)? Cdc42 JNK Enigma Ras Grb2-Shc-(p)Y 1062 Gab1 Grb2-(p)Y 1096 p51 p9 p43 Y 1015 (p)-plcγ DAG IP 3 production PKC ERK CREB, etc PI3K Akt Ca 2+ release Figure1.3: GDNF-GFRα-RET signaling. The GDNF family of ligands (GDNF, Neurturin (NTN), Artemin (ART) and Persephin (PSP) bind to the GFRα family of receptors and the receptor-ligand complex binds to RET and activate downstream signaling molecules. 22

39 Figure 1.4: RET is rearranged to form the RET/PTC family of oncogenes. RET/PTC forms share the intracellular portion of RET including the tyrosine kinase domain and are fused to different N-terminal donor genes.the breakpoint location is indicated by a black arrow. The N-terminal donor genes and corresponding PTC names are listed. The extracellular domain (EC), transmembrane domain (TM), tyrosine kinase domain (TK), coiled-coil domain (CC), cysteine residues involved in disulfide bonds in PTC2 dimerization (C18, C39), ring finger domain (RF), b1 and b2 box domains (b1, b2), phd finger domain (phd), and the bromodomain (Br) are indicated. Modified from (Jhiang 2000), *(Corvi et al. 2000), **(Saenko et al. 2003) 23

40 EC TM TK RET N-terminal donor gene H4 CC TK RET/PTC1 RIα C18 C39 TK RET/PTC2 ELE1 CC TK RET/PTC3 r2 r3 r1 ELE1 CC TK RET/PTC4 RFG5 CC x 4 TK RET/PTC5 htif1 RF b1 b2 CC TK RET/PTC6 RFG7 RF b1 b2 CC phdbr TK RET/PTC7 ELKS CC x 6 TK RET/PTC8 (ELKS-RET) RET/PCM-1 TK * RET/PCM-1 RFP CC TK ** Delta rfp/ret 24

41 Adapter protein Phosphotyrosine binding site RET RET/PTC1 RET/PTC2 RET/PTC3 Grb10/ PLC γ Shc/Enigma Table 1.1: Tyrosine conversion chart showing the conversion of tyrosine residues numbering between RET, RET/PTC1, RET/PTC2 and RET/PTC3. 25

42 Gene Glial cell-derived neurotrophic factor (GDNF) Neurturin (NTN) Artemin (ART) Persephin (PSP) GFR -1 GFR -2 GFR -3 GFR -4 RET proto-oncogene Chromosomal location 5p13.1-p p13.3 1p33-p32 19p q26 8p q31.1-q31.3 3q36 * 10q11.2 Expression Brain, thyroid, lung, kidney, GI tract Brain, PNS, thyroid, heart, lung, GI tract, kidney, liver Brain Brain, heart, kidney, liver Brain, PNS, thyroid, heart, lung, GI tract, kidney, liver Brain, PNS, thyroid, heart, lung, GI tract, kidney, liver PNS, thyroid, heart, lung, GI tract, kidney, liver Brain, heart, testis * Brain, PNS, thyroid, heart, lung, GI tract, kidney, liver Modified from Hoff et al., Annu Rev Physiol, 2000, 62:377 *(Masure et al. 2000) Table 1.2: Chromosomal location and expression of GDNF ligands and GFR-α family of receptors. 26

43 Adapter protein Ret form Methodology Cell line Shc RET/PTC2 Yeast two hybrid system GST pulldown NIH/3T3 1 RET (+ GDNF) Immunoprecipitation NIH/3T3 2 RET/MEN2A Immunoprecipitation NIH/3T3 3 Enigma RET/PTC2 GST pulldown 293 HEK 1 Grb2 RET/PTC2 Immunoprecipitation, Y/F mutagenesis COS7 4 RET (+ GDNF) Immunoprecipitation NIH/3T3 4 p85 (PI-3 kinase) RET/MEN2A Immunoprecipitation, GST pulldown PC12 5 Rat-1 6 FRS-2 EGFR-RET chimera Immunoprecipitation NIH/3T3 7, PC Cl 3 7 RET-MEN2A GST pulldown 293 HEK 7 RET (+ GDNF) GST pulldown SK-N-SH, TGW, SK-N- MC, NIH/3T3 8 Dok-2, dok-4, dok- 5 RET (+GDNF) Flag immunoprecipitation 293 HEK 9 Table 1.3: Proteins that bind to py1062 of various forms of RET. GST glutathione S- transferase, EGFR Epidermal growth factor receptor. 1 (Durick et al. 1998) 2 (Besset et al. 2000) 3 (Asai et al. 1996) 4 (Alberti et al. 1998) 5 (De Vita et al. 2000) 6 (Segouffin-Cariou and Billaud 2000) 7 (Melillo et al. 2001) 8 (Kurokawa et al. 2001) 9 (Grimm et al. 2001) 27

44 CHAPTER 2 FORSKOLIN, 8-Br-cAMP, AND CATALYTIC PKA EXPRESSION IN THE NUCLEUS INCREASE RADIOIODIDE UPTAKE AND NIS PROTEIN LEVELS IN RET/PTC1 EXPRESSING CELLS INTRODUCTION RET/PTC1 is a chimeric oncogene that is expressed in thyroid follicular cells and leads to the development of papillary thyroid carcinomas. RET/PTC1 consists of the N-terminus of the H4 gene that contains a leucine zipper domain, fused to the intracellular portion of RET that contains the tyrosine kinase domain. RET/PTC1 undergoes constitutive dimerization, leading to autophosphorylation of specific tyrosine residues. These phosphotyrosines serve as docking sites to recruit and activate several downstream signaling molecules implicated in cell proliferation and differentiation, such as Ras, PLCγ, and Akt (see review Jhiang 2000). In addition, expression of RET/PTC1 has been demonstrated to reduce camp-mediated signaling events (Wang et al. 2003). 28

45 In thyroid follicular cells, thyroid stimulating hormone (TSH)-mediated camp signaling is the major regulator of proliferation and differentiation. Recently, Wang et al. reported that inducible RET/PTC3 expression reduced TSHR mrna levels and decreased camp levels by blocking adenylyl cyclase activity directly (Wang et al. 2003). Furthermore, RET/PTC3 reduced DNA synthesis stimulated by 8-Br-cAMP, indicating that RET/PTC also acts at site(s) distal to adenylyl cyclase. Thus, they concluded that acute expression of RET/PTC decreases TSH-mediated growth by interfering with TSH signaling at multiple levels. In addition to inducing proliferation, RET/PTC has also been shown to reduce thyroid-specific gene expression, resulting in dedifferentiation. RET/PTC1 has been reported to reduce expression of the sodium/iodide symporter (NIS) in cultured rat thyroid cells (Trapasso et al. 1999) and also reduces radioiodide accumulation in the thyroid tissue of transgenic mice with thyroid targeted RET/PTC1 expression (Jhiang et al. 1996). Furthermore, RET/PTC1 has been reported to downregulate rat NIS promoter activity in NIH/3T3 cells stably expressing RET/PTC1 (Tong et al. 1997). Several lines of evidence suggest that Ras is the major downstream signaling molecule of RET/PTC that mediates thyroid cell dedifferentiation. Knauf et al. have reported that activation of the SHC-Ras-MAPK pathway is required for RET/PTC3 to reduce NIS mrna levels (Knauf et al. 2003). It has also been demonstrated that expression of constitutively active Ras is sufficient to reduce the accumulation of radioiodide in thyroid cells (Monaco et al. 1995). Interestingly, Gallo et al. have reported that v-ras induces thyroid dedifferentiation by inhibiting nuclear accumulation of catalytic PKA (cpka) (Gallo et al. 1992). Taken together, these studies suggest that RET/PTC, most likely via 29

46 Ras activation, may reduce expression of thyroid-specific genes by interfering with camp/pka signaling. However, the ability of camp/pka signaling to increase NIS expression and radioiodide accumulation in RET/PTC1-expressing cells has not been previously investigated. In this study, we report that forskolin, which increases intracellular camp levels, was sufficient to increase radioiodide uptake and NIS expression in RET/PTC1 expressing cells. Similarly, 8-Br-cAMP, a camp agonist, was able to increase radioiodide uptake activity in RET/PTC1 expressing cells. Furthermore, RET/PTC1 inhibits nuclear localization of catalytic PKA, and forced expression of catalytic PKA in the nucleus increases NIS expression and function in RET/PTC1 expressing cells. These results suggest that RET/PTC mediates NIS reduction by interfering TSH-cAMP- PKA signaling pathway and these effects are reversed by enhancing camp-pka pathways. 30

47 MATERIALS AND METHODS Cell Culture PC Cl 3 immortalized rat thyroid cells were maintained in Coon s modified F-12 media (Irvine Scientific) with 5% calf serum, 2mM glutamine, 1% Penicillin- Streptomycin (Invitrogen), 10 mm NaHCO 3 and 6H hormone mixture (1 mu/ml bovine TSH, 10 µg/ml bovine insulin, 10 nm hydrocortisone, 5 µg/ml transferrin, 10 ng/ml somatostatin and 2 ng/ml L-glycyl-histidyl-lysine). Phoenix retroviral producer cells were cultured in DMEM supplemented with 10% FBS (Invitrogen) and 1% Penicillin- Streptomycin (Invitrogen). Reagents, DNA constructs, retrovirus production and generation of PC Cl 3 stable cell lines Forskolin and 8-Br-cAMP were purchased from Sigma. GFP-cPKA, GFPcPKAnes and GFP-cPKAnls plasmids were constructed as described elsewhere (Bok et al. 2003). RET/PTC1was cloned into the plncx vector (Clontech). Phoenix retroviral packaging cells were transiently transfected with plnc-ret/ptc1 by the calcium phosphate precipitation method. Twenty-four hours after transfection, the media was changed and the cells were incubated at 32 o C for optimal virus production. After 48 hours, the media was harvested, centrifuged at 1000 rpm to pellet producer cells, and 31

48 then filtered through a 0.22µm filter. The filtered viral supernatant was aliquoted and stored at 80 o C. PC Cl 3 parental cells (3 x 10 5 ) were seeded in 60-mm plates and were transduced 24 hours later with 1 ml of RET/PTC1 viral supernatant. Forty-eight hours after infection, the media was changed to media containing 400 µg/ml G418 (Invitrogen) for selection for 5 days. Stable clones were isolated and expanded in media containing 200 µg/ml G418 and were screened for RET/PTC1 expression by Western blot analysis. PC Cl 3 Tet-On cells were generated by transducing PC Cl 3 cells with CMV-Tet-On retrovirus and selecting stable clones with 400 µg/ml G418. PC Cl 3 Tet- On cells were screened for Dox-inducible expression with TRE-Luciferase. A clonal cell population with highest induction was infected with TRE-RET/PTC1retrovirus to generate Doxycycline-inducible PC Cl 3 Tet-On RET/PTC1cell lines. Dox-inducible RET/PTC1expression was confirmed by Western blot analysis. Quantitative real-time Reverse Transcriptase-PCR Total RNA was isolated using TRIzol reagent (GibcoBRL) from PC Cl 3 parental, PC Cl 3 RET/PTC1 and PC Cl 3 Tet-On TRE-PTC1 (untreated or treated with 2 µg/ml doxycycline) cell lines, according to the manufacture s protocol. One microgram of RNA was treated with 1 U of DNase 1, (GibcoBRL) followed by quantitative real-time RT-PCR analysis using a one-step QuantiTect SYBR Green RT-PCR Kit (Qiagen) in a LightCycler system (Roche). The TSHR primers used in the reaction were: 32

49 TSHRF: 5 aat gga aca aag ctg gat gc 3 TSHRR: 5 agt gag gtg gag gaa gct ca 3 SYBR Green fluorescence was measured after each extension step in order to monitor amplification. RT-PCR products were analyzed by measuring their annealing temperatures and by gel electrophoresis to verify their specificity and identity. GAPDH was used to normalize the expression level of TSHR. Western blot analysis Western blot for rat NIS was performed as described below. Membrane fractions were prepared from each cell line as described elsewhere (Jhiang et al. 1998), resolved on a 7.5% polyacrylamide gel and transferred to a nitrocellulose filter. The filter was blocked with 5% dry milk at room temperature for 1 hour and then probed with PA716 anti-rat NIS antibody at 1:1500 dilution (a kind gift from Dr. Bernard Rousset,INSERM, Lyon, France). The secondary antibody was donkey anti-rabbit IgG- HRP (1:5000) followed by ECL detection. The intensity of the Western blot signals was quantitated using NIH Image software. To determine equal protein loading, the blots were probed with an antibody against the V-ATPase E subunit (a kind gift from Dr. B.S. Lee, The Ohio State University) at 1:1000 dilution followed anti-rabbit secondary antibody conjugated to HRP. The signal was detected by ECL. Alternatively, the blots were stripped in stripping buffer (62.5 mm Tris-HCl ph 6.8, 100 mm β- mercaptoethanol and 2% SDS) at 50 o C for 1 hour and probed with insulin receptor β 33

50 antibody (Santa Cruz, CA) at a dilution of 1:500 followed by anti-rabbit IgG-HRP. Detection of signal was by ECL. Fifty µg of total cell lysates were subjected to Western blotting and probed with rabbit anti-thyroglobulin antibody (a kind gift from Dr. Paul Kim, University of Cincinnati, Cincinnati, OH) at a dilution of 1:1000, followed by anti-rabbit IgG-HRP at a dilution of 1:2500. Detection was performed using ECL. To determine equal protein loading, the blots were probed with mouse monoclonal PLCγ antibody (Santa Cruz) at 1:500 dilution followed by anti-mouse IgG-HRP secondary antibody (Cell Signaling Technology) and ECL detection. Radioiodide uptake assay Radioiodide uptake (RAIU) assay was performed essentially as described elsewhere (La Perle et al. 2002). 7x10 4 cells were seeded per well in a 24-well plate. When applicable, cells were treated with either forskolin or 8-Br-cAMP at the indicated doses for indicated time periods after which RAIU assay was performed. PC Cl 3 parental and RET/PTC1 expressing cells were transiently transfected with GFPcPKAnls using FuGene6 (Roche). Twenty-four hours after transfection, RAIU assay was performed. Briefly, 2 µci 125 I-Na diluted in 0.5 mm NaI to a final concentration of 5 µm was added to each well followed by incubation at 37 o C for 30 minutes. The cells were then washed with ice-cold HBSS (Invitrogen) twice and lysed in 95% ethanol. The ethanol lysates were counted in a gamma counter (Cobra Quantum, Packard). Additions of 3 µm sodium perchlorate reduced radioiodide uptake activity to less than 1000 cpm 34

51 in both PC Cl 3 parental and RET/PTC1 cells (data not shown). The counts per minute (cpm) were normalized by cell number (1 x10 5 cells). Statistical analysis was performed using the paired t-test (GraphPad Software, CA). Detection of GFP fluorescence PC Cl 3 parental and RET/PTC1 expressing cells were seeded on coverslips in 60-mm plates. Twenty four hours after seeding, cells were transiently transfected with GFP-cPKA, GFP-cPKAnes or GFP-cPKAnls using FuGene6 (Roche). Forskolin treatment (10 µm) was performed 24 hours after transfection for 12 hours. Forty-eight hours after transfection, the cells were washed with PBS and fixed in 4% paraformaldehyde for 15 minutes at room temperature. After fixation, the coverslips were mounted on glass slides using aqueous mounting media (Biomeda). GFP fluorescence was detected using a Zeiss Axioskop equipped with a 40x objective lens. Only intact cells were scored for nuclear or cytoplasmic GFP fluorescence. 35

52 RESULTS RET/PTC1 reduces TSH receptor mrna levels in stable and inducible RET/PTC1 expressing cells TSH receptor mrna levels were reduced 22.4% in PC Cl 3 RET/PTC1 expressing cells compared to PC Cl 3 parental cells. Acute expression of RET/PTC1 reduced TSH receptor mrna levels to a greater extent (36.7%) than stable expression of RET/PTC1 (Figure 2.1). Our data supports the finding reported by Wang et al. who showed that acute expression of RET/PTC3 reduced TSH receptor mrna levels (Wang et al. 2003). RET/PTC1 mediated NIS reduction can be rescued by Forskolin or 8-Br-cAMP In agreement with others (Trapasso et al. 1999), we showed that stable expression of RET/PTC1 reduced radioiodide uptake (Figure 2.2) and NIS protein levels (Figure 2.3) in PC Cl 3 rat thyroid cells. Forskolin, an adenylyl cyclase activator that increases intracellular camp levels, was able to increase RAIU in RET/PTC1 expressing cells in a temporal (Figure 2.2A) and dose-dependent manner (Figure 2.2B). It is of great interest to note that 10 µm forskolin treatment for 12 hours was sufficient to increase RAIU in RET/PTC1 expressing cells to the same level as the parental PC Cl 3 cells. In contrast, forskolin treatment did not further increase RAIU in PC Cl 3 parental cells. The increase of RAIU by forskolin in RET/PTC1 expressing cells is most 36

53 likely due to an increase in NIS expression (Figure 2.3). Forskolin treatment also increased Tg expression in RET/PTC1 expressing cells. For PC Cl 3 parental cells, forskolin slightly increased Tg expression but not NIS expression. The camp agonist 8-Br-cAMP also increased RAIU in a temporal (Figure 2.4A) and dose-dependent (Figure 2.4B) manner. However, the extent of increase in RAIU by 8-Br-cAMP appears to be less than that of forskolin. Furthermore, 8-BrcAMP slightly decreased RAIU in PC Cl 3 parental cells. RET/PTC1 decreases nuclear accumulation of catalytic PKA While the mechanism underlying NIS reduction by RET/PTC1 is not fully understood, Ras, a major downstream signaling molecule of RET/PTC1, has been shown to inhibit nuclear accumulation of cpka (Feliciello et al. 1996). To investigate the effect of RET/PTC1 on cpka nuclear accumulation, PC Cl 3 parental and RET/PTC1 expressing cells were transiently transfected with GFP tagged catalytic PKA (GFP-cPKA), GFP-cPKA containing a nuclear export sequence (GFP-cPKAnes) or GFP-cPKA containing a nuclear localization sequence (GFP-cPKAnls). PC Cl 3 parental cells showed mainly nuclear accumulation of GFP-cPKA while PC Cl 3 RET/PTC1 cells showed mainly cytosolic localization of GFP-cPKA. As expected, GFP-cPKAnes had cytosolic localization while GFP-cPKAnls had nuclear accumulation in both parental and RET/PTC1 cells (Figure 2.5). 37

54 Forskolin increases cpka nuclear accumulation in RET/PTC1 expressing cells To investigate whether forskolin can increase cpka nuclear accumulation in RET/PTC1 expressing cells, PC Cl 3 parental and RET/PTC1 expressing cells were transiently transfected with GFP-cPKA, GFP-cPKAnes, or GFP-cPKAnls followed by forskolin treatment. In the absence of forskolin treatment, cpka nuclear accumulation was found in 80% of transfected PC Cl 3 parental cells, but in only 7% of transfected RET/PTC1 expressing cells (Figure 2.6 and Table 2.1). For PC Cl 3 parental cells, forskolin further increased cpka nuclear accumulation to 90% transfected cells. In comparison, forskolin increased cpka nuclear accumulation from 7% to 60% in transfected RET/PTC1 expressing cells (Figure 2.6 and Table 2.1). Transient expression of GFP-cPKAnls is sufficient to increase RAIU and NIS expression in RET/PTC1 expressing cells We investigated whether forced expression of GFP-cPKAnls is sufficient to increase RAIU and NIS expression in RET/PTC1 expressing cells. As shown in Figure 2.7A, transient expression of GFP-cPKAnls increased RAIU in RET/PTC1 cells, yet did not increase RAIU significantly in PC Cl 3 parental cells. We further demonstrated that NIS reduction in RET/PTC1 expressing cells was reversed by transient expression of GFP-cPKAnls, while NIS protein levels in parental cells was not changed (Figure 2.7B). In comparison, transient expression of GFP-cPKAnes did not significantly increase RAIU in either parental or RET/PTC1 expressing cells (data not shown). 38

55 DISCUSSION The mechanism by which RET/PTC reduces NIS expression has not previously been fully elucidated. Recently, Knauf et al. reported that RET/PTC3 reduced NIS mrna levels through the Ras-MAPK pathway (Knauf et al. 2003), and Wang et al. demonstrated that acute expression of RET/PTC3 decreased TSH receptor mrna levels and reduced camp levels in PC Cl 3 cells (Wang et al. 2003). Considering the fact that elevation of serum TSH levels is effective in inducing radioiodide uptake activity in some thyroid tumors (see review by (Mazzaferri and Kloos 2002)) and that Ras has been shown to inhibit nuclear localization of cpka, we hypothesize that, similar to Ras signaling, RET/PTC1 reduces NIS expression and function by interfering with the TSH-cAMP-PKA signaling pathway. Our hypothesis is supported by this study showing that RET/PTC1-reduced NIS expression and function could be reversed by stimulating the camp-pka pathway. Several reports have provided evidence that Ras mediates thyroid dedifferentiation by inhibiting nuclear accumulation of catalytic PKA. Gallo et al. reported that v-ras inhibited nuclear localization of the catalytic subunit of PKA via protein kinase C (PKC) (Gallo et al. 1992). Feliciello et al. showed that juxtanuclear localization of RIIβ is essential to maintain camp-dependent differentiation in rat thyroid cells, and that Ras activation induce cytosolic translocation of RIIβ resulting in decreased cpka nuclear accumulation (Feliciello et al. 1996). Our study provides direct evidence that RET/PTC, similar to Ras, decreases cpka nuclear accumulation (Figure 2.5). Furthermore, increased nuclear accumulation of cpka by forskolin treatment 39

56 (Figure 2.6 and Table 2.1) or forced expression of nuclear localized cpka (Figure 2.7) can increase NIS expression in PC Cl 3 RET/PTC1 expressing cells. In thyroid cells, RET/PTC has been reported to confer TSH-independent proliferation, loss of differentiation markers, and apoptosis (Santoro et al. 1993; Castellone et al. 2003). Wang et al. recently reported that acute expression of RET/PTC interferes with TSHstimulated proliferation at various levels (Wang et al. 2003). Similarly, we showed that stable expression of RET/PTC1 reduces NIS expression by interfering with camp-pka signaling. In addition, elevation of camp or forced expression of nuclear localized cpka was sufficient to increase NIS expression in RET/PTC1-expressing cells. The fact that nuclear accumulation of cpka was increased by forskolin treatment in RET/PTC1 expressing cells (Figure 2.6) suggests that signaling downstream of adenylyl cyclase was not affected by RET/PTC1 to reduce NIS expression. Recently, it has been reported that NIS and TPO, but not Tg, expression are lost in mice with defects in TSH or TSH receptor (Postiglione et al. 2002). Thus, the authors concluded that TSH signaling is necessary for NIS and TPO expression but not for Tg expression (Postiglione et al. 2002; De Felice et al. 2004). In agreement with this finding, our data show that forskolin significantly increases NIS expression, but only moderately increases Tg expression, in RET/PTC1 expressing cells (Figure 2.3). Signaling pathways involved in TSH-mediated proliferation and differentiation in thyroid cells have been extensively studied (see reviews by (Rivas and Santisteban 2003; De Felice et al. 2004)). It appears that TSH-mediated proliferation is affected by many other factors such as IGF-1 and serum (see review by (Kimura et al. 2001)). In comparison, this study and the study by Postiglione et al. indicate that NIS expression 40

57 in thyroid cells appears to be critically dependent on TSH-cAMP-PKA signaling pathway. Similar to most human tumors carrying RET/PTC1, the RET/PTC1 expressing PC Cl 3 cells remain differentiated, expressing Tg and NIS, albeit at reduced levels. In addition, the finding that NIS expression and radioiodide uptake activity in RET/PTC1 expressing cells is increased by stimulating camp/pka signaling pathways is similar to human cancers responsive to TSH stimulation. The fact that NIS expression/function is increased by elevation of serum TSH levels in these thyroid tumors suggests that the defects underlying NIS reduction in these tumors are reversible and mainly contributed by factors interfering TSH signaling. For tumors refractory to TSH-stimulated radioiodide uptake, in which defect(s) may occur at TSH receptor, efforts should be made to directly stimulate camp-pka signaling pathways. 41

58 A. TSHR/GAPDH % parental RET/PTC1 B. TSHR/GAPDH % 0 Dox 2 µg/ml Dox Figure 2.1: TSH receptor mrna levels are decreased in PC Cl 3 cells with (A) stable and (B) acute RET/PTC1 expression. TSH receptor mrna levels are normalized by GAPDH mrna levels. 42

59 Figure 2.2: (A) Forskolin increases radioiodide uptake within 12 hours of treatment. 125 I uptake is expressed as counts per minute (cpm) per 10 5 cells. RAIU of forskolin treated RET/PTC1 cells in comparison to DMSO treated cells was statistically significant (* p< 0.05). (B) Forskolin increases radioiodide uptake in RET/PTC1 expressing cells in a dose-dependent manner. 125 I uptake is expressed as counts per minute (cpm) per 10 5 cells. RAIU of forskolin treated RET/PTC1 cells in comparison to DMSO treated cells was statistically significant (* p< 0.05). 43

60 A. RAIU (cpm/10 5 cells) FK : Parental * hrs * hrs RET/PTC1 B. RAIU (cpm/10 5 cells) * * * * FK: - 10 µm - 1 µm 2 µm 5 µm 10 µm Parental RET/PTC1 44

61 Parental RET/PTC1 10 µm FK: Tg 300 kda PLCγ1 155 kda NIS 87 kda V-ATPase E subunit 31 kda Figure 2.3: Forskolin increases NIS protein levels in RET/PTC1 expressing cells but not in PC Cl 3 parental cells. In comparison, forskolin increases thyroglobulin (Tg) protein level in both RET/PTC1 expressing cells and PC Cl 3 parental cells. Cells were treated with either DMSO or 10 µm forskolin for 12 hours. The cytosolic fractions were used for Tg Western blot analysis while membrane fractions were used for NIS Western blot analysis. Equal loading was determined by reprobing the blot with mouse anti- PLCγ antibody (for Tg) or with an antibody against an integral membrane protein V- ATPase E subunit (for NIS). 45

62 Figure 2.4: (A) 8-Br-cAMP increases radioiodide uptake in RET/PTC1 expressing cells in a temporal manner. 125 I uptake is expressed as counts per minute (cpm) per 10 5 cells. RAIU of 8-Br-cAMP treated RET/PTC1 cells in comparison to control cells was statistically significant (* p<0.05). (B) 8-Br-cAMP increases radioiodide uptake in RET/PTC1 expressing cells in a dosedependent manner. 125 I uptake is expressed as counts per minute (cpm) per 10 5 cells. RAIU of 8-Br-cAMP treated RET/PTC1 cells in comparison to control cells was statistically significant (* p< 0.05). 46

63 A RAIU (cpm/10 5 cells) * Br-cAMP: - 1 mm - 12 hrs 24 hrs Parental RET/PTC1 B RAIU (cpm/10 5 cells) * Br-cAMP: - 1 mm µm 500 µm 1 mm Parental RET/PTC1 47

64 GFP - cpka GFP- cpkanes GFP- cpkanls Parental RET/PTC1 Figure 2.5: (A) Nuclear accumulation of GFP-tagged cpka is reduced in RET/PTC1 expressing cells compared to parental cells. Magnification: 400x. GFP-tagged cpka with nuclear export sequence (nes) or nuclear localization sequence (nls) were included as controls. The results were consistent in three independent experiments. 48

65 GFP - cpka (DMSO) GFP-cPKA (+ 10 µm FK) Parental RET/PTC1 Figure 2.6: Nuclear accumulation of cpka is increased by forskolin in RET/PTC1 expressing cells. Cells were transiently transfected with GFP-tagged catalytic PKA (GFP-cPKA) and treated with DMSO or 10 µm forskolin for 12 hours prior to GFP detection. Magnification: 400x. The results were consistent in three independent experiments. 49