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Molecular Endocrinology, doi:10.1210/me.2004-0168
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Molecular Endocrinology 18 (11): 2672-2684
Copyright © 2004 by The Endocrine Society

Regulation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT1-Dependent Genes by RET/PTC (Rearranged in Transformation/Papillary Thyroid Carcinoma) Oncogenic Tyrosine Kinases

Eun Suk Hwang, Dong Wook Kim, Jung Hwan Hwang, Hye Sook Jung, Jae Mi Suh, Young Joo Park, Hyo Kyun Chung, Jung Hun Song, Ki Cheol Park, Su Hyeon Park, Hwan-Jung Yun, Jin Man Kim and Minho Shong

Laboratory of Endocrine Cell Biology (E.S.H., D.W.K., J.H.H., H.S.J., J.M.S., H.K.C., J.H.S., K.C.P., S.H.P., J.M.K., M.S.), National Research Laboratory Program, Department of Internal Medicine, Department of Internal Medicine (Y.J.P.), Seoul National University College of Medicine, Seoul 151-742, Korea; and Department of Pathology (H.-J.Y.), Chungnam National University School of Medicine, Daejeon 301-721, Korea

Address all correspondence and requests for reprints to: Minho Shong, Laboratory of Endocrine Cell Biology, Department of Internal Medicine, Chungnam National University School of Medicine, 640 Daesadong Chungku Taejon 301-721, Korea. E-mail: minhos{at}cnu.ac.kr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chimeric RET/PTC (rearranged in transformation/papillary thyroid carcinoma) oncoproteins are constitutively active tyrosine kinases found in thyroid papillary carcinoma and nonneoplastic Hashimoto’s thyroiditis. Although several proteins have been identified to be substrates of RET/PTC kinases, the pathogenic roles played by RET/PTC in malignant and benign thyroid diseases and the molecular mechanisms that are involved are not fully understood. We found that RET/PTC expression phosphorylates the Y701 residue of STAT1, a type II interferon (IFN)-responsive protein. RET/PTC-mediated signal transducer and activator of transcription 1 (STAT1) phosphorylation requires RET/PTC kinase activity to be intact but other tyrosine kinases, such as Janus kinases or c-Src, are not involved. RET/PTC-induced STAT1 transcriptional activation was not inhibited by suppressor of cytokine signaling-1 or -3, or protein inhibitors of activated STAT3 [(protein inhibitor of activated STAT (PIAS3)], but PIAS1 strongly repressed the RET/PTC-induced transcriptional activity of STAT1. RET/PTC-induced STAT1 activation caused IFN regulatory factor-1 expression. We found that STAT1 and IFN regulatory factor-1 cooperated to significantly increase transcription from type IV IFN-{gamma}responsive promoters of class II transactivator genes. Significantly, cells stably expressing RET/PTC expressed class II transactivator and showed enhanced de novo membrane expression of major histocompatibility complex (MHC) class II proteins. Furthermore, RET/PTC1-bearing papillary thyroid carcinoma cells strongly expressed MHC class II (human leukocyte-associated antigen-DR{alpha}) genes, whereas the surrounding normal tissues did not. Thus, RET/PTC is able to phosphorylate and activate STAT1. This may lead to enhanced MHC class II expression, which may explain why the tissues surrounding RET/PTC-positive cancers are infiltrated with lymphocytes. Such immune response-promoting activity of RET/PTC may also relate to the development of Hashimoto’s thyroiditis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE RET (REARRANGED in transformation) protooncogene encodes a membrane receptor that expresses tyrosine kinase activity in tissues of neural crest origin, including the sympathetic ganglia, the adrenal medulla, thyroid C cells, and the excretory system of the developing kidney (1). Glial cell line-derived neurotropic factor, neurturin, artemin, and persephin have recently been shown to be RET ligands (2, 3). Gain-of-function mutations of the RET protooncogene are associated with cancer syndromes such as multiple endocrine neoplasia types 2A and 2B (MEN2A and 2B) and familial medullary thyroid carcinoma (4, 5).

RET/PTC (RET/papillary thyroid carcinoma) chimeric tyrosine kinases are formed by the rearranged fusion of the RET tyrosine kinase domain to the 5'-end sequences of different donor genes (6, 7). Of the several forms of RET/PTC, RET/PTC1, and RET/PTC3 are the most frequently found in papillary thyroid carcinomas (7). RET/PTC tyrosine kinases are constitutively active and trigger the activation of signaling pathways by interacting with and phosphorylating several signaling molecules such as Shc, Grb2, fibroblast growth factor receptor substrate 2, phospholipase C, downstream of kinase, phosphoinositide-dependent kinase 1, and the signal transducer and activator of transcription 3 (STAT3) (8, 9, 10, 11, 12). The pathogenic activities of RET/PTC may depend on the presence of an intact catalytic domain and the interaction motifs that are located in the C-terminal region (13).

RET/PTC gene rearrangements are observed not only in papillary thyroid carcinomas but also in nonneoplastic Hashimoto’s thyroiditis (14). Hashimoto’s thyroiditis can be considered as a representative autoimmune thyroid disease, whereas papillary thyroid carcinoma is the most common type of malignant thyroid tumor. Interestingly, recent clinical observations showed that the phenotypes of papillary thyroid carcinoma and Hashimoto’s thyroiditis are closely related in terms of their histology and immunohistochemical staining patterns, and more importantly, share the same molecular profile (15). Hashimoto’s thyroiditis is also frequently associated with submicroscopic foci of papillary thyroid cancer (16, 17). These observations suggest that RET/PTC activation may play a role in the pathogenesis of not only differentiated papillary thyroid carcinomas but also thyroid-specific autoimmune responses.

Thyroid epithelial cells have been observed to aberrantly express major histocompatibility complex (MHC) class II molecules in autoimmune thyroid diseases (18). The expression of MHC class II molecules has long been considered to be a factor that contributes to the development and amplification of autoimmune thyroid diseases (18). In this study, we showed that RET/PTC tyrosine kinase phosphorylates and activates STAT1. The activation of STAT1 results in the serial induction of STAT1-responsive genes encoding IRF-1 [interferon (IFN) regulatory factor-1], class II transactivator (CIITA), and MHC class II. These observations suggest that RET/PTC is able not only to activate the signaling pathways for cell transformation but also to activate key molecules such as STAT1 involved in immunomodulatory pathways.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tyrosine Phosphorylation of STAT1 by RET/PTC
STAT1 and STAT3 are activated by the phosphorylation of their Y701 and Y705 tyrosine residues, respectively, by the nonreceptor tyrosine kinases Janus kinases (JAKs), and c-Src (19, 20). STAT3 has also been reported to be phosphorylated on the Y705 residue by RET/PTC1 and RET/PTC3 (12). To determine whether STAT1 can also be phosphorylated by RET/PTC chimeras, we first transfected TPC1 human thyroid papillary thyroid carcinoma cells, which bear the RET/PTC1 rearrangement (21), with a STAT1 expression plasmid and determined STAT1 phosphorylation statusby Western blot analysis using anti-RET and phospho-RET (Tyr 905) antibodies, which respectively recognize total RET/PTC1 and phosphorylated RET/PTC1. This analysis confirmed that TPC1 cells express total RET/PTC1 and phosphorylated RET/PTC1, suggesting that this cell line bears significant amounts of activated RET/PTC tyrosine kinase (Fig. 1AGo). Use of a phospho-specific anti-STAT1 antibody indicated that the Y701 residue of exogenous STAT1 introduced by transfection was markedly phosphorylated. Moreover, the Y701 residue of endogenous STAT1 was also phosphorylated (Fig. 1AGo, lane 1). When ARO thyroid cancer cells, which do not bear a RET/PTC rearrangement, were analyzed similarly, the phosphorylation of the Y701 residue of endogenous STAT1 was not detected (Fig. 1Go, B and C, lane 1). Thus, RET/PTC1 expression leads to phosphorylation of the Y701 residue of STAT1.



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  		Fig. 1. Tyrosine Phosphorylation and Activation of Endogenous and Exogenous STAT1 by RET/PTC

A, Whole-cell lysates of TPC1 cells cultured in six-well plates and transfected with a STAT1 expression plasmid were subjected to 10% SDS-PAGE and immunoblot analysis using the antibodies indicated. B, Whole-cell lysates of ARO cells cultured in six-well plates and transfected with various doses of the RET/PTC1 expression plasmid were subjected to 10% SDS-PAGE and immunoblot analysis using the antibodies indicated. C, Whole-cell lysates of ARO cells cultured in six-well plates and transfected with expression plasmids expressing WT RET/PTC3 or the kinase-deficient RET/PTC3 K/M mutant were subjected to 10% SDS-PAGE and immunoblot analysis using the antibodies indicated. The figures represent results from three or more independent experiments.

 
To confirm that RET/PTC phosphorylates endogenous STAT1, RET/PTC1 was introduced into ARO cells (Fig. 1BGo). This increased the level of Y701-phosphorylated endogenous STAT1 (Fig. 1BGo, lane 1 vs. lanes 2–4) without concurrently changing the total level of STAT1. In addition, when wild-type (WT) RET/PTC3 was expressed in ARO cells, it also induced tyrosine phosphorylation of STAT1 (Fig. 1CGo, lane 1 vs. lanes 2 and 3). However, the kinase-deficient form of RET/PTC3, RET/PTC3 K/M, which contains a substitution of lysine 284 with methionine, could not induce Y701 phosphorylation of STAT1 (Fig. 1CGo, lanes 5 and 6). The phosphospecific antibody that specifically recognizes the phosphorylated S727 residue of STAT1 also did not bind to the lysates of any of the RET/PTC-expressing cells (data not shown). In summary, RET/PTC1 and 3 expressing cells, but not kinase-deficient RET/PTC cells, show phosphorylation of the Y701 residue of STAT1. These observations suggest that the intact tyrosine kinase activity of RET/PTC is required for the phosphorylation of the Y701 residue of STAT1.

To confirm that RET/PTC expression can lead to the phosphorylation of the Y701 residue of exogenous STAT1, RET/PTC1 was coexpressed with WT or mutant (Y701F) STAT1 in NIH3T3 cells. IFN-{gamma} treatment of the NIH3T3 cells, which served as a positive control, revealed that endogenous STAT1 is phosphorylated on the Y701 residue by this treatment (Fig. 2AGo, lane 2). Unlike human ARO cells, mouse NIH3T3 cells showed a very low level of endogenous phosphorylated STAT1 after RET/PTC transfection. However, RET/PTC1 transfection did induce the Y701 phosphorylation of endogenous or exogenous WT STAT1 (Fig. 2AGo, lane 3 and 4), although it did not induce the Y701 phosphorylation of mutant STAT1-Y701F (Fig. 2AGo, lane 6). Thus, the Y701 residue of STAT1 is targeted when RET/PTC is expressed.



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  		Fig. 2. Tyrosine Phosphorylation of Exogenous STAT1 and Induction of IRF-1 by RET/PTC

A, NIH3T3 cells were cultured in six-well plates and cotransfected with RET/PTC1 or RET/PTC3 together with plasmids expressing WT human STAT1 or the STAT1-Y701F mutant (500 ng/well). The cell lysates were then subjected to 10% SDS-PAGE and immunoblot analysis using the antibodies indicated. B, NIH3T3 cells were cultured in six-well plates and transfected with the plasmids expressing RET/PTC3 or the RET/PTC3 K/M mutant (500 ng/well). The lysates were then subjected to 10% SDS-PAGE and immunoblot analysis using the antibodies indicated. The blots shown represent results from three or more independent experiments.

 
STAT1 activation in cytokine signaling pathways induces IRF-1 expression (22, 23). Thus, we analyzed the effect of STAT1 activation in RET/PTC-expressing cells on IRF-1 expression. We found that IFN-{gamma} treatment and RET/PTC3 transfection of WT STAT1expressing NIH3T3 cells can both induce IRF-1 expression (Fig. 2BGo). However, when these cells were transfected with the kinase-deficient RET/PTC3 mutant (RET/PTC3 K/M), IRF-1 was not expressed (Fig. 2BGo). Transfection with RET/PTC1 also induced IRF-1 expression in NIH3T3 cells (data not shown). Thus, the induction of IRF-1 expression under these conditions requires RET/PTC and its kinase activity.

Activation of STAT1 by RET, MEN2A, MEN2B, and RET/PTC
Cotransfection of ARO cells with increasing amounts of the RET/PTC3 expression plasmid led to the activation of the promoter of the 8xGAS reporter, which contains multiple IFN-{gamma} activation site (GAS) elements that are bound by STAT1 (Fig. 3AGo). However, cotransfection with kinase-deficient RET/PTC3 did not increase the reporter activity of the 8xGAS construct (Fig. 3AGo). When ARO cells were cotransfected with MEN2A and 2B gain-of-function mutants of RET, STAT1 was activated but the STAT1-mediated increase in luciferase activities was much less than that achieved by RET/PTC (Fig. 3BGo). Thus, RET/PTC activates STAT1-mediated transcriptional activity by phosphorylation of the Y701 residue of STAT1.



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  		Fig. 3. Activation of a STAT1-Responsive Reporter by RET/PTC

A, ARO cells cultured in six-well plates were cotransfected with 300 ng/well of the 8xGAS luciferase reporter plasmid and with either RET/PTC3 or RET/PTC3 K/M mutant expression plasmid (300 ng/well) as indicated. B, ARO cells cultured in six-well plates were cotransfected with 300 ng/well of the 8xGAS luciferase reporter plasmid and with the indicated expression plasmids (300 ng/well). C, ARO cells cultured in six-well plates were cotransfected with 300 ng/well of the 8xGAS luciferase reporter plasmid and the RET/PTC1, Jak2 and v-Src expression plasmids (300 ng/well) and then treated with PD98059 (50 µM), PP1 (5 µM), or AG490 (100 nM). After 12 h, the cells were lysed and luciferase activity was measured. The results represent the means ± SD from three or more experiments.

 
Because STAT proteins are known to be phosphorylated on tyrosine residues by JAK kinases (24) and Src kinases (20), we investigated whether JAK2 and Src kinases are involved in RET/PTC-mediated STAT1 tyrosine phosphorylation. As shown in Fig. 3CGo, RET/PTC1 increases 8xGAS reporter activity. In addition, JAK2 increased 8xGAS reporter activity. Similarly, v-Src transfection also increased 8xGAS reporter activity. JAK2 and v-Src-mediated activation of 8xGAS reporter activity was significantly suppressed by the specific inhibitors, AG490 and PP1, respectively. However, PP1, an inhibitor of c-Src-related kinases and EGF-R kinase, did not affect the RET/PTCinduced increase in 8xGAS reporter activity (Fig. 3CGo). In addition, an inhibitor of JAK2 tyrosine kinase, AG490, had no effect on RET/PTC-mediated STAT1 activation (Fig. 3CGo). Thus, neither JAK2 nor other c-Src-related kinases are involved in RET/PTC-mediated STAT1 tyrosine phosphorylation.

A recent study indicated that the phosphorylation of the Y1062 residue of c-Ret (corresponding to Y451 of RET/PTC1) mediates Ras/ERK activation through interactions with several adapters (25). To determine whether Ras/ERK activation modulates RET/PTCinduced 8xGAS reporter activity, ARO cells were cotransfected with the RET/PTC1 and 8xGAS luciferase constructs and the cells were treated with PD98059, a MAPK-specific inhibitor. PD98059 did not significantly inhibit RET/PTC1-mediated activation of the 8xGAS promoter (Fig. 3DGo). Thus, the Ras/ERK pathway is not involved in RET/PTC-mediated STAT1 activation.

Roles Played by Suppressor of Cytokine Signaling (SOCS) and Protein Inhibitor of Activated STAT (PIAS) in RET/PTC-Mediated STAT1 Activation
The JAK and STAT signaling pathways are negatively regulated by several molecules, including SH2domain containing phosphatase-1 (26), SOCS (27), and PIAS (28, 29) protein families. A SOCS family member, SOCS-1, also termed JAB (Jak-binding protein) (30) or SSI-1 (STAT-induced STAT inhibitor 1) (31), was first identified through its ability to inhibit JAK2. SOCS-3 is induced by hormones and cytokines and may participate in inhibiting responses to leukemia inhibitory factor (32), GH (32), leptin (33), and IL-2 (34) by blocking JAK1 activity. To determine whether SOCS are induced by RET/PTC, NIH3T3 cells were transfected with RET/PTC1 or RET/PTC3 and their extracts were examined by Northern blot analysis with SOCS1 and SOCS3 cDNA probes. The expression of RET/PTC1 and RET/PTC3 increased SOCS-1 and SOCS-3 RNA levels in NIH3T3 cells (Fig. 4AGo).



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  		Fig. 4. Down-Regulation of RET/PTC-Mediated STAT1-Responsive Reporter Activity by PIAS1

A, NIH3T3 cells cultured in six-well plates were cotransfected with RET/PTC1 or RET/PTC3 expression plasmid (500 ng/well). P, control vector (pcDNA3.1 vector, lane 1). Total RNA was isolated 24 h after transfection and subjected to Northern analysis (20 µg/lane) using SOCS1, SOCS3, and ß-actin cDNA probes as described in Materials and Methods. The amount of RNA in each lane was monitored using ß-actin. The results shown are representative of three or more independent experiments. B, ARO cells cultured in six-well plates were cotransfected with 300 ng/well of the 8xGAS luciferase reporter plasmid, the TEL-JAK2 and RET/PTC1 expression plasmids (300 ng/well) and either the SOCS-1 or the SOCS3 expression plasmid at concentrations of 300 ng/well or 500 ng/well. C, ARO cells cultured in six-well plates were cotransfected with 300 ng/well of the 8xGAS luciferase reporter plasmid, the RET/PTC1 expression plasmid (300 ng/well), and increasing concentrations (100, 300, or 800 ng/well) of the PIAS1 expression plasmid. D, ARO cells cultured in six-well plates were cotransfected with 300 ng/well of the 8xGAS luciferase reporter plasmid, the RET/PTC1 expression plasmid (300 ng/well), and increasing concentrations (100, 300, or 800 ng/well) of the PIAS3 expression plasmid. After 12 h, the cells were lysed and luciferase activity was measured. The results represent the means ± SD from three or more experiments.

 
To observe the effects of SOCS-1 and SOCS-3 on RET/PTC-mediated STAT1 activation, ARO cells were cotransfected with RET/PTC, 8xGAS and either SOCS-1 or SOCS-3, and reporter activities were examined (Fig. 4BGo). Increasing amounts of either SOCS-1 or SOCS-3 suppressed the activation of STAT1 by TEL-JAK2. However, increasing amounts of either SOCS-1 or SOCS-3 did not alter the activation of STAT1 by RET/PTC1 (Fig. 4BGo) or RET/PTC3 (data not shown). This supports the notion that JAK is not involved in RET/PTC-induced STAT1 activation.

Members of the recently identified PIAS protein family interact with several different nuclear proteins. PIAS1 and PIAS3 bind to STAT1 and STAT3, respectively, and inhibit their action (28, 29). We found by Northern blot analysis that, unlike SOCS-1 and SOCS-3, PIAS1 and PIAS3 are not induced in ARO cells by cotransfection with RET/PTC1 or RET/PTC3 (data not shown). However, cotransfection of ARO cells with RET/PTC, 8xGAS, and either PIAS1 or PIAS3 revealed that PIAS1 but not PIAS3 inhibited the activation of STAT1 by RET/PTC1 (Fig. 4Go, C and D) and RET/PTC3 (data not shown). Because the expression of PIAS1 and PIAS3 did not alter the autophosphorylation of RET/PTC1 and RET/PTC3 (data not shown), it is unlikely that PIAS1 and PIAS3 act by inhibiting the catalytic activities of these RET/PTC tyrosine kinases. Thus, PIAS-1 can down-regulate the STAT1-mediated transcriptional activity induced by RET/PTC tyrosine kinases.

Regulation of IRF-1 and the CIITA Gene by RET/PTC
STAT1 and IRF-1 induce the transcriptional activation of the IFN-{gamma}-responsive type IV promoter of the CIITA gene by binding to cis-acting elements in its promoter, which contains GAS (–133 bp ~ –142 bp), E box (–126 bp ~ –131 bp), and IRF (–55 bp ~ –66 bp) motifs (35). The activation of the CIITA gene leads to the expression of MHC class II molecules. The two IFN-{gamma}-activated transcription factors STAT1 and IRF-1 bind the GAS and IRF elements, respectively (35, 36). To determine whether RET/PTC can mediate the up-regulation of the type IV CIITA promoter, WT, deletion mutants, and mutant reporters based on this promoter were constructed (Fig. 5Go, A and D) and transfected into NIH3T3 cells along with RET/PTC1. When present in NIH3T3 cells on its own, the full-length construct, hCIITA 1.7 WT, showed very low transcriptional activity. However, IFN-{gamma} treatment, which served as a positive control, markedly increased this activity (Fig. 5BGo). Cotransfection with RET/PTC1 also resulted in a dose-dependent increase in CIITA promoter activity (Fig. 5BGo). However, when RET/PTC1 was cotransfected with hCIITA-D3, hCIITA-D5, and hCIITA-D6, which lack the proximal GAS/IRF elements, the CIITA promoter activity was significantly lower compared with WT promoter activity (Fig. 5CGo). These results collectively demonstrate that RET/PTC1 activation of the CIITA gene is dependent on the GAS/IRF elements contained within a 154-bp fragment in the type IV CIITA promoter, which supports the notion that STAT1 and IRF-1 are involved in the regulation of the CIITA promoter by RET/PTC.



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  		Fig. 5. Regulation of the CIITA Promoter by RET/PTC

A, Schematic representation of the WT and deletion mutant constructs of the CIITA promoter. B, NIH3T3 cells were cultured in six-well plates and transiently transfected with 300 ng/well of the CIITA promoter (hCIITAp1.7 WT) and increasing concentrations of the RET/PTC1 expression plasmid as indicated. C, NIH3T3 cells cultured in six-well plates were cotransfected with 300 ng/well of the WT or deletion CIITA promoter constructs together with the control vector (pCDNA3.1) or the RET/PTC1 expression plasmid (300 ng/well). D, Schematic illustration of the mutant sequences in the mutant CIITA promoter constructs. NIH3T3 cells cultured in six-well plates were cotransfected with 300 ng/well of either the WT or a mutant CIITA promoter construct together with the control vector (pCDNA3.1) or the RET/PTC1 expression plasmid (300 ng/well). The cells were harvested after 12–24 h and assayed for luciferase activity. The results represent the means ± SD from three or more experiments.

 
To define further the individual contributions of the proximal GAS, the E box, and the proximal IRF elements in RET/PTC-induced CIITA promoter activity, NIH3T3 cells were cotransfected with RET/PTC1 and one of a series of type IV CIITA promoter constructs that bear mutations in each (GAS, E box, or IRF) or several (GAS + E box or GAS + IRF) of these elements (Fig. 5DGo). Mutation of only one of the elements (i.e. GAS, E box, or IRF) was sufficient to decrease RET/PTC1-induced CIITA promoter activity, whereas combined mutations (i.e. GAS+E box or GAS+IRF) resulted in further decreases. (Fig. 5DGo). These findings suggest that the GAS, E-box, and IRF motifs are critical for the induction of CIITA promoter activity by RET/PTC1.

We then investigated whether STAT1 or IRF-1 are involved in the induction of the CIITA gene by RET/PTC1. First, the dependence of CIITA induction on STAT1 activation was assessed by using STAT1 (+/–) and STAT1 (–/–) mouse embryonic fibroblasts (MEFs) (36). When these MEFs were cotransfected with RET/PTC1 and the hCIITAp1.7 (WT) CIITA promoter construct, it was found that although RET/PTC1 increased CIITA promoter activity in STAT1 (+/–) MEFs, it could not do so in STAT1-deficient MEFs (Fig. 6AGo). The dependence of CIITA induction on IRF-1 was then assessed by using IRF1 (+/–) and IRF-1 (–/–) MEFs. The cotransfection of these cells with RET/PTC1 and hCIITAp1.7 (WT) revealed that RET/PTC1 increased CIITA promoter activity in IRF1 (+/–) MEFs but that RET/PTC1-induced CIITA promoter activity was significantly reduced in IRF1 (–/–) MEFs (Fig. 6BGo). IFN-{gamma} treatment induced CIITA promoter activity in STAT1 (+/–) and IRF1 (+/–) MEFs but to a lesser extent in STAT1(–/–) and IRF1(–/–) MEFs. Moreover, RET/PTC3 was also unable to increase the promoter activity of hCIITA p1.7 (WT) in STAT1 (–/–) MEFs (data not shown). These data confirm that STAT1 and IRF-1 are involved in the transcriptional activation of the CIITA promoter induced by RET/PTC.



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  		Fig. 6. Requirement for STAT1 and IRF1 in RET/PTC-Mediated CIITA Induction

MEFs were prepared as described in Materials and Methods. Cells cultured in six-well plates were cotransfected with 300 ng/well of the CIITA promoter (hCIITAp1.7 WT) and various RET/PTC1 doses as indicated. The positive control was treated with mouse IFN-{gamma} for 12 h. The cells were harvested 24 h after cotransfection and assayed for their luciferase activity. The results represent the mean ± SD from three or more experiments.

 
De Novo Induction of MHC Class II Antigen by RET/PTC Activation
Because the above findings suggest that RET/PTC induces the transcriptional activation of the CIITA gene, we examined MHC class II antigen expression in NIH3T3 cell lines that stably express active RET/PTC1 tyrosine kinase. All four stable NIH3T3 cell clones expressed RET/PTC1, exhibited autophosphorylation of RET/PTC1, and showed significantly higher IRF-1 levels than control cells (Fig. 7AGo). The transient transfection of ARO cells with RET/PTC3 also induced CIITA and MHC class II (DR{alpha}) RNA expression (Fig. 7BGo). FACS analysis with MHC class II-specific antibodies was then performed to confirm that RET/PTC1 induced MHC class II protein expression. IFN-{gamma} treatment of NIH3T3 cells increased the level of MHC class II expression and similarly, the stable RET/PTC1-expressing cells showed high levels of MHC class II expression on their surface (Fig. 7CGo). In contrast, MHC class II expression of the stable cell line that expressed the kinase-deficient form of RET/PTC1 was no different to that of the untransfected control (Fig. 7CGo).



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  		Fig. 7. The Expression of MHC Class II (DR{alpha}) in RET/PTC1 Stable Cells

A, Stable NIH3T3 cells expressing RET/PTC1 were generated as described in the Experimental Procedures. Whole cell lysates were resolved by 10% SDS-PAGE and examined by immunoblot analysis with the indicated antibodies. The four stable NIH3T3 cell clones expressed RET/PTC1 (P, pcDNA3.1 vector, lane 1). B, Total RNAs of the control vector- (pcDNA3.1 vector, lane 1) and RET/PTC1-transfected ARO cells (lane 2) were isolated and subjected to Northern analysis (20 µg/lane) using class II and ß-actin cDNA probes. The CIITA IV mRNA levels were assessed by RT-PCR with specific primers as described in Materials and Methods. The results were standardized with respect to GAPDH values. C, The control vector (pcDNA3.1 vector), RET/PTC1- and RET/PTC1 K/M mutant-expressing stable cells were stained using anti-I-Ab antibody or the isotype control. The cells were analyzed using a Becton Dickinson (Franklin Lakes, NJ) FACScan.

 
To determine whether RET/PTC-positive papillary thyroid carcinoma cells also show in vivo induction of MHC class II antigens, tumor specimens were subjected to immunohistochemical staining with an MHC class II-specific antibody [human leukocyte antigen (HLA)-DR{alpha}]. The after tumor tissues from 12 different patients were tested: follicular adenoma (three patients), RET/PTC-negative papillary thyroid carcinoma (six patients), and RET/PTC-positive papillary thyroid carcinoma (three patients). RT-PCR analysis did not detect any RET/PTC1, RET/PTC2, or RET/PTC3 in the follicular adenoma and RET/PTC-negative papillary thyroid carcinoma samples. The RET/PTC-positive papillary thyroid carcinoma cells showed strongly diffuse immunoreactivity with the HLA-DR{alpha}-chain monoclonal antibody, whereas the normal thyroid follicular cells located adjacent to the tumor showed no MHC class II immunoreactivity (Fig. 8Go). The tumor cells of three of the six RET/PTC-negative papillary thyroid carcinomas and all three benign follicular adenomas also did not exhibit significant MHC class II immunoreactivity (Fig. 8Go). The tumor cells of the remaining three RET/PTC-negative papillary thyroid carcinomas showed scanty levels of MHC class II expression. These observations suggest that the presence of RET/PTC rearrangements in papillary thyroid carcinomas is related to the aberrant expression of MHC class II antigen by these tumors.



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  		Fig. 8. The Expression of MHC Class II (DR{alpha}) in RET/PTC-Positive Papillary Thyroid Carcinoma Cells

Immunohistochemical staining was performed using a mouse antihuman HLA-DR{alpha}-chain monoclonal antibody on papillary thyroid carcinoma tissue as described in Materials and Methods. A, Adenoma; N, normal thyroid follicle cells; T, papillary thyroid carcinoma. Tumor tissues for immunohistochemical staining were obtained from 12 different patients: follicular adenoma (three patients), RET/PTC-negative papillary thyroid carcinoma (six patients), RET/PTC-positive papillary thyroid carcinoma (three patients). The photomicrographs show representative histologic findings.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study, the expression of RET/PTC was found to lead to the phosphorylation of the Y701 tyrosine residue of STAT1. The tyrosine kinase activity of RET/PTC was required for this. We then investigated the mechanism by which RET/PTC induces STAT1 phosphorylation. Because JAK1 and JAK2 have been reported to play crucial roles in IFN-{gamma}-induced Y701 phosphorylation and activation of STAT1 (19), we assessed the effect of the JAK2 inhibitor AG490 on RET/PTCinduced STAT1 activity. This revealed that JAK2 is not involved in RET/PTC-induced STAT1 phosphorylation. Overexpression of WT JAK2 or the dominant-negative JAK2 K882E mutant also did not hamper the activation of STAT1 by RET/PTC (data not shown). Other studies have indicated that the Y701 residue of STAT1 can also be phosphorylated by members of other protein tyrosine kinase families, such as the Src family (20). For example, the nonreceptor tyrosine kinases c-Src and v-Src are involved in the growth factor-dependent and growth factor-independent activation of STAT1, STAT3, and STAT5 (37, 38). However, we found by using the Src inhibitor PP1 that Srcs are not involved in the RET/PTC-induced phosphorylation of STAT1. In addition, although RET/PTC is known to activate Ras/ERK (25), the MEKK inhibitor PD98059 did not significantly reduce the RET/PTC-mediated increase in STAT1 transcriptional activity. In a previous study, we showed that RET/PTC interacts physically with STAT3 (12). Thus, it is possible that RET/PTC directly phosphorylates and activates STAT1 and that this activation process does not require JAKs or c-Src kinases. However, immunoprecipitation experiments have failed to show that STAT1 and RET/PTC interact. It may be that the transient nature of this interaction or the very weak binding between RET/PTC and STAT1 (or both) makes it difficult to demonstrate this interaction by immunoprecipitation analyses.

How the activation of STAT1 by RET/PTC could lead to oncogenesis is not clear. In fact, we speculate that it may not be oncogenic at all. In our previous report, we showed that RET/PTC is also able to activate STAT3 in cellular transformation and to induce several genes, including those encoding vascular endothelial growth factor, cyclin D1, and intercellular adhesion molecule-1 (12). Because STAT3 participates in the cellular transformation and induction of genes that are involved in tumor progression, it is plausible that Src-mediated or RET/PTC-mediated STAT3 activation can lead to oncogenesis. In contrast, activated STAT1 proteins specifically recognize conserved STAT-responsive elements in the promoter of the cyclin-dependent kinase inhibitor p21WAF1CIP1 gene and thereby regulate the induction of p21 mRNA (39). In addition, STAT1 is also required for the efficient constitutive expression of caspases Ice, Cpp32, and Ich-1 in human fibroblasts and is involved in regulating apoptosis (40). Thus, activation of the STAT1 protein may be essential for the suppression of growth and for apoptosis in response to cytokines. Consequently, it is unlikely that the constitutive activation of STAT1 is oncogenic. However, it may be that the activation of STAT3 by RET/PTC, which promotes cell growth, survival, and transformation, overpowers the growth suppressing effect of STAT1 activation by RET/PTC during the process of transformation. The greater affinity of RET/PTC for STAT3 than for STAT1 might allow a more profound activation of STAT3, thereby allowing STAT3-mediated cellular effects to predominate.

Recent studies showed that expression of RET/PTC1 and RET/PTC3 induce apoptosis of rat thyroid PC CL 3 cells (41, 42). The promotion of thyroid cell death was found to depend both on the kinase activity of RET/PTC and on the phosphorylation of the Y1062 residue that maps to the carboxy-terminus of the RET protein. Our observation that RET/PTC may activate the proapoptotic protein STAT1 suggests that this may be how RET/PTC can lead to thyroid cell apoptosis. This question remains to be evaluated.

We found that the RET/PTC-mediated Y701 phosphorylation of STAT1 induced significant levels of STAT1 transcriptional activity as measured by using the 8xGAS reporter construct. The gain-of-function c-RET mutants, MEN2A and MEN2B, were also able to increase the STAT1-responsive activities in the reporter constructs but less potently than RET/PTC (Fig. 3BGo). These observations suggest that RET/PTC, which is located in the cytoplasm, is constitutively better in activating STAT1 than the receptor tyrosine kinases MEN2A and MEN2B.

The genes of SOCS family members were initially identified as targets for induction by JAK-STAT signaling (27, 30, 31). Subsequently, SOCS proteins were shown to be negative regulators of JAK- and STAT-mediated signal transduction. SOCS-1 reportedly impairs tyrosine phosphorylation of JAK2 and inhibits both STAT3 and STAT5 activation (27, 30, 31). We found that expression of RET/PTC induces SOCS-1 and SOCS-3, probably through the activation of endogenous STAT1 or STAT3, or both. However, cotransfection with SOCS-1 or SOCS-3 did not inhibit RET/PTC-induced increase of STAT1-responsive promoter activity. These findings suggest that SOCS-1 and SOCS-3 may not be able to inhibit the tyrosine kinase activity of RET/PTC. Other negative regulatory proteins are members of a newly identified family of negative regulators, the PIAS protein family (28, 29), which interact with transcriptional mediators of cytokine action (i.e. STATs) to suppress their DNA-binding activity. PIAS1 but not PIAS3 was shown to inhibit RET/PTC-mediated STAT1 transcriptional activity. Thus, PIAS1 can down-regulate the effect of RET/PTC on STAT1 activation.

The Y701 phosphorylation of STAT1 by JAK1 and JAK2 in response to type II IFNs is critical for the activation of STAT1-dependent genes, which are essential for immune responses (19, 22, 23). Some of the STAT1 downstream effects, such as antiviral responses, are mediated by IRF-1 (22, 23). We found that expression of WT RET/PTC induces IRF-1 and that kinase-deficient RET/PTC was unable to induce IRF-1 expression. Thus, IRF-1 is involved in the transcriptional activation mediated by RET/PTC-activated STAT1 molecules. It is known that the up-regulation of the CIITA promoter requires the cooperative actions of STAT1, IRF-1, and upstream stimulating factor-1 in response to IFN-{gamma} (35). We found that, like the response to IFN-{gamma}, RET/PTC-mediated STAT1 activation and IRF-1 induction increase CIITA promoter activity in a cooperative manner.

We have shown here that RET/PTC-expressing papillary thyroid carcinoma cells strongly express MHC II molecules, unlike papillary thyroid carcinoma cells that do not express RET/PTC. This may explain why the tissues surrounding RET/PTC-positive cancers are infiltrated with lymphocytes (43) and why transplantation of RET/PTC3-expressing thyroid tumors into naive mice results in leukocytic infiltrates and the generation of RET/PTC3-specific T cells (44). Thus, under specific circumstances, RET/PTC-positive cells may initiate immune reactions. This may also be relevant in the development of Hashimoto’s thyroiditis, as the RET/PTC rearrangements are also found in nonneoplastic Hashimoto’s thyroiditis and thyroid epithelial cells have been observed to aberrantly express MHC class II molecules in autoimmune thyroid diseases (17, 18). It is possible that RET/PTC may in general induce strong and aberrant MHC II expression and that this influences the maintenance of peripheral tolerance or the activation of immune cells. This may then lead to the massive infiltration of inflammatory cells into the thyroid, a characteristic of Hashimoto’s thyroiditis. The inappropriate expression of MHC class II antigen in nonlymphoid cells has long been considered to be important for the initiation or amplification of organ-specific autoimmune diseases, including Hashimoto’s thyroiditis and type 1 diabetes (17, 18). It will be of interest to examine whether RET/PTC-expressing cells show changes in costimulatory molecules such as B7. Thus, in conclusion, the RET/PTC-induced activation of STAT1 and the resulting expression of CIITA may explain why RET/PTC-induced papillary thyroid carcinoma and Hashimoto’s thyroiditis share several features in common, and why the two diseases sometimes coexist.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Cultures and Reagents
The papillary cancer cell line TPC1, which bears the RET/PTC1 rearrangement, was provided by Dr. M. Takahashi (Nagoya University, Nagoya, Japan) and grown in RPMI 1640 medium [RPMI 1640 medium base (Sigma-Aldrich Corp., St. Louis, MO) supplemented with 2 g/liter sodium bicarbonate, 0.14 mM nonessential amino acids, 1.4 mM sodium pyruvate, and 10% fetal bovine serum (pH 7.2)], unless otherwise specified. The ARO and NIH3T3 cells were cultured as previously described (11, 12). MEFs were prepared from IRF-1(–/–) (45) and STAT1(–/–) (36) fetal mice (129S6/SvEv-STATtm1, Taconic, Germantown, NY) on d 13.5 of gestation (E13.5) (vaginal plug detection was designated as E0.5). The media and cell culture reagents and materials were purchased from Invitrogen Life Technologies (Gaithersburg, MD), Sigma (St. Louis, MO), Fisher Scientific (Fairlawn, NJ), Corning, Inc. (Corning, NY), and Hyclone Laboratories, Inc. (Logan, UT). Mouse recombinant IFN-{gamma} was from R&D Systems, Inc. (Minneapolis MN). Antibodies specific for RET and IRF-1 were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and antibodies specific for STAT1 (total), phospho-STAT3 (Y705) and phospho-RET(Y905) were purchased from Cell Signaling Technology (Beverly, MA). The anti-upstream stimulating factor 1 antibody was obtained from Upstate Biotechnology Inc. (Charlottesville, VA). Fluorescein isothiocyanate-conjugated mouse antimouse I-Ab monoclonal antibody (AF6–120.1, Pharmingen, San Diego, CA), the isotype control IgG1 (BD Biosciences, Heidelberg, Germany), and mouse antihuman HLA-DR{alpha} chain monoclonal antibody (TAL.1B5, DAKO, Carpinteria, CA) were used.

Plasmid Constructs
pcDNA3-RET/PTC1 (iso9), pcDNA3-RET/PTC3 (iso9) and the mutant constructs pcDNA3-RET/PTC1-K147M and pcDNA3-RET/PTC3-K284M have been reported previously (11, 12). The 8xGAS reporter construct was obtained from Dr. A. E. Horvai (46), whereas the WT and mutant STAT1 constructs were from Dr. J. Bromberg (Rockefeller University, New York, NY). The expression vectors SOCS1 and SOCS3 were provided by Dr. R. Starr (27), whereas PIAS1 and PIAS3 were kindly provided by Dr. K. Shuai (28, 29). The type IV CIITA promoter constructs were from Dr. E.N. Benveniste (35). The c-RET, MEN2A and MEN2B expression plasmids were kindly provided by Dr. M. Takahashi (1, 4).

Immunoblot Analysis
Total cell lysates were obtained from cells that had been treated with IFN-{gamma} or transfected with RET/PTC. Cells were centrifuged, washed with PBS, and lysed at 0 C for 30 min in lysis buffer [20 mM HEPES (pH 7.4), 2 mM EGTA, 50 mM ß-glycerol phosphate, 1% Triton X-100, 10% ß-glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM Na3VO4, and 5 mM NaF]. The protein content was determined using the Bio-Rad dye binding microassay, and 20 µg of protein per lane was electrophoresed on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel after boiling for 5 min in SDS sample buffer. Proteins were blotted onto Hybond ECL membranes (Amersham Biosciences, Piscataway, NJ). After electroblotting, the membranes were blocked with Tris-buffered saline and Tween 20 [10 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% Tween 20] containing 5% milk and incubated with the primary antibody diluted in blocking buffer for 1 h. The primary antibody dilutions were those recommended by the manufacturer. Membranes were then washed, incubated with the appropriate second antibody (1:3000) in blocking buffer for 1 h, and rewashed. Blotted proteins were detected using the enhanced chemiluminescence detection system (New England Biolab, Boston, MA).

RNA Isolation, Northern Analysis, and RT-PCR
Total cellular RNA was isolated using standard procedures and Northern analysis was performed as previously described (47). The hybridization probes for class II (DR{alpha}), SOCS-1, and SOCS-3 were the purified inserts of pCR2.1-MHC class II (DR{alpha}), pEF-SOCS-1, and pEF-SOCS-3, respectively. All probes were radiolabeled using a random priming protocol (Amersham Pharmacia Biotech, Arlington Heights, IL). For RT-PCR, first-strand cDNA was synthesized using reverse transcriptase (Invitrogen Life Technologies, Grand Island, NY). PCR was performed using AmpliTaq DNA polymerase (PerkinElmer, Norwalk, CT) in a PerkinElmer 9700 thermocycler. The PCR conditions were as follows: predenaturation at 94 C for 20 sec, followed by 30 cycles of denaturation at 94 C for 15 sec, annealing at 59 C for 45 sec, and elongation at 72 C for 1 min. The primers used were mouse CIITA, 5'-GAGACTGCATGCAGGCAGCA-3' (sense) and 5'-TCCTTAACAGTGATGCCGACC-3' (antisense).

Transient and Stable Transfection
The cells were transfected using the LipofectAMINE method (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. After the cells were allowed to recover for 12–24 h, they were washed with PBS and lysed with 100 µl of the lysis buffer containing 40 mM Tricine (pH 7.8), 50 mM NaCl, 2 mM EDTA, 1 mM MgSO4, 5 mM dithiothreitol, and 1% Triton X-100. For the luciferase assay, the transfected cells were harvested and the extracts were assayed in triplicate for their luciferase activity. Light intensity was measured using a luminometer (Berthold, Bad Wildbad, Germany). The luciferase activity was integrated over a 10-sec period. The firefly luciferase values were standardized to the Renilla values.

Stable transfectants were selected in a medium containing G418 (800 µg/ml) and the clones were grown routinely in DMEM containing 10% FBS and G418 (800 µg/ml).

Flow Cytometry
Aliquots of NIH3T3 cells (1 x 106 cells) that showed stable expression of RET/PTC1 were washed three times in an isotonic cold PBS buffer (supplemented with 0.5% BSA) after trypsin/EDTA treatment and incubated for 30 min at 4 C with fluorescein isothiocyanate-conjugated mouse antimouse I-Ab monoclonal antibody (AF6–120.1, Pharmingen) (San Diego CA). The isotype mouse IgG1 (BD Biosciences, Heidelberg, Germany) was used as a control. After this incubation, any unreacted anti-I-Ab antibody was removed by washing and the cells were resuspended in 200 µl of PBS buffer for final flow cytometric analysis. The cells were analyzed using a Becton Dickinson FACScan.

Immunohistochemistry
Papillary thyroid carcinoma tissue was obtained from patients who provided written informed consent. RT-PCR was carried out to detect RET/PTC1 and RET/PTC3 expression in the papillary thyroid carcinoma tissue as shown previously (48). The following tumor tissues for immunohistochemical staining were obtained from 12 different patients: follicular adenoma (three patients), RET/PTC-negative papillary thyroid carcinoma (six patients), and RET/PTC-positive papillary thyroid carcinoma (three patients). The tumor tissues were fixed routinely (in 10% buffered formalin) and embedded in paraffin. Immunohistochemistry was performed according to previously reported protocols (48) using a 1/200 dilution of the monoclonal mouse antihuman HLA-DR{alpha}-chain antibody and a DAKO Envision kit. A negative control was used wherein the primary antibody was not used.


   ACKNOWLEDGMENTS
 
The authors extend their thanks to Dr. A. E. Horvai (The California University, La Jolla, CA) and Dr. E. N. Benveniste (The Alabama University, Birmingham, AL) for their gifts of the promoter constructs 8xGAS and type IV CIITA, respectively, and to Dr. J. Bromberg (The Rockefeller University, New York, NY) and Dr. S. M. Jhiang (Ohio University, Columbus, Ohio) for providing the expression vectors for STAT1 and RET/PTC, respectively. We also thank Dr. R. Starr (The Walter and Eliza Hall Institute for Medical Research, Victoria, Australia) and Dr. K. Shuai (UCLA, Los Angeles, CA) providing SOCS and PIAS expression vectors, respectively, and Dr. M. Takahashi (Nagoya University, Nagoya, Japan) for providing the TPC1 cells and c-RET, MEN2A and MEN2B expression plasmids. In addition, we thank Ms. Youngmi Kang for her secretarial assistance during the preparation of this manuscript.


   FOOTNOTES
 
This work was supported by National Research Laboratory Program (M1-0104-00-0014), Ministry of Science and Technology, Korea.

E.S.H. and D.W.K. contributed equally to this work.

Abbreviations: CIITA, Class II transactivator; GAS, IFN-{gamma} activated site; IFN, interferon; IRF, IFN regulatory factor; JAK, Janus kinase; MEFs, mouse embryonic fibroblasts; MHC, major histocompatibility complex; PTC, papillary thyroid carcinoma; PIAS, protein-inhibitor of activated STAT; RET, rearranged in transformation; SDS, sodium dodecyl sulfate; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; WT, wild-type.

Received for publication April 21, 2004. Accepted for publication July 27, 2004.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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