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*ACETYLCYSTEINE
*COPPER, ELEMENTAL
*HYDROGEN PEROXIDE
*THYROGLOBULIN
Endocrinology Vol. 141, No. 12 4373-4382
Copyright © 2000 by The Endocrine Society

ARTICLES

Regulation of Thyroid Follicular Cell Function by Intracellular Redox-Active Copper1

Atsushi Iseki, Fukushi Kambe, Kenji Okumura, Tetsuo Hayakawa and Hisao Seo

Department of Endocrinology and Metabolism (A.I., F.K., H.S.), Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan; and Department of Internal Medicine II (A.I., K.O., T.H.), Nagoya University School of Medicine, Nagoya 466-8550, Japan

Address all correspondence and requests for reprints to: Fukushi Kambe, Department of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Furo-cho, Chikusa-ku, Nagoya University, Nagoya 464-8601, Japan. E-mail: kambe{at}riem.nagoya-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pyrrolidine dithiocarbamate (PDTC) is a metal-chelating compound that exerts prooxidant or antioxidant effects and is widely used to study redox regulation of cell function. In the present study, we investigated effects of PDTC on the function of rat thyroid follicular FRTL-5 cells. Treatment of the cells with PDTC resulted in a marked decrease in Pax-8 messenger RNA level and its DNA-binding activity. This decrease was associated with a significant reduction in thyroperoxidase (TPO) messenger RNA level. Expression of TTF-1 and thyroglobulin was not affected by PDTC. Treatment with PDTC also decreased DNA-binding activity of p53, a tumor suppressor protein, and increased cell proliferation rates. These changes were not observed by the treatment with another antioxidant, N-acetyl-Lcysteine, suggesting that the metal-chelating, prooxidant property of PDTC is responsible for its effects. Indeed, the intracellular level of copper was significantly increased by PDTC. Treatment with bathocuproinedisulfonic acid, a noncell-permeable chelator of Cu1+, abrogated the copper increase by PDTC and its effects on Pax-8 and TPO expression as well as on p53 binding. Taken together, these results indicate that the intracellular level of redox-active copper is crucial for Pax-8 and TPO expression and for proliferation of thyroid follicular cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID follicular cells are specialized epithelial cells that concentrate iodide and incorporate it into the thyroglobulin (TG) molecule, which is subsequently hydrolyzed to release thyroid hormones. These processes require cell-type specific gene products, which include TG, thyroid peroxidase (TPO), the receptor for the thyroid-stimulating hormone (TSH), and sodium/iodide symporter. The expression of TG and TPO genes is cooperatively regulated by three distinct types of thyroid-enriched transcription factors, Pax-8, TTF-1, and TTF-2 (1).

Thyroid hormone synthesis is tightly regulated by TSH, the prime regulator for thyroid function: the transcription of the TG, TPO, TSH receptor and sodium/iodide symporter genes is under control of TSH (2, 3, 4, 5, 6). In addition, production of H2O2, which is necessary for iodide oxidation at the step of iodide incorporation into TG, is also regulated by TSH (7, 8). Furthermore, TSH regulates proliferation of thyroid follicular cells (9, 10).

In various types of cells, it has been demonstrated that the cellular reduction-oxidation (redox) status modulates various aspects of cellular function (11). Oxidative stress can induce cell proliferation as well as growth inhibition or cell death. It has been also shown that change in intracellular redox status regulates the function of transcription factors, leading to alteration in gene expression (12, 13). Recently, we and others have demonstrated that the DNA-binding activities of Pax-8, TTF-1, and TTF-2 are regulated posttranslationally by redox state of the molecule (14, 15, 16, 17). It has been also demonstrated in some cells that reactive oxygen intermediates (ROIs) such as H2O2 and O2- act as an intracellular signal-transducing molecule to regulate cellular function and proliferation (18, 19, 20).

Based on these findings, we postulated that H2O2 or other ROIs, generated in response to TSH, might play a role in regulating the function of thyroid follicular cells. In the present study, we therefore investigated the effects of antioxidants, which scavenge ROIs, on expression of Pax-8, TTF-1, TG, and TPO, and on proliferation of rat thyroid FRTL-5 cells. We used two kinds of antioxidants, pyrrolidine dithiocarbamate (PDTC) and N-acetyl-L-cysteine (NAC). NAC enters cells readily and scavenges ROIs directly or indirectly by its conversion to L-cysteine and by increasing intracellular glutathione (21). PDTC contains two thiol moieties and is thus generally recognized as an antioxidant (22). However, it has been recently shown that PDTC can chelate and transport redox-active metal ions into cells, and exert prooxidant effect on rat thymocytes (23). The present study will show that PDTC and NAC exert their effects on the function and proliferation of thyroid cells with a conspicuous difference that could be attributable to the prooxidant effect of PDTC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Pyrrolidine dithiocarbamate (PDTC) and bathocuproinedisulfonic acid (BCS) were purchased from Sigma (St. Louis, MO). N-acetyl-L-cysteine (NAC) was purchased from Roche Molecular Biochemicals (Mannheim, Germany). Other chemicals were obtained from Sigma, unless otherwise stated.

Cell culture
FRTL-5 cells (ATCC CRL8305), which were less than 10 passages after the purchase from ATCC were cultured in Coon’s modified Ham’s F-12 medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 5% heat-inactivated calf serum (Life Technologies, Inc.) and with six hormones (6H) including hydrocortisone (10 nM), transferrin (5 µg/ml), somatostatin (10 ng/ml), glycyl-L-histidyl-L-lysine acetate (10 ng/ml), bovine TSH (1 mU/ml) and insulin (10 µg/ml). The F-12 medium contains 2.5 ng/ml of CuSO4 5H2O and 830 ng/ml of FeSO4 7H2O. The cells were treated with 10 µM to 80 µM PDTC or 5 mM to 20 mM NAC for various lengths of time. In some experiments, the cells maintained in the TSH-deprived medium (5H) for 5 days were used.

Northern blot analysis
Total RNA was extracted from FRTL-5 cells by the method of Chomczynski and Sacchi (24). Northern blot and hybridization were carried out as described previously (25). In brief, total RNA (15 µg) was fractionated in 0.8% agarose gels, and then was transferred onto a nylon membrane (Gene Screen Plus; NEN Life Science Products, Boston, MA). Heat-denatured complementary DNA (cDNA) probes were labeled with [32P]dCTP using the random primed DNA labeling kit (Roche Molecular Biochemicals). Preparation of cDNAs for rat Pax-8, TTF-1, TPO, TG, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were described previously (26, 27). After hybridization and wash, the membrane was subjected to imaging by Fujix Bioimage Analyzer (BAS 2000; Fuji Photo Film Co., Ltd., Tokyo, Japan) to quantify radioactivities of bands. The membrane was then autoradiographed.

Preparation of antirat Pax-8 antiserum
The polypeptide named PACP (CSAPPTSATAFDHL) spanning amino acid residues 445–457, C-terminal region of rat Pax-8, was synthesized and purified to more than 95% purity by HPLC. Additional cysteine (in italic) was introduced at N-terminal of PACP to facilitate conjugation with a carrier protein. PACP was mixed with methylated bovine albumin (28): 180 µl of PACP (10 mg/ml in 0.15 M NaCl), 720 µl of methylated bovine albumin (2.5 mg/ml in 0.15 M NaCl) and 300 µl of 0.15 M NaCl were mixed and incubated at 25 C for 30 min. The mixture was stored at -30 C as 100 µl aliquots.

Three female rabbits (Nihon Hakushoku-shu) weighing around 2.5 kg were purchased from Nippon Bio-Supply Center (Tokyo, Japan). We used 150 µg PACP for each immunization. One hundred µl of PACP mixture, 400 µl of 0.15 M NaCl, and 500 µl of complete Freund adjuvant (Difco Laboratories, Detroit, MI) were completely mixed to emulsion using glass syringes. After preimmune blood was drawn from an ear vein, the emulsion (1 ml) was injected intradermally into 10 to 15 separate positions on the back. Freshly prepared emulsion was used for booster immunizations at 2, 4, and 8 weeks after the first injection. Blood samples were collected 4, 6, 8, and 9 weeks after the first immunization. Antibody titer was assessed by dot blot ELISA (enzyme-linked immunosorbent assay) as described (29). All the sera obtained after the last bleeding exhibited specific binding to PACP and were successfully used for immunoprecipitation and supershift analysis in electrophoretic mobility shift assay.

Electrophoretic mobility shift assay (EMSA)
Preparation of whole cell extracts and nuclear extracts, and procedures of EMSA were described previously (27). Protein concentration of the extracts was determined by a microassay kit (Bio-Rad Laboratories, Inc., Richmond, CA) using BSA as a standard. The nucleotide sequence of EMSA probes are as follows: oligo P which contains recognition sites for Pax-8/TTF-1 in the rat TPO promoter (1), 5'-TGTCTAAGCTTGAGTGGGCATCAGA-3'; oligo EN, which contains a recognition site for Pax-8 in the E2 region of the human TPO enhancer (14, 30), 5'-CTAGAACCAGGGATTCTTCACACTTCATAGAGCACCTCTAG-3'; oligo Z, which contains a recognition site for TTF-2 in the rat TPO promoter (1), 5'-AGAAATACTAAACAAACAGAATCG-3'; oligo K, which contains a recognition site for TTF-2 in the rat TG promoter (31), 5'-TGACTAGCAGAGAAAACAAAGTGAGC-3'; GADD45, which contains a p53-binding site in the promoter of growth-arrest DNA-damage inducible gene 45 (32), 5'-AGCAGAACATGTCTAAGCATGCTGGGCTCG-3'. The probes were prepared by annealing sense- and antisense-oligonucleotides and by extending the nucleotides with Klenow enzyme in the presence of [32P]dCTP. Preparation of oligo Ct, which was used for the competition experiment was described previously (14). Whole cell extract (20 µg) or nuclear extract (10 µg) was preincubated in a final volume of 20 µl containing 40 mM HEPES-KOH (pH 7.9), 75 mM KCl, 0.2 mM EDTA-NaOH (pH 8.0), 10% glycerol, 0.5 mM DTT, and 2 µg poly (dI-dC) (Amersham Pharmacia Biotech, Uppsala, Sweden). In some experiments, DTT was omitted. After 15-min incubation on ice, 0.03 pmol of a 32P-labeled probe and 3 pmol of competitor oligonucleotide for competition experiment were added, and incubated for 30 min at 25 C. In experiments using anti-Pax-8 antiserum or anti-p53 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), it was added after this incubation, and further incubated for 1 h at 4 C. The reaction mixture without dye was loaded directly onto a 4% polyacrylamide gel (30: 1, acrylamide: bisacrylamide) in 45 mM Tris-HCl (pH 8.0), 45 mM boric acid and 1 mM EDTA-NaOH (pH 8.0) and electrophoresed at 160 V at 4 C. The gel was dried on filter paper and then autoradiographed.

Metabolic labeling and immunoprecipitation
Metabolic labeling was carried out 12 h after treatment with PDTC (40 µM) or NAC (20 mM). Detailed procedure was reported previously (33). In brief, the cells were preincubated in methionine-free Eagle’s MEM (Nissui Pharmaceutical Co., Tokyo, Japan) for 2 h and then incubated in methionine-free MEM containing [35S]methionine (Amersham Pharmacia Biotech, Arlington Heights, IL) and incubated for an additional 4 h. Immunoprecipitation was performed using antirat Pax-8 antiserum and Staphylococcus aureus protein A (Pansorbin; Calbiochem, San Diego, CA). The precipitates were subjected to 12% SDS-PAGE. After fixation, gels were incubated in an enhancer (Amplify, Amersham Pharmacia Biotech) for fluorography, dried, and exposed to BAS 2000 system. The gel was then subjected to fluorography.

Transfection
The 395-bp rat TPO promoter sequence (the position 1 to 395 in EMBL/DDBJ accession number x61170) was cloned by PCR, and ligated to the upstream of luciferase gene in pGL2-basic plasmid (Promega Corp., Madison, WI). The plasmid was named pTPOpro. The construction of rat Pax-8-expressing plasmid (pPAX8) was described in our previous report (14). In brief, the full-length cDNA for rat Pax-8 was ligated into mammalian expression vector pRC/RSV5.2 (Invitrogen Corp., San Diego, CA). FRTL-5 cells were grown in 6H media in 12-well plates. Transient transfections were carried out using calcium-phosphate precipitation method with 1 µg of reporter plasmid pTPOpro or promoter-less plasmid pGL2-basic together with or without 1 µg of pPAX8. In all the experiments, 0.1 µg of pSVß-galactosidase plasmid (Promega Corp.) was cotransfected, and the total amount of plasmid DNA was kept constant by addition of pRC/RSV5.2 carrying no cDNA insert. After overnight exposure to the DNA-CaPO4 precipitate, the medium was replaced with the fresh medium containing 40 µM PDTC. After a 30-h incubation, the cells were lysed by addition of 100 µl glycylglycine buffer (25 mM glycylglycine pH 7.8, 15 mM MgSO4, 4 mM EGTA, 1 mM DTT) containing 0.2% Triton x100. Aliquots (20 µl) were mixed with 300 µl of glycylglycine buffer containing 2 mM ATP and 15 mM potassium phosphate. One hundred microliters of glycylglycine buffer containing 0.2 mM D-luciferin was injected to the mixture using automatic device in LUMAT LB9501 (Berthold, Germany) and luciferase activity was measured for 10 sec. The ß-galactosidase activity was measured using Luminescent ß-gal detection kit (CLONTECH Laboratories, Inc., Palo Alto, CA) and LUMAT LB9501 according to the instruction from the supplier. The luciferase activities were normalized by ßgalactosidase activities.

Assays for cell proliferation
Cell proliferation was assessed by WST-1 assay and 3H-thymidine incorporation method. WST-1 assay was performed using Cell Counting Kit (Dojindo, Kumamoto, Japan). FRTL-5 cells (3 x 103/well) were plated in 96-well microplates. After preincubation for 48 h, they were exposed to various concentrations of PDTC or NAC for 24 h. After the exposure, 10 µl of a solution of the Cell Counting Kit was added to the wells, followed by the incubation for 2 h. This solution contains water-soluble tetrazolium salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1). In viable cells, WST-1 is enzymatically converted into a water soluble and noncytotoxic formazan dye. Optical densities at 450 nm of the media were measured using a microplate reader (Bio-Rad Laboratories, Inc. Richmond, CA) (34, 35).

3H-thymidine incorporation was performed as follows. Cells were plated in 12-well culture plates at a density of 5 x 104 cells per well, and then treated with various concentration of PDTC or NAC for 12 h. During the last 2 h, 10 µCi/ml of 3H-thymidine (37MBq/ml; NEN Life Science Products) was added to the medium. The incorporation of 3H-thymidine was determined after precipitation of acid-insoluble material with ice-cold 10% trichloroacetic acid. The acid-insoluble material was dissolved in 2% SDS and counted in a liquid scintillation counter.

H2O2 determination
Concentration of H2O2 in cell culture media was determined by homovanillic acid fluorescence assay (7) (8) (36). FRTL-5 cells were maintained in 5H media in 12-well culture plates. The medium was then replaced with 0.85 ml/well HEPES-buffered medium (pH 7.4) containing 1 mU TSH alone or in combination with 300 U/ml catalase (purified from mouse liver, Sigma), 40 µM PDTC, and 20 mM NAC. After a 2-h incubation, 440 µM homovanillic acid and 0.5 U/ml horseradish peroxidase were added to the medium. The HEPES-buffered medium consisted of 10 mM HEPES, 134 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.0 mM CaCl2, 2.5 mM NaHCO3, 5 mM glucose, and 0.1% BSA. After 45-min incubation at 37 C, the dishes were chilled on ice to terminate the reaction. A portion of the medium was collected, and fluorescence of the medium was measured using a Hitachi 650–10S fluorescence spectrophotometer (Hitachi Co. Ltd., Tokyo, Japan). The excitation wavelength was set at 315 nm, and the emission wavelength at 425 nm. A standard curve was prepared by measuring the samples containing a different amount of H2O2. The activity of horseradish peroxidase per se was not affected by the presence of 40 µM PDTC or 20 mM NAC (data not shown).

Determination of intracellular concentration of copper and iron
FRTL-5 cells cultured in 6H medium were treated with PDTC (40 µM) alone or together with BCS (0.4 mM) for 12 h. After washing the cells with PBS several times, the whole cell extracts were prepared as described previously (14). Copper and iron contents were determined by using a Hitachi Z6100 atomic absorption spectrophotometer (Hitachi), and corrected by protein contents.

Statistical analysis
Statistical analysis was carried out by using one-way ANOVA followed by Fisher’s protected least significant difference analysis. The P value less than 0.05 is considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PDTC decreases TPO and Pax-8 messenger RNA (mRNA)
FRTL-5 cells cultured in the presence of six hormones (6H) were treated with various concentrations of PDTC or NAC for 12 h, and mRNA levels of TPO, TG, Pax-8, TTF-1, and GAPDH were determined by Northern blot analysis. As shown in Fig. 1Go, single bands of TPO mRNA (3.7 kb in size), TG mRNA (8.5 kb), Pax-8 mRNA (3.0 kb), and TTF-1 mRNA (2.5 kb) were detected in the control, nontreated cells. The sizes of these mRNAs were comparable to the previous reports (26, 37).



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  		Figure 1. Effect of PDTC and NAC on mRNA levels of TPO, TG, Pax-8, TTF-1, and GAPDH in FRTL-5 cells. The cells were treated with various concentrations of PDTC or NAC for 12 h. Total RNA (15 µg per lane) was subjected to Northern blot analysis. Representative autoradiographs are shown in the lower panels. Closed and open arrowheads indicate the positions of 28S and 18S ribosomal RNA, respectively. One experiment was performed using duplicate flasks. Similar results were obtained from a separate experiment. After the radioactivities of the bands were measured by BAS; 2000 system, the mRNA levels were expressed as percentage of the level of untreated cells. Values are expressed as mean ± SE (n = 4). *, P < 0.05.

 
Treatment with 40 µM PDTC for 12 h markedly decreased the levels of Pax-8 and TPO mRNAs to 32% and 25% of the control cells, respectively. PDTC 80 µM also decreased these mRNA levels. However, it did not alter the levels of TG and TTF-1 mRNAs. In contrast, GAPDH mRNA was significantly increased by PDTC.

Surprisingly, treatment of the cells with 20 mM NAC had only a marginal effect on TPO mRNA and no effect on Pax-8 mRNA. Contrary to the effect of PDTC, NAC decreased GAPDH mRNA.

As shown in Fig. 2Go, the decreased level of TPO mRNA was apparent at 12 h after treatment with 40 µM PDTC, whereas it increased GAPDH mRNA as early as at 3 h, followed by a gradual increase. TG mRNA levels were not altered at any time point studied. These results demonstrate that PDTC specifically reduces Pax-8 and TPO mRNA, and increases GAPDH mRNA level in FRTL-5 cells.



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  		Figure 2. Time-course of PDTC effect on mRNA levels of TPO, TG, and GAPDH. FRTL-5 cells were treated with PDTC (40 µM) as indicated. Total RNA (15 µg per lane) was subjected to Northern blot analysis. Representative autoradiographs are shown. One experiment was performed in duplicate flasks. Similar results were obtained from a separate experiment.

 
To confirm the effects of PDTC and NAC on Pax-8 expression, the cells treated with 40 µM PDTC or 20 mM NAC for 12 h were pulse labeled with 35S-methionine for 4 h, and newly synthesized Pax-8 was immunoprecipitated with antirat Pax-8 antiserum. As shown in Fig. 3Go, a protein with a molecular weight of approximately 50 kDa was detected in nontreated control cells, being compatible with the estimated molecular weight of murine Pax-8 (38). However, this protein was not precipitated with preimmune serum. Treatment with PDTC markedly decreased Pax-8 synthesis, which is compatible with the decrease in the mRNA level. Note that NAC had almost no effect on the synthesis.



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  		Figure 3. PDTC inhibits de novo protein synthesis of Pax-8. FRTL-5 cells were treated with PDTC (40 µM) or NAC (20 mM) for 12 h, and they were metabolically labeled with 35S-methionine for 4 h. The experiment was performed using duplicate flasks. The immunoprecipitates by anti-Pax-8 antiserum and preimmune serum (pre) were subjected to SDS-PAGE. The bands of Pax-8 are indicated by closed arrowhead. *, Nonspecific bands.

 
Effects of PDTC on DNA-binding activities of thyroid-enriched transcription factors
Previous study demonstrated that a minimum promoter of the rat TPO gene contains several binding sites for Pax-8, TTF-1, and TTF-2 (1). Effects of PDTC on DNA-binding activities of these factors to the promoter sequences were analyzed by EMSA.

We at first characterized the binding of the transcription factors to oligo P, which corresponds to the P region of the rat TPO promoter. As shown in Fig. 4AGo, three protein/DNA complexes with different mobility were detected by EMSA using this oligonucleotide as a probe (lane 1). These three complexes were displaced by excess amount of cold oligo P (lane 2). When oligo Ct, which has a complete TTF-1 binding site in rat TG promoter but lacks Pax-8 binding site, was used as a competitor, the slowest-migrating complex was completely displaced (lane 3), indicating that this complex represents TTF-1/oligo P complex. Unrelated competitor oligo K, which is recognized by TTF-2, did not displace any of the complexes (lane 4). When antirat Pax-8 antiserum was added to the EMSA reaction buffer, the complex in the middle disappeared, and a supershifted complex as indicated by an asterisk appeared (lane 5), indicating that the middle complex represents Pax-8/oligo P complex. The fastest-migrating complex was not characterized.



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  		Figure 4. Effect of PDTC on the DNA-binding activities of Pax-8, TTF-1 and TTF-2. A, Whole cell extracts prepared from FRTL-5 cells in 6H medium were subjected to EMSA using oligo P as a probe, which corresponds to the promoter region of the rat TPO gene. Lane 1: extract alone; lanes 2 to 4: the presence of excess amount of competitor, oligo P, oligo Ct and oligo K, respectively; lane 5: the presence of anti-Pax-8 antiserum. B, Time-course of PDTC effect on DNA-binding of TTF-1 and Pax-8 is shown. The cells were treated with PDTC (40 µM) as indicated. The extracts were subjected to EMSA using oligo P as a probe. C, Time-course of PDTC effect on DNA-binding of Pax-8 is shown. The same extracts in Panel B were subjected to EMSA using oligo EN as a probe, which correspond to the human TPO enhancer sequence. D and E, Effect of PDTC on TTF-2 binding is shown. The same extracts in Panel B were subjected to EMSA using oligo Z (D) and oligo K (E) as probes. Oligo Z and oligo K correspond to a TTF-2 binding site in rat TPO and TG promoters, respectively.

 
We then examined the time-course of PDTC effect on DNA-binding of Pax-8 and TTF-1 (Fig. 4BGo). At 6 h after PDTC (40 µM), a substantial decrease in the Pax-8 binding was detected, and the reduction continued until 12 h, whereas TTF-1 binding was only marginally affected. The reduction of Pax-8 binding by PDTC was reproduced using another probe, oligo EN (Fig. 4CGo), which had been shown to bind with Pax-8 predominantly (14). The Pax-8 binding was decreased at 6 and 12 h after PDTC. In contrast, 20 mM NAC had no effect on the Pax-8 binding (data not shown). It should be noted that reduction of Pax-8 binding precedes the decrease in TPO mRNA level. On the other hand, TTF-2 binding to oligo Z and K (1) was not affected by PDTC (Fig. 4Go, D and E). These results strongly suggest that attenuation of DNA-binding of Pax-8 to the promoter is involved in the decreased expression of TPO mRNA by PDTC.

We previously demonstrated that the reduction of Pax-8 is required for its full binding activity (14). To examine whether PDTC affects the redox status of Pax-8, the cell extracts were prepared without DTT from the cells treated with or without PDTC, and subjected to EMSA in the absence of DTT. Pax-8 in the extracts prepared with or without DTT bound to oligo EN to a similar extent (data not shown), indicating that it is fully reduced in the presence of TSH and that PDTC does not affect the redox status of Pax-8.

Overexpression of Pax-8 restores TPO promoter activities
To confirm the involvement of Pax-8 in the decreased expression of TPO gene by PDTC, we examined whether introduction of Pax-8-expressing plasmid could restore the TPO promoter activity by transient transfection study (Fig. 5Go). When TPO promoter-luciferase plasmid (pTPOpro) was transfected into FRTL-5 cells, the luciferase activity was markedly increased above that of promoterless plasmid (pGL2-basic). Treatment of the transfected cells with PDTC (40 µM) significantly decreased the luciferase activity. Cotransfection of Pax-8-expressing plasmid (pPAX8) resulted in the restoration of the TPO promoter activities, indicating that the decreased TPO gene expression by PDTC is caused by the reduced expression of Pax-8.



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  		Figure 5. Overexpression of Pax-8 restores the TPO promoter activity. FRTL-5 cells were treated with or without PDTC (40 µM) for 12 h, and the cells were transfected with pGL2-basic or pTPOpro plasmid together with or without pPAX8 plasmid. The luciferase activities in the cell lysates were determined by a luminometer and normalized with ß-galactosidase activities. The experiment was carried out in triplicate. The data were expressed in arbitrary luciferase units relative to those of the cells transfected with pGL2-basic, and presented as mean ± SD. *, P < 0.05 vs. pGL2-basic-transfected cells. Similar result was obtained from a separate experiment.

 
PDTC stimulates proliferation of FRTL-5 cells
As mentioned above, PDTC increases the level of GAPDH mRNA in FRTL-5 cells. Because the increase in GAPDH mRNA has been shown to be associated with cell growth (39), we examined effect of PDTC on proliferation of FRTL-5 cells. As shown in Fig. 6AGo, treatment with 40 µM and 80 µM PDTC for 24 h significantly increased the cell number when compared with that of nontreated cells. 3H-thymidine incorporation was also increased by PDTC (Fig. 6BGo). In contrast, NAC at the concentration of 10 mM or 20 mM had no effects on the cell growth.



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  		Figure 6. Effect of PDTC on the proliferation of FRTL-5 cells. A, FRTL-5 cells were treated with 40 µM or 80 µM PDTC, or 10 mM or 20 mM NAC for 24 h. After the treatment, WST-1 assay was performed. The data are presented as percentage of the level of untreated cells (CONT), and expressed as mean ± SD (n = 6). *, P < 0.05 vs. CONT. B, FRTL-5 cells were treated with 40 µM or 80 µM PDTC, or 10 mM or 20 mM NAC for 12 h. During the last 2 h, 3H-thymidine was added to the medium. The incorporation of 3H-thymidine was determined by a liquid scintillation counter after precipitation of acid-insoluble material with trichloroacetic acid. The data are presented as percentage of the level of untreated cells (CONT), and expressed as mean ± SD (n = 3). *, P < 0.05 vs. CONT.

 
Because it has been recently shown that PDTC decreases the DNA-binding activity of p53, a tumor-suppressor protein, in the breast cancer cell line (40), we studied the PDTC effect on p53 binding in FRTL-5 cells. As shown in Fig. 7AGo, when the nuclear extracts prepared from FRTL-5 cells were subjected to EMSA using GADD45 as a probe, a single band of protein/DNA complex was detected. This band was completely displaced by the excess amount of cold GADD45 oligonucleotide, and the density of the band was decreased by addition of anti-p53 antibody, indicating that the single band represents p53/GADD45 complex. Because p53 has been shown to bind DNA as a homotetramer, the antibody might prevent the tetramer formation or its DNA binding itself.



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  		Figure 7. Effect of PDTC on p53 binding. A, Nuclear extracts prepared from FRTL-5 cells were subjected to EMSA using oligo GADD45 as a labeled probe. To characterize the protein/DNA complex, excess amount of cold GADD45 probe and anti-p53 antibody were added to EMSA reaction buffer in lanes 2 and 4, respectively. B, FRTL-5 cells were treated with 40 µM PDTC for various lengths of time. Nuclear extracts were subjected to EMSA using oligo GADD45 as a labeled probe.

 
We then examined PDTC effect on the DNA binding of p53. The extracts prepared from FRTL-5 cells treated with 40 µM PDTC for various length of time were subjected to EMSA. As shown in Fig. 7BGo, DNA-binding activity of p53 was decreased as early as 3 h after PDTC. In contrast, 20 mM NAC had no effect on p53 binding (data not shown).

Does H2O2 play a role in modulation of thyroid function?
PDTC and NAC are generally recognized as antioxidants. FRTL-5 cells are demonstrated to produce H2O2 under control of TSH (7) (8). We therefore examined the capacity of these antioxidants to scavenge H2O2. Homovanillic acidfluorescence assay was employed to determine H2O2 content in medium. As shown in Fig. 8AGo, TSH increased the generation of H2O2 by more than 2-fold when compared with the level of the cells cultured in the absence of TSH (5H). Addition of purified catalase, which metabolizes H2O2 into H2O, into the medium markedly reduced the fluorescence, indicating that more than 90% of the fluorescence is derived from the H2O2 generated by the cells. Treatment of the cells with TSH along with 40 µM PDTC or 20 mM NAC almost completely scavenged the generated H2O2. Considering the different effects of PDTC and NAC on TPO and Pax-8 expression and on the cellular proliferation, it is suggested that the effects of PDTC cannot be attributed to its H2O2-scavenging action.



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  		Figure 8. H2O2 does not mediate the PDTC action. A, FRTL-5 cells cultured in 5H medium were treated with or without 1 mU TSH in the presence or absence of 300 U/ml catalase, 40 µM PDTC, and 20 mM NAC for 45 min. Concentration of H2O2 in the medium was determined by homovanillic acid fluorescence assay. Values are presented as percentage of the level of untreated cells (TSH minus), and expressed as mean ± SD (n = 3). Similar results were obtained from two separate experiments. B, FRTL-5 cells were cultured in the 5H medium, and exposed to 40 µM PDTC and 20 mM NAC for 12 h. The total RNA (15 µg/lane) was subjected to Northern blot analysis using TPO, Pax-8 and GAPDH as probes.

 
Effect of PDTC on the expression of TPO, Pax-8, and GAPDH was studied in FRTL-5 cells cultured in the 5H medium where the cells produce low level of H2O2 (Fig. 8BGo). Even in the absence of TSH, PDTC significantly decreased the levels of TPO and Pax-8 mRNAs and increased GAPDH mRNA. In contrast, NAC had no effects. These results again suggest that H2O2-scavenging action of PDTC does not account for its effect on TPO and Pax-8 expression and on the cell proliferation.

Noncell-permeable chelator of copper (BCS) inhibits PDTC effects
It has been shown that PDTC has not only antioxidant property but also prooxidant property. As a prooxidant, PDTC binds and transports extracellular redox active copper into the cells (23). We thus determined intracellular levels of copper in FRTL-5 cells. As shown in Table 1Go, treatment of the cells with PDTC (40 µM) for 12 h resulted in a significant increase in the intracellular copper. Treatment of the cells with 0.4 mM BCS which is a noncell-permeable chelator of Cu1+ (41) and thereby inhibits PDTC-dependent transport of Cu1+, prevented the increase in the intracellular copper by PDTC. In contrast, intracellular iron levels were not altered by PDTC and BCS.


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  		Table 1. Determination of the intracellular copper and iron contents in FRTL-5 cells after treatment with PDTC and BCS

 
We then examined effect of BCS on PDTC-induced decrease in TPO mRNA level. As shown in Fig. 9AGo, the treatment with BCS abrogated the PDTC-induced decrease in TPO and Pax-8 mRNA levels in a dose-dependent manner. Note that BCS alone had no effect on the mRNA levels. This result indicates that elevation of intracellular Cu1+ concentration causes the decreased expression of TPO and Pax-8 genes.



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  		Figure 9. BCS reverses the PDTC effect. A, FRTL-5 cells cultured in 6H medium were treated with or without 40 µM PDTC in the presence or absence of various concentrations of BCS for 12 h. Total RNA (15 µg per lane) was subjected to Northern blot analysis using TPO or Pax-8 cDNA as probes. B, The cells were treated with or without 40 µM PDTC in the presence or absence of 0.8 mM BCS for 12 h. The nuclear extracts were subjected to EMSA using GADD45 as a probe. C, The nuclear extracts prepared from FRTL-5 cells were treated with various concentrations of CuSO4, and subjected to EMSA using GADD45 as a probe.

 
Effect of BCS on p53 binding was also studied. As shown in Fig. 9BGo, the PDTC-induced decrease in p53 binding was reversed by the treatment with BCS, indicating that the reduced binding of p53 is also due to elevation of intracellular Cu1+. To test whether the increase in copper ion affects p53 binding, nuclear extracts from FRTL-5 cells were incubated with various concentrations of CuSO4 in vitro. As shown in Fig. 9CGo, p53 binding was almost completely inhibited by addition of CuSO4 at a concentration of 100 µM, suggesting that the direct interaction of copper with p53 reduces its DNA binding.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Effects of PDTC on function and proliferation of thyroid cells
The present study demonstrated that treatment of FRTL-5 cells with PDTC markedly decreased the expression of Pax-8 and TPO mRNAs. It was also shown that the DNA-binding activity of Pax-8 to the TPO promoter sequence was reduced by PDTC treatment. Because the binding of other transcription factors, TTF-1 and TTF-2, were not altered by PDTC, it is strongly suggested that the reduction of TPO mRNA is caused by the decreased transcription of the TPO gene due to reduced amount of Pax-8. This hypothesis was supported by the fact that transfection of the Pax-8-expressing plasmid alone restored the TPO promoter activity in the cells treated with PDTC. Because no difference in Pax-8 binding activity was detected in the extracts prepared with or without DTT from PDTC-treated cells, it is also suggested that PDTC does not alter the redox status of Pax-8 molecule.

Interestingly, despite similar organizations of cis-regulatory elements for Pax-8, TTF-1 and TTF-2 in the TPO and TG promoters (1), the expression of TG mRNA was not affected by PDTC. This might indicate that the TPO gene expression is more tightly regulated by Pax-8 than TG gene. A previous report that showed that transfection of a plasmid expressing Pax-8 activates TPO promoter better than TG promoter (1), supports this notion. It was also shown by the in situ hybridization method using human specimens of the follicular adenomas that Pax-8 mRNA level correlated well with that of TPO mRNA (42).

On the other hand, PDTC treatment of FRTL-5 cells increased GAPDH mRNA levels and stimulated cell proliferation rates. It was also demonstrated that PDTC induced a decrease in the DNA-binding activity of p53. Because it has been shown that the role of p53 in G1 arrest is largely dependent on transcriptional activation of its target gene p21 (43), and that p53-deficient cells exhibit accelerated proliferation rates (44), the reduction of p53 binding might be responsible for the PDTC-dependent promotion of growth of FRTL-5 cells.

Difference in PDTC and NAC effects on thyroid cells
It was reported that H2O2 acts as an intracellular signal-transducing molecule for platelet-derived growth factordependent cell proliferation in vascular smooth muscle cells (18). Therefore, we postulated a similar role of H2O2 in thyroid follicular cells and expected that PDTC and NAC would have similar effects on thyroid cell functions by scavenging H2O2. To our surprise, the effect of PDTC was different from that of NAC: PDTC promoted cell growth and inhibited the TPO and Pax-8 expression, whereas NAC had almost no effects on these parameters. Because PDTC and NAC scavenged the cellular H2O2 to a similar extent, TSH-induced production of H2O2 seems not to play a role in thyroid cell growth and in Pax-8 and TPO expression.

Increase in intracellular copper by PDTC
PDTC also acts as a chelator of ions and exerts a prooxidant effect by transporting the external redox-active ions into cells. We thus examined the alteration in intracellular contents of copper and iron by PDTC. It was clearly shown that PDTC increased the intracellular concentration of copper, whereas iron contents were not altered. The copper increased by PDTC must be a form of Cu1+, because BCS, a noncell-permeable chelator of Cu1+, inhibited the PDTC-dependent transport of copper. Similar observations were reported in thymocytes and breast cancer cell line MCF-7. In thymocytes, it was shown that PDTC increases intracellular copper and induces oxidative stress, resulting in toxic effects on the cells including apoptotic cell shrinkage and chromatin fragmentation (23). In MCF-7 cells, PDTC increases the intracellular copper, and down-regulates p53 DNA-binding activity (40).

Consistent with the latter report, the present study demonstrated that PDTC induced a decrease in p53 binding activity and that BCS abrogated the effects of PDTC, indicating the causal effect of the increase in intracellular Cu1+ in FRTL-5 cells. Our study further showed that the PDTCdependent reduction of TPO and Pax-8 expression was also reversed by BCS treatment. However, it remains to be studied how Cu1+ decreases the expression of Pax-8. Copper is an essential transition metal that plays an important role in function of some transcription factors and enzymes. Intracellular copper exists in several oxidation status, which can change from one redox status to another under physiological conditions. This redox cycling is responsible for physiological and toxic potential of this metal. Recently, it was shown that Pax-8 mRNA levels are under control of TSH (45, 46). It is thus speculated that Cu1+ might affect redox-sensitive molecules in the intracellular signaling cascades of TSH.

Modification of p53 by PDTC
Recent study demonstrated that p53 directly binds with Cu1+, and loses its DNA-binding activity in vitro (47). Thus, the observed PDTC effect on p53 in the present study might be explained by a direct interaction between p53 and Cu1+. Accordingly, our in vitro study demonstrated that CuSO4 at as low as 100 µM inhibits the p53 binding.

Another possible mechanism for PDTC effect on p53 is that PDTC might disrupt the structure of DNA-binding domain of p53 by chelating zinc ion because it was demonstrated that zinc ion is required for the formation of normal structure of the domain (48). It was also reported that PDTC promotes the oxidation of cysteine residue in p53 and inhibits p53-mediated transactivation (49). Thus, redox modification of cysteine residue in p53 could result in loss of its binding ability.

Physiological role of intracellular copper
It was reported that the average copper level in normal human hepatic tissues is 11.43 ± 4.74 µg/g of tissue (180 ± 74 µM), and it increases in the cirrhotic tissues (15.53 ± 5.90 µg/g) (50). The present study demonstrated that an increase in the intracellular copper levels from 10.6 ± 1.4 µg/g to 16.0 ± 1.2 µg/g by PDTC affected the thyroid-specific gene expressions and proliferation. Furthermore, the treatment of the cell extracts with 100 µM CuSO4 markedly reduced the p53 binding activities. These results indicate that the change in intracellular copper levels in a physiological range influences the function of thyroid follicular cells.

In conclusion, the present study demonstrated that intracellular copper levels and its redox state are crucial for the TPO and Pax-8 expressions, and p53 function. Recently, it has been shown that the loss-of-function mutation in the p53 gene, usually found in the region for the DNA-binding domain, affects thyroid differentiation phenotypes: introduction of mutated p53 into differentiated cell line abolished TPO and TG expression as well as Pax-8 and TTF-2 expression, and induced TSH-independent cell growth (51). Conversely, introduction of wild-type p53 into undifferentiated thyroid carcinoma cell line induced reexpression of TPO gene (52). Collectively, it is suggested that the redox regulation including intracellular copper levels and its redox cycle plays a role in control and maintenance of the differentiated phenotype and growth of thyroid follicular cells by modulating the p53 function.


   Acknowledgments
 
We are indebted to Dr. Devanand Sarkar for his critical review of the manuscript.


   Footnotes
 
1 This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan. Back

Received July 6, 2000.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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