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Forskolin, 8-Br-3',5'-Cyclic Adenosine 5'-Monophosphate, and Catalytic Protein Kinase A Expression in the Nucleus Increase Radioiodide Uptake and Sodium/Iodide Symporter Protein Levels in RET/PTC1-Expressing Cells

The Ohio State Biochemistry Program (A.V., S.M.J.), Department of Physiology and Cell Biology (A.V., D.K.M., S.M.J.), Medical Scientist Program (D.K.M.), The Ohio State University, Columbus, Ohio 43210; and Department of Biological Sciences (S.H.G.), University of Iowa, Iowa City, Iowa 52242-1324

Address all correspondence and requests for reprints to: S. M. Jhiang, Department of Physiology and Cell Biology, The Ohio State University, 304 Hamilton Hall, 1645 Neil Avenue, Columbus, Ohio 43210. E-mail: jhiang.1{at}osu.edu.

	Abstract

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RET/PTC1, a thyroid-specific oncogene, has been reported to down-regulate sodium/iodide symporter (NIS) expression and function in vitro and in vivo. Recently, RET/PTC1 has been shown to interfere with TSH signaling at multiple levels in thyroid cells. The objective of this study was to investigate whether RET/PTC1-mediated NIS reduction can be rescued by activating cAMP-protein kinase A (PKA) pathways. We showed that both forskolin and 8-Br-cAMP increase radioiodide uptake and NIS protein in RET/PTC1-expressing cells to the same extent as the parental PC Cl 3 cells. We found that RET/PTC1 decreases nuclear localization of catalytic PKA, and forskolin treatment was able to counteract this RET/PTC1 effect. Furthermore, transient expression of catalytic PKA in the nucleus increased radioiodide uptake and NIS protein in RET/PTC1-expressing cells. Taken together, these studies suggest that RET/PTC1 down-regulates NIS expression by interrupting TSH/cAMP signaling, and this RET/PTC1 effect can be reversed by activating cAMP-PKA pathways.

	Introduction

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RET/PTC1 IS A chimeric oncogene that is expressed in thyroid follicular cells and leads to the development of papillary thyroid carcinomas. RET-PTC1 protein 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, phospholipase C (PLC), and AKT (see review, Ref.1). In addition, expression of RET/PTC1 has been demonstrated to reduce cAMP-mediated signaling events (2).

In thyroid follicular cells, TSH-mediated cAMP signaling is the major regulator of proliferation and differentiation. Recently, Wang et al. (2) reported that inducible RET/PTC3 expression reduced TSHR mRNA levels and decreased cAMP levels by blocking adenylyl cyclase activity directly. 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 (3) and also reduces radioiodide accumulation in the thyroid tissue of transgenic mice with thyroid targeted RET/PTC1 expression (4). Furthermore, RET/PTC1 has been reported to down-regulate rat NIS promoter activity in NIH/3T3 cells stably expressing RET/PTC1 (5). Several lines of evidence suggest that Ras is the major downstream signaling molecule of RET/PTC that mediates thyroid cell dedifferentiation. Knauf et al. (6) have reported that activation of the SHC-Ras-MAPK pathway is required for RET/PTC3 to reduce NIS mRNA levels. It has also been demonstrated that expression of constitutively active Ras is sufficient to reduce the accumulation of radioiodide in thyroid cells (7). Interestingly, Gallo et al. (8) have reported that v-Ras induces thyroid dedifferentiation by inhibiting nuclear accumulation of the protein kinase A (PKA) catalytic subunit (cPKA). Taken together, these studies suggest that RET/PTC, most likely via 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 (RAIU) and NIS expression in RET/PTC1-expressing cells. Similarly, 8-Br-cAMP, a cAMP agonist, was able to increase RAIU activity in RET/PTC1-expressing cells. Furthermore, RET/PTC1 inhibits nuclear localization of cPKA, and forced expression of cPKA in the nucleus increases NIS expression and function in RET/PTC1-expressing cells. These results suggest that RET/PTC mediates NIS reduction by interfering with the TSH-cAMP-PKA signaling pathway, and these effects are reversed by enhancing cAMP-PKA signaling.

	Materials and Methods

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Cell culture

PC Cl 3-immortalized rat thyroid cells were maintained in Coon’s modified F-12 medium (Irvine Scientific, Santa Ana, CA) with 5% calf serum, 2 mM glutamine, 1% Penicillin-Streptomycin (Invitrogen, Carlsbad, CA), 10 mM NaHCO₃, 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% fetal bovine serum (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 (St. Louis, MO). Green fluorescence protein (GFP)-cPKA, GFP-cPKAnes, and GFP-cPKAnls plasmids were constructed as described elsewhere (9). RET-PTC1 was cloned into the pLNCX vector (Clontech, Palo Alto, CA). Phoenix retroviral packaging cells were transiently transfected with pLNC-RET/PTC1 by the calcium phosphate precipitation method. Twenty-four hours after transfection, the medium was changed, and the cells were incubated at 32 C for optimal virus production. After 48 h, the media was harvested, centrifuged at 1000 rpm to pellet producer cells, and then filtered through a 0.22-µm filter. The filtered viral supernatant was aliquoted and stored at –80 C.

PC Cl 3 parental cells (3 x 10⁵) were seeded in 60-mm plates and were transduced 24 h later with 1 ml RET-PTC1 viral supernatant. Forty-eight hours after infection, the medium was changed to medium containing 400 µg/ml G418 (Invitrogen) for selection for 5 d. Stable clones were isolated and expanded in medium containing 200 µg/ml G418 and were screened for RET-PTC1 expression by Western blot analysis. PC Cl 3 stable clones transduced with empty retrovirus were not generated, because we have found that retroviral infection does not alter NIS or TSH receptor (TSHR) expression in PC Cl 3 cells (data not shown).

Western blot analysis

Western blot for rat NIS was performed as described below. Membrane fractions were prepared from each cell line as described elsewhere (10), 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 h and then probed with PA716 antirat NIS antibody at 1:1500 dilution (a kind gift from Dr. Bernard Rousset, Institut National de la Santé et de la Recherche Médicale, Lyon, France). The secondary antibody was donkey antirabbit IgG-horseradish peroxidase (HRP) (1:5000) followed by enhanced chemiluminescence (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 by antirabbit 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 ß-mercaptoethanol; and 2% sodium dodecyl sulfate) at 50 C for 1 h and probed with insulin receptor ß-antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a dilution of 1:500 followed by antirabbit IgG-HRP. Detection of signal was by ECL.

Fifty micrograms of total cell lysates were subjected to Western blotting and probed with rabbit antithyroglobulin antibody (a kind gift from Dr. Paul Kim, University of Cincinnati, Cincinnati, OH) at a dilution of 1:1000 followed by antirabbit 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 PLC antibody (Santa Cruz) at 1:500 dilution followed by antimouse IgG-HRP secondary antibody (Cell Signaling Technology, Beverly, MA) and ECL detection.

RAIU assay

RAIU assay was performed essentially as described elsewhere (11), and 7 x 10⁴ 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 GFP-cPKAnls using FuGene6 (Roche). Twenty-four hours after transfection, RAIU assay was performed. Briefly, 2 µCi ¹²⁵I-Na was diluted in NaI at a final concentration of 5 µM. The cells were incubated with ¹²⁵I-Na/NaI mixture at 37 C for 30 min. The cells were then washed with ice-cold Hanks’ balanced salt solution (Invitrogen) twice and lysed in 95% ethanol. The ethanol lysates were counted in a -counter (Cobra Quantum; Packard, Boston, MA). Addition of 3 µM sodium perchlorate reduced RAIU activity to less than 1000 cpm in both PC Cl 3 parental and RET/PTC1 cells (data not shown). The counts per minute were normalized by cell number (1 x 10⁵ cells). Statistical analysis was performed using the paired t test (GraphPad Software, San Diego, 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, Indianapolis, IN). Forskolin treatment (10 µM) was performed 24 h after transfection for 12 h. Forty-eight hours after transfection, the cells were washed with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. After fixation, the coverslips were mounted on glass slides using aqueous mounting medium (Biomeda, Foster City, CA). GFP fluorescence was detected using a Zeiss Axioskop (Thornwood, NY) equipped with a x40 objective lens. Only intact cells were scored for nuclear or cytoplasmic GFP fluorescence.

	Results

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RET/PTC1-mediated NIS reduction can be rescued by forskolin or 8-Br-cAMP

In agreement with others (3), we showed that stable expression of RET/PTC1 reduced RAIU (Fig. 1A) and NIS protein levels (Fig. 1B) 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 dose-dependent manner (Fig. 1A). It is of great interest to note that 10 µM forskolin treatment for 12 h was sufficient to increase RAIU in RET/PTC1-expressing cells to the same level as in 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 likely due to an increase in NIS expression (Fig. 1B). Forskolin treatment also increased cellular thyroglobulin (Tg) levels in RET/PTC1-expressing cells. For PC Cl 3 parental cells, forskolin slightly increased cellular Tg level but not NIS protein level. It is important to note that because PC Cl 3 cells presumably do not form follicles, most of the Tg may be found in the cultured medium.

View larger version (40K): [in this window] [in a new window]   FIG. 1. Forskolin increases RAIU and NIS protein level in RET/PTC1-expressing cells. A, Forskolin (FK) increases RAIU in RET/PTC1-expressing cells in a dose-dependent manner. PC Cl 3 parental cells were treated with DMSO control (–) or 10 µM forskolin for 12 h, whereas PC Cl 3 RET/PTC1 cells were treated with DMSO (–) or 1, 2, 5, 10 µM forskolin for 12 h followed by RAIU assay. 125I uptake is expressed as counts per minute (cpm) per 105 cells. RAIU of forskolin-treated RET/PTC1 cells, in comparison with DMSO-treated cells, was statistically significant (*, P < 0.05). B, Forskolin increases NIS protein level in RET/PTC1-expressing cells but not in PC Cl 3 parental cells. In comparison, forskolin increases cellular 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 h. The cytosolic fractions were used for Tg Western blot analysis, whereas membrane fractions were used for NIS Western blot analysis. Equal loading was determined by reprobing the blot with mouse anti-PLC antibody (for Tg) or with an antibody against an integral membrane protein V-ATPase E subunit (for NIS).

View larger version (39K): [in this window] [in a new window]   FIG. 2. 8-Br-cAMP increases RAIU in RET/PTC1-expressing cells in a temporal and dose-dependent manner. A, Cells were treated with water (–) or 1 mM 8-Br-cAMP for either 12 or 24 h. RAIU of 8-Br-cAMP-treated RET/PTC1 cells, in comparison with control cells, was statistically significant (*, P < 0.05). B, PC Cl 3 parental cells were treated with water (–) or 1 mM 8-Br-cAMP for 24 h, whereas PC Cl 3 RET/PTC1 cells were treated with water (–) or 100 µM, 500 µM, or 1 mM for 24 h followed by RAIU assay. 125I uptake is expressed as counts per minute per 105 cells. RAIU of 8-Br-cAMP-treated RET/PTC1 cells, in comparison with control cells, was statistically significant (**, P < 0.05).

Although 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 (12). To investigate the effect of RET/PTC1 on cPKA nuclear localization, PC Cl 3 parental and RET/PTC1-expressing cells were transiently transfected with GFP-tagged cPKA (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 localization of GFP-cPKA, whereas PC Cl 3 RET/PTC1 cells showed mainly cytosolic localization of GFP-cPKA. As expected, GFP-cPKAnes had cytosolic accumulation, whereas GFP-cPKAnls had nuclear localization in both parental and RET/PTC1 cells (Fig. 3A).

View larger version (25K): [in this window] [in a new window]   FIG. 3. Forskolin increases cPKA nuclear localization in RET/PTC1-expressing cells. A, Nuclear localization of transiently expressed GFP-tagged cPKA (GFP-cPKA) is reduced in RET/PTC1-expressing cells compared with parental cells. GFP-cPKAnes and GFP-cPKAnls were included as controls to show forced nuclear exclusion (nes) and nuclear inclusion (nls), respectively, in both parental and RET/PTC1-expressing cells. Magnification, x400. B, Nuclear localization of cPKA is increased by forskolin in RET/PTC1-expressing cells. Transiently transfected cells were treated with DMSO or 10 µM forskolin for 12 h before GFP detection. Magnification, x400. Results in A and B were consistent in three independent experiments. The mean ± SD for B is tabulated in C.

To investigate whether forskolin can increase cPKA nuclear localization 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 localization was found in 80% of transfected PC Cl 3 parental cells but in only 7% of transfected RET/PTC1-expressing cells (Fig. 3, B and C). For PC Cl 3 parental cells, forskolin further increased cPKA nuclear localization to 90% of transfected cells. In comparison, forskolin increased cPKA nuclear localization from 7% to 60% in transfected RET/PTC1-expressing cells (Fig. 3, B and C).

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 Fig. 4A, transient expression of GFP-cPKAnls did increase 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, whereas NIS protein levels in PC Cl 3 parental cells was not changed (Fig. 4B). In comparison, transient expression of GFP-cPKAnes did not significantly increase RAIU in either parental or RET/PTC1-expressing cells (data not shown).

View larger version (38K): [in this window] [in a new window]   FIG. 4. Transient expression of GFP-cPKAnls is sufficient to increase RAIU and NIS protein levels in RET/PTC1-expressing cells. A, PC Cl 3 parental and RET/PTC1-expressing cells were untransfected (–) or transiently transfected with GFP-cPKAnls followed by RAIU assay. 25I uptake is expressed as counts per minute per 105 cells. RAIU of transfected RET/PTC1 cells, in comparison with untransfected cells, was statistically significant (*, P < 0.05). B, Western blot for NIS in PC Cl 3 parental and RET/PTC1-expressing cells that were untransfected or transiently transfected with GFP-cPKAnls. To ensure equal loading, the blot was stripped and probed with an antibody against insulin receptor ß (IRß).

	Discussion

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Several reports have provided evidence that Ras mediates thyroid dedifferentiation by inhibiting nuclear accumulation of cPKA. Gallo et al. (8) reported that v-Ras inhibited nuclear localization of the cPKA via protein kinase C. Feliciello et al. (12) showed that juxtanuclear localization of RIIß is essential to maintain cAMP-dependent differentiation in rat thyroid cells, and that Ras activation induced cytosolic translocation of RIIß, resulting in decreased cPKA nuclear accumulation. Our study provides direct evidence that RET/PTC, similar to Ras, decreases cPKA nuclear accumulation (Fig. 3A), and that increased nuclear accumulation of cPKA by forskolin treatment (Figs. 1 and 3, B and C) or forced expression of nuclear localized cPKA (Fig. 4) increases NIS expression.

In thyroid cells, RET/PTC has been reported to confer TSH-independent proliferation, loss of differentiation markers, and apoptosis (14, 15). Wang et al. recently reported that acute expression of RET/PTC interferes with TSH-stimulated proliferation at various levels (2). 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 (Fig. 3B) suggests that signaling downstream of adenylyl cyclase was not affected by RET/PTC1 to reduce NIS expression. However, both Wang et al. (2) and our unpublished data show that TSHR mRNA levels are decreased in PC Cl 3 RET/PTC1-expressing cells.

Recently, it has been reported that NIS and TPO, but not Tg, expression are lost in mice with defects in TSH or TSHR (16). Thus, the authors concluded that TSH signaling is necessary for NIS and TPO expression but not for Tg expression (16, 17). In agreement with this finding, our data show that forskolin significantly increases NIS protein levels, but only moderately increases cellular Tg levels, in RET/PTC1-expressing cells (see Fig. 1B).

Signaling pathways involved in TSH-mediated proliferation and differentiation in thyroid cells have been extensively studied (see review, Refs.17 and 18). It appears that TSH-mediated proliferation is affected by many other factors, such as IGF-I and serum (see review, Ref.19). In comparison, this study and the study by Postiglione et al. (16) indicate that NIS expression in thyroid cells appears to be critically dependent on the TSH-cAMP-PKA signaling pathway.

Similar to most human tumors carrying RET/PTC1, the RET/PTC1-expressing PC Cl3 cells remain differentiated, expressing Tg and NIS, albeit at reduced levels. In addition, the finding that NIS expression and RAIU 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 with TSH signaling. For tumors refractory to TSH-stimulated RAIU, in which defect(s) may occur at TSHR, efforts should be made to directly stimulate cAMP-PKA signaling pathways.

	Acknowledgments

 
We thank Dr. Bernard Rousset for providing the PA716 rat NIS antibody and Dr. Paul Kim for providing the PC Cl 3 rat thyroid cell line and the rat Tg antibody.

	Footnotes

 
This work was supported by National Institutes of Health Grant R01 CA60074 (to S.M.J.).

Abbreviations: cPKA, Catalytic PKA; DMSO, dimethylsulfoxide; ECL, enhanced chemiluminescence; GFP, green fluorescence protein; HRP, horseradish peroxidase; NIS, sodium/iodide symporter; PKA, protein kinase A; PLC, phospholipase C ; RAIU, radioiodide uptake; Tg, thyroglobulin; TSHR, TSH receptor.

Received July 19, 2004.

Accepted September 17, 2004.

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