Thyroid-Specific Gene Expression Is Differentially Influenced by ...

0013-7227/00/$03.00/0 Endocrinology Copyright © 2000 by The Endocrine Society

Vol. 141, No. 3 Printed in U.S.A.

Thyroid-Specific Gene Expression Is Differentially Influenced by Intracellular Glutathione Level in FRTL-5 Cells* R. LONIGRO, D. DONNINI, D. FABBRO, G. PERRELLA, G. DAMANTE, F. S. AMBESI IMPIOMBATO, AND F. CURCIO Dipartimento di Patologia e Medicina Sperimentale e Clinica, and Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Genetica (R.L., D.F., G.D.), Universita` degli Studi di Udine, 33100 Udine, Italy ABSTRACT Alteration of the redox potential has been proposed as a mechanism influencing gene expression. Reduced glutathione (GSH) is one of the cellular scavengers involved in the regulation of the redox potential. To test the role that GSH may play in thyroid cells, we cultured a differentiated rat thyroid cell strain (FRTL-5) in the presence of Lbuthionine-(S,R)-sulfoximine (BSO). BSO affects GSH synthesis by irreversibly inhibiting ␥-glutamylcysteine synthetase (EC 6.3.2.2), a specific enzyme involved in GSH synthesis. BSO-treated FRTL-5 cells show a great decrease in the GSH level, whereas malondialdehyde increases in the cell culture medium as a sign of lipid peroxidation. In these conditions the activity of two thyroid-specific promoters, thyroglobulin (Tg) and thyroperoxidase (TPO), is strongly reduced in transient transfection experiments. As both Tg and TPO promoters depend upon the thyroid-specific transcription factors, thyroid-specific transcription factor-1 (TTF-1) and Pax-8 for full transcriptional activity, we tested whether reduction of GSH concentration impairs the activity of these transcription factors. After BSO treatment of FRTL-5 cells, both transcription factors fail to trans-activate the respective chimerical targets, C5 and B-cell specific activating protein promoters, containing, respectively, multimerized TTF-1- or Pax-8-

binding sites only as well as the Tg and TPO natural promoters. Northern analysis revealed that endogenous Tg messenger RNA (mRNA) expression is also reduced by BSO treatment, whereas endogenous TPO expression is not modified. Furthermore, the Pax-8 mRNA steady state concentration does not change in BSO-treated cells, whereas TTF-1 mRNA slightly decreases. Immunoblotting analysis of FRTL-5 nuclear extracts does not show significant modification of the Pax-8 concentration in BSO-treated cells, whereas a decrease of 25% in TTF-1 protein is revealed. Furthermore, BSO treatment decreases the DNA-binding activity to the respective consensus sequence of both transcription factors. Finally, different mechanisms seem to act on TTF-1 and Pax-8 functional impairment in BSOtreated cells. Indeed, with a lowered GSH concentration, the overexpressed Pax-8 still activates transcription efficiently, whereas, on the contrary, the overexpressed TTF-1 does not recover its transactivation capability when the respective chimerical target sequences are used (C5 and BSAP). When the natural Tg and TPO promoter sequences are used, overexpression of Pax-8 parallels the effect on both promoters observed using the chimeric target sequences, whereas overexpression of TTF-1 increases TPO promoter transcriptional activity only. (Endocrinology 141: 901–909, 2000)

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LTERATION of the redox potential is known to accompany a variety of pathological states in different cell types and tissues (1– 4). The cellular redox potential has been proposed as a regulatory mechanism influencing gene expression in bacterial cells as well as in eukaryotic cells (5, 6). Transmembrane or intracellular redox signaling can influence eukaryotic gene expression (7, 8), either by inducing the nuclear translocation or by modifying the DNA-binding activity of housekeeping as well as tissue-specific trans-acting factors (9 –14). Many agents may induce oxidative stress, and the cellular defense mechanisms may be grouped in two categories: enzymatic (i.e. superoxide dismutase, catalase, thioredoxin) and nonenzymatic [i.e. glutathione (GSH)] scavengers. Among these scavengers, GSH plays a pivotal role (15–17). Thyroid-specific gene expression has been extensively studied, and cell-specific as well as ubiquitous tran-

scription factors have been identified in the thyroid (18 –21). Thyroglobulin (Tg) and thyroperoxidase (TPO) genes are two of the most studied thyroid-specific genes, and their promoters seem to be similar in the organization of cis-acting elements (22, 23). Indeed, Tg and TPO gene expression appear to be mediated by the combination of the same thyroidspecific transcription factors, TTF-1, TTF-2, and Pax-8. Tg and TPO gene expressions are sensitive to TSH (24, 25). TSH is a unique hormone that induces the production of hydrogen peroxide in the iodination and coupling reaction for thyroid hormone synthesis (26, 27). Hence, the efficiency of antioxidant defense mechanisms might be critical for thyroid cells. Recently, in some noncellular systems, both TTF-1 and Pax-8 have been found to be affected in their DNA-binding activity by the oxidation of cystein residues (13, 14). In the present study we used FRTL-5, a differentiated rat thyroid follicular cell strain (28), to test whether the GSH concentration influences the expression of thyroid-specific genes.

Received August 19, 1999. Address all correspondence and requests for reprints to: Dr. Renata Lonigro, Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Genetica, Universita` degli Studi di Udine, P. le M. Kolbe 4, 33100 Udine, Italy. E-mail: [email protected]. * This work was supported in part by grants from the Agenzia Spaziale Italiana, Centro Nazionale Ricerche (Target Project on Biotechnology), M.U.R.S.T.

Materials and Methods Intracellular GSH assay and lipid peroxidation assay The cellular concentration of GSH was assayed using the GSH-400, a commercial colorimetric assay kit (Bioxitech, Paris, France). Cells were

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harvested by trypsinization and counted. Duplicate aliquots of 4 ⫻ 106 cells were collected by centrifugation, and pellets were resuspended in 500 ␮l 5% (wt/vol) metaphosphoric acid. Pellets were homogenized using a Teflon pestle and then centrifuged (3000 ⫻ g, 4 C, 8 min). The assay was performed on 100-␮l aliquots of the centrifugation supernatants according to the manufacturer’s instructions. Malondialdehyde (MDA) production was measured in the culture medium of the same cells used for the GSH assay, using LPO-586, a commercial colorimetric assay kit (Bioxytech), following the manufacturer’s instruction.

Promoters, plasmids, and probes Tg natural minimal promoter is inserted in front of the chloramphenicol acetyltransferase (CAT) complementary DNA (cDNA) sequence (pTg-CAT plasmid) (19). TPO natural minimal promoter is inserted in front of the luciferase (LUC) cDNA sequence (pTPO-LUC plasmid) (22). The thyroid hormone response element (TRE) promoter is a 5 times multimerized activating protein-1 consensus sequence inserted in front of the CAT cDNA sequence (pTRE-CAT plasmid; provided by Prof. Di Lauro, Naples, Italy). The pC5-CAT construct consists of the C site sequence of the Tg promoter recognized by TTF-1 transcription factor and multimerized five times in front of the E1 TATA box in the pE1-CAT plasmid (29). The B-cell specific activating protein-CAT construct contains two copies of the CD19 BSAP site recognized by Pax-8 and inserted into the p⌬␦71-CAT plasmid (30). pCMV-␤Gal plasmid has a cytomegalovirus (CMV) promoter driving the ␤-galactosidase (␤Gal) cDNA transcription. The pRSV-CAT plasmid has a Rous sarcoma virus (RSV) promoter driving the CAT cDNA transcription. Tg-, TPO-, TTF-1-, Pax-8-, and glyceraldehyde-3-phosphate dehydrogenase (GPDH)-specific probes are restricted fragments of the respective cDNAs.

Cell cultures and transient transfection assay FRTL-5 cells were cultured at 37 C in chemically defined Coon’s modified F-12 medium (Sigma, St. Louis, MO) supplemented with 5% calf serum and six hormones (31). For the assays, cells were plated at 1.5 ⫻ 106/plate in 100-mm dishes with or without the addition of 0.5 mm BSO. Forty-eight hours later, cells were transfected using the calcium phosphate coprecipitation method (32) with pTg-CAT, pTPO-LUC, pC5E1, pBSAP-CAT, pTRE-CAT (10 ␮g), or pRSV-CAT (2 ␮g) reporter plasmid and 2 ␮g pCMV-␤Gal plasmid to monitor the transfection efficiency. In the promoter trans-activation experiments, 10 ␮g of each plasmid were cotransfected with 2 ␮g of a CMV-driven TTF-1 or Pax-8 expression vector, respectively (29, 33). Forty-eight hours after transfection, cells were washed twice with PBS, then collected and pelleted, and cell extracts were prepared by three cycles of freeze/thawing. CAT and ␤Gal expressions were measured using commercial kits (Roche Molecular Biochemicals). LUC activity was measured as previously described (34).

Northern analysis For Northern analysis FRTL-5 cells were grown in the same conditions used in the transfection experiments, with or without the addition of 0.5 mm BSO. Cells were cultured in these conditions for 4 days, and the culture medium was changed every 2 days. Total RNA was extracted by the acid-phenol extraction method (35). Ten micrograms of total RNA were separated on formaldehyde denaturing gel, blotted on a Hybond-N nylon membrane (Amersham Pharmacia Biotech, Arlington Heights, IL), and probed with Tg-, TPO-, TTF-1-, and Pax-8-specific cDNA sequences. Endogenous RNA expression was normalized for GPDH expression. All probes were [␣-32P]deoxy-CTP labeled by the random priming method (36).

TTF-1 half-life assay FRTL-5 cells were plated and cultured in the same conditions adopted for the transfection experiment, with or without the addition of 0.5 mm BSO. The culture medium was changed every 2 days. After 4 days, 10 ␮g/ml actinomycin D (Sigma) were added to the culture medium, and both BSO-treated and untreated cells were collected every hour. Total RNA extraction and TTF-1 Northern analysis were performed as pre-

FIG. 1. GSH and MDA determinations in BSO-untreated and BSOtreated FRTL-5 cells. A, GSH concentration expressed as micromoles per 106 cells ⫾ SD and measured in cell extracts (see Materials and Methods): gray rectangle, GSH level in FRTL-5 cells cultured for 4 days in the absence of BSO (BSO: ⫹); black rectangle, GSH level in FRTL-5 cells cultured for 4 days in presence of 0.5 mM BSO (BSO: ⫺; P ⬍ 0.001). B, MDA level expressed as micromoles per 106 cells ⫾ SD and measured in the medium (see Materials and Methods): gray rectangle, MDA concentration in the medium of BSO untreated FRTL-5 cells; black rectangle, MDA concentration in the medium of BSO treated (0.5 mM) FRTL-5 cells (P ⬍ 0.001). The results are the average of three independent experiments, each in duplicate dishes. viously described. The same amount of RNA was analyzed at each time point after actinomycin D treatment.

Immunoblotting assay Nuclear extracts were obtained according to the method of Tell et al. (37). The protein concentration of the nuclear extracts was measured according to the Bradford method (38). Nuclear extracts from BSOtreated or untreated FRTL-5 cells (10 ␮g) were electrophoresed in a 10% SDS-PAGE minigel. The gels were transferred to nitrocellulose membrane (Schleicher & Schuell, Inc., Keene, NH). The membranes were saturated by incubation at 4 C overnight with 10% nonfat dry milk in PBS/0.1% Tween-20. Then, they were incubated with the anti-TTF-1 and anti-Pax-8 antibodies (provided by Prof. R. Di Lauro, Naples, Italy) for

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FIG. 2. Effects of different BSO concentrations on GSH level and Tg and TPO promoter activities. A, GSH concentration expressed as micromoles per 106 cells ⫾ SD and measured in cell extracts (see Materials and Methods) at different doses of BSO. B, Transient expression assay of Tg and TPO promoters at different doses of BSO. Tg promoter drives CAT gene expression; TPO promoter drives LUC gene expression. The transcriptional activity of the promoters is normalized for the CMV-driven ␤Gal gene expression of a cotransfected pCMV-␤Gal construct, and it is expressed as a percentage of promoter activity. Tg, Tg promoter; TPO, TPO promoter. Bars, The data shown represent the average of three independent transfection assays ⫾ SD, each in duplicate dishes. 60 min at room temperature. After three washes with PBS/0.1% Tween20, they were incubated with an antirabbit Ig coupled to peroxidase (Sigma). After 60 min of incubation at room temperature, the membranes were washed several times with PBS/0.1% Tween-20, and the blots were developed using the enhanced chemiluminescence method (Amersham Pharmacia Biotech).

Mobility shift assay For mobility shift assays the FRTL-5 cells were grown in the same conditions used for the transfection experiments, with or without the addition of 0.5 mm BSO. Nuclear extracts were prepared as previously reported (37). Five micrograms of nuclear extracts were incubated with DNA in a buffer containing 20 mm Tris-HCl (pH 7.6), 75 mm KCl, 10 ␮g/ml calf thymus DNA, and 10% glycerol for 30 min at room temperature. Oligonucleotides harboring monomeric C5 and BSAP sequences were used as probes. The sense strands of these oligonucleotides are: C5, 5⬘-CCCAGTCAAGTGTTCTT-3⬘; and BSAP, 5⬘-ACCCATGGTTGAGTGCCCT-3⬘. Oligonucleotides were labeled at the 5⬘-end using polynucleotide kinase (Amersham Pharmacia Biotech) and [␥-32P]ATP and annealed with respective complementary strand. A 100-fold molar excess of cold competitor oligonucleotides, TTF-1 specific (D1) and Pax-8 specific (C␤)

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FIG. 3. Transient expression assay of Tg and TPO gene promoters. Tg and TRE promoters drive CAT gene expression; TPO promoter drives LUC gene expression. The transcriptional activity of the promoters is normalized for the CMV-driven ␤Gal gene expression of a cotransfected pCMV-␤Gal construct and is expressed as the CAT/␤Gal ratio. Tg, Tg promoter; TPO, TPO promoter; RSV, RSV promoter; BSO: ⫺, BSO-untreated cells; BSO: ⫹, BSO-treated cells. Bars, The data shown represent the average of six independent transfection assays ⫾ SD, each in duplicate dishes. Gray rectangles, Transcriptional activity of the promoters in the absence of BSO; black rectangles, transcriptional activity of the promoters in the presence of BSO. See Materials and Methods for details. Tg promoter, P ⬍ 0.001; TPO promoter, P ⬍ 0.05; RSV promoter, P ⫽ NS; TRE, P ⬍ 0.005. (39), was used where indicated. At the end of the binding reaction samples were loaded on a 7.5% native polyacrylamide gel and run at 4 C in 0.5 ⫻ Tris-borate-EDTA buffer.

Densitometric analysis Specific signals obtained in the Northern, immunoblotting, and mobility shift retardation assays were quantified using a computerized phosphorimager (GS-525 Molecular Imager System, Bio-Rad Laboratories, Inc., Richmond, CA).

Expression of data and statistics Data are presented as the mean ⫾ sd and were analyzed by Student’s t test.

Results BSO treatment of FRTL-5 cells decreases the intracellular GSH concentration

To test whether reduction of the GSH concentration influences thyroid-specific gene expression we used BSO to irreversibly block the ␥-glutamylcysteine synthetase activity, thus reducing GSH synthesis. As shown in Fig. 1A, BSO treatment decreases the GSH concentration (1.64 ⫾ 0.33 ␮mol/106 cells in BSO-treated samples vs. 13.03 ⫾ 1.89 ␮mol/106 cells in untreated samples). An increase in MDA has been reported to be an indication of lipid peroxidation during oxidative stress (40). Under the same experimental conditions, the MDA concentration was increased (0.58 ␮mol/106 cells in BSO-treated samples vs. 0.37 ␮mol/106 cells in untreated samples; Fig. 1B). Hence,

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FIG. 4. Steady state level of endogenous Tg and TPO gene expression. A, Northern analysis: Tg, Tg mRNA; TPO, TPO mRNA; GPDH, GPDH mRNA; (⫺), BSO-untreated cells, (⫹), BSO-treated cells. B, mRNA signal quantification obtained at the computerized phosphorimager. Tg and TPO mRNA signals are normalized for GPDH signal, and in the BSO-treated cells (⫹) are reported as a percentage of the levels detected in the BSO-untreated cells (⫺). The data shown in this figure represent the average of three independent experiments ⫾ SD, each in duplicate dishes. Tg, P ⬍ 0.01; TPO, P ⫽ NS.

Dose response of BSO effect on GSH level and Tg and TPO promoter activities

In Fig. 2A are shown the effects of different doses of BSO on the GSH concentration in FRTL-5 cells, and in Fig. 2B are shown the corresponding Tg and TPO promoter activities. Both promoters were already sensitive to the lowest BSO concentration used (0.01 mm), decreasing their transcriptional activities to 40% and 65%, respectively, vs. that in BSO-untreated cells. In subsequent experiments we decided to use the BSO concentration of 0.5 mm because it is the most effective in decreasing TPO promoter activity, and it does not affect cell viability. Transcriptional activity of Tg and TPO promoters is downregulated in BSO-treated FRTL-5 cells

FIG. 5. Transient expression assay of C5 and BSAP promoters in BSOuntreated and BSO-treated cells. The two promoters assayed drive CAT gene expression. The transcriptional activity of the promoters is normalized for the CMV-driven ␤Gal gene expression of a cotransfected pCMV-␤Gal construct and is expressed as the CAT/␤Gal ratio. C5, TTF1-dependent chimerical promoter; BSAP, Pax-8-dependent chimerical promoter. BSO: ⫺, BSO-untreated cells; BSO: ⫹, BSO-treated cells. Bars, The data shown represent the average of four independent transfection assays ⫾ SD, each in duplicate dishes. Gray rectangles, Transcriptional activity of the promoters in absence of BSO; black rectangles, transcriptional activity of the promoters in presence of BSO. See Materials and Methods for details. C5, P ⬍ 0.05; BSAP, P ⬍ 0.01.

BSO treatment of FRTL-5 cells decreases the GSH concentration and causes an oxidative injury, as suggested by the increased MDA concentration. Cell counts were performed with trypan blue to measure viability.

In the constructs, pTg-CAT and pTPO-LUC (thyroidspecific promoters of Tg and TPO genes) are inserted in front of CAT cDNA and LUC cDNA, respectively. Their ability to drive transcription was assayed by transient transfection experiments in FRTL-5 cells cultured with or without BSO (Fig. 3). Tg transcriptional activity was 4 –5 times lower in the presence of 0.5 mm BSO. TPO transcriptional activity was reduced to an even greater degree, being almost 10 times lower in BSO-treated than in BSO-untreated cells. This phenomenon is specific, as the transcriptional activity of the viral RSV promoter does not appear to be affected by BSO treatment, whereas the nonthyroid-specific TRE promoter activity is increased 2 times in BSO-treated cells (Fig. 3). Furthermore, the behavior of TRE promoter activity supports the MDA increase, further suggesting an oxidative injury of the cells derived by reduction of GSH (41). Transfection efficiency does not appear to be influenced by BSO treatment, as the values of ␤Gal assays are similar with or without BSO (data not shown).

GSH CONTROLS THYROID GENE EXPRESSION Endogenous Tg and TPO gene expressions are influenced differently by reduction of the GSH concentration in FRTL5 cells

To test whether the endogenous expression of Tg and TPO genes is sensitive to reduction of the GSH concentration, we measured the steady state level of Tg and TPO messenger RNA (mRNA) in BSO-treated or untreated FRTL-5 (Fig. 4A) under the same experimental conditions as those used for the transient transfection assays. BSO treatment of FRTL-5 cells reduced the Tg mRNA level to almost 60% of that in BSOuntreated cells (Fig. 4B). On the other hand, expression of endogenous thyroperoxidase was not affected, as its mRNA level was not influenced by BSO in thyroid cells. Therefore, although the behavior of endogenous Tg gene expression is in agreement with the exogenous minimal promoter activity, endogenous TPO gene expression is not. TTF-1 and Pax-8 transcription factors are involved in BSOinduced down-regulation of Tg and TPO promoter transcriptional activities

TTF-1 and Pax-8 transcription factors are essential for the full transcriptional activity of Tg and TPO promoters. To address the question of whether TTF-1 and/or Pax-8 are involved in the down-regulation of Tg and TPO promoter activities in BSO-treated cells, FRTL-5 cells were transfected with constructs containing only the TTF-1 or Pax-8 specific

FIG. 6. Steady state level of endogenous TTF-1 and Pax-8 gene expression. A, Northern analysis: TTF-1, TTF-1 mRNA; Pax-8, Pax-8 mRNA; GPDH, GPDH mRNA; (⫺), BSO-untreated cells; (⫹), BSO-treated cells. B, mRNA signal quantification obtained at the computerized phosphorimager. TTF-1 and Pax-8 mRNA signals are normalized for GPDH signal, and in the BSOtreated cells (⫹), data are reported as a percentage of the concentration found in BSO-untreated cells (⫺). The data shown represent the averages of three independent experiments ⫾ SD, each in duplicate dishes. TTF-1 P ⬍ 0.05; Pax-8, P ⫽ NS. C, TTF-1 mRNA halflife. As detailed in Materials and Methods, cells were treated with actinomycin D for the time indicated (hours ⫹ act.), and the same amount of RNA was analyzed for TTF-1 signal at each time point of actinomycin D exposure. The decrease in TTF-1 mRNA in the actinomycin-treated cells was evaluated using the computerized phosphorimager and is reported in the figure as a percentage of the TTF-1 mRNA level detected in the actinomycin-untreated cells (point 0). The data represent the average of two independent experiments, each in duplicate dishes. White squares, RNA from BSO-untreated cells; black squares, RNA from BSOtreated cells.

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binding sites (C5 and BSAP, respectively) inserted in front of basal promoters. We evaluated the ability of these chimeric promoters to drive the transcription in GSH-impoverished FRTL-5 cells. A 4 –5 times transcriptional reduction of both promoters was found when cells were treated with BSO (Fig. 5). These results strongly suggest that reduction of the GSH concentration influences the amount and/or the activity of TTF-1 and Pax-8 on the chimeric targets. Reduction of GSH concentration differently influences TTF1 and Pax-8 expression and reduces the DNA-binding capability of both transcription factors

To test whether the decrease in GSH affects the expression of TTF-1 and Pax-8, we performed Northern blot analysis in BSO-treated and untreated FRTL-5 cells. The steady state level of TTF-1 mRNA was reduced by almost 30% in BSO-treated cells (Fig. 6, A and B), indicating that the reduction of GSH influences the expression of this transcription factor. Furthermore, the interference might be at the transcriptional level, as no change in the TTF-1 mRNA half-life was observed (Fig. 6C). On the contrary, the Pax-8 mRNA steady state level was not significantly influenced by BSO treatment of the cells (Fig. 6, A and B). Immunoblot analysis performed with nuclear extracts of FRTL-5 cells, treated and untreated with BSO, did not reveal detectable differences in the Pax-8 protein concen-

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FIG. 7. TTF-1 and Pax-8 immunoblotting analysis and mobility shift assays in BSO-treated (4 days) and BSO-untreated cells. A, Top, Immunoblotting analysis of Pax-8 protein in BSO-untreated (⫺) and BSO-treated (⫹) cells. A, Bottom, Pax-8 DNA-binding activity to BSAPradiolabeled oligonucleotide in BSO-untreated (⫺) and BSO-treated (⫹) cells. Bound, Pax-8 DNA-binding activity; Comp, competitor; C␤, Pax-8-specific cold competitor; C5, Pax-8 nonspecific competitor. B, Top, Immunoblotting analysis of TTF-1 protein in BSO-untreated (⫺) and BSO-treated (⫹) cells. B, Bottom, TTF-1 DNA-binding activity to the radiolabeled monomeric consensus sequence derived by C5, in BSOuntreated (⫺) and BSO-treated (⫹) cells. Bound, TTF-1 DNA-binding activity; Comp, competitor; D1, TTF-1-specific cold competitor; BSAP, TTF-1 nonspecific competitor.

tration (Fig. 7A, top), whereas it revealed a reduction of almost 25% in the TTF-1 protein concentration (Fig. 7B, top). Furthermore, the mobility shift assay performed with the same nuclear extracts and double strand oligonucleotides harboring Pax-8 and TTF-1 monomeric consensus sequences, respectively (BSAP and C5 consensus sequences), revealed a reduction of the DNA-binding capability of both transcription factors (Fig. 7, A and B, bottom) when BSO was used. The ability of Pax-8 DNA to bind to BSAP

sequence is reduced to almost 57%, and the TTF-1 DNA binding capability to C sequence is reduced to almost 64%. Different mechanisms interfere with the abilities of TTF-1 and Pax-8 to activate transcription in GSH-impoverished thyroid cells

Recently, the expression level of a transcription factor active during mouse development has been demonstrated to be finely

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controlled; even 20% variation in the protein concentration dramatically affects the wild-type mouse phenotype (42). It is conceivable that a similar sensitivity to TTF-1 exists in FRTL-5 cells. To test whether the reduction of TTF-1 expression observed in BSO-treated cells is the only factor responsible for the impaired TTF-1 transcriptional activity, TTF-1 expression was increased by transfecting in FRTL-5 cells a CMV-driven expression vector, and the ability of the overexpressed protein to activate transcription activating the specific target promoter C5 in BSOtreated and untreated cells was tested. The transfection of a CMV-TTF-1 construct strongly increased the transcriptional activity of C5 promoter when no BSO was added (Fig. 8-BSO). In BSO-treated cells, however, the overexpressed TTF-1 fails to trans-activate C5 promoter (Fig. 8, ⫹BSO) suggesting that other mechanisms also interfere with TTF-1 protein, functionally impairing its ability to activate the transcription of specific target genes in GSH-impoverished cells. Interestingly, when a similar experiment was performed with Pax-8, the overexpressed Pax-8 protein efficiently increased transcription from BSAP promoter in both BSO-treated and untreated FRTL-5 cells (Fig. 8). To further investigate the roles of TTF-1 and Pax-8 in GSHimpoverished cells, we overexpressed both transcription factors in cotransfection experiments with Tg and TPO natural minimal promoters in the presence of BSO. The results show that the overexpressed TTF-1 protein is not able to trans-activate the Tg promoter, as it does using the C5 chimeric sequence, whereas it trans-activates the TPO promoter (Fig. 9). The overexpressed Pax-8 protein trans-activates both natural promoters, although with different strength, as it does using the BSAP

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FIG. 9. Differential transcriptional activation of Tg and TPO natural promoters in BSO-treated cells by the overexpressed TTF-1 and Pax-8 proteins. BSO-treated FRTL-5 cells were transfected with pTg-CAT or pTPO-LUC promoters. Activator, Promoter activity in the absence (⫺) and presence of a cotransfected TTF-1 or/and Pax-8 expression vectors. Promoter activity is expressed as the fold activation of Tg and TPO promoters over their basal level measured in BSO-treated cells and in the absence of activators. White square, Tg promoter activity; gray squares, TPO promoter activity; bars, The data shown represent the average of two independent transfection assays ⫾ SD, each in duplicate dishes.

chimeric sequence. Together, these results indicate that BSO treatment affects TTF-1 and Pax-8 transcriptional activities by different mechanisms. Discussion

FIG. 8. Differential transcriptional activation of C5 and BSAP promoters in BSO-treated cells by the overexpressed TTF-1 and Pax-8 proteins, respectively. BSO-untreated and BSO-treated FRTL-5 cells (BSO: ⫺ and ⫹, respectively) were transfected with constructs in which the CAT gene expression depends upon C5 or BSAP promoters. Activator, Promoter activity in the absence (⫺) and presence of a cotransfected TTF-1 or Pax-8 expression vector. Promoters activity is expressed as the fold activation of C5 and BSAP promoters over their basal levels in BSO-untreated cells and in the absence of activators. Bars, The data shown represent the average of four independent transfection assays ⫾ SD, each in duplicate dishes.

Our data show that in the differentiated rat thyroid follicular cell strain FRTL-5, a decline in the GSH concentration is accompanied by oxidative stress (as suggested by the increase in the MDA concentration) as well as by an impairment of some thyroid-specific gene expression. To our knowledge, these results represent the first correlation between the GSH intracellular concentration and the expression of thyroid-specific genes. It has been previously reported that the DNA-binding activity of TTF-1 and Pax-8 is sensitive to alterations of the redox potential in noncellular systems. Indeed, our data show that the transcriptional activity of Tg and TPO natural minimal promoters is reduced in transient transfection experiments. Both TTF-1 and Pax-8 seem to be involved in this phenomenon, because at a low GSH concentration, their effects on chimeric specific promoters are greatly reduced compared with those at a normal GSH concentration. However, different mechanisms may be involved in the regulation of endogenous Tg and TPO transcription, as the endogenous concentration of Tg mRNA was reduced in BSOtreated, GSH-impoverished cells, whereas for the TPO mRNA concentration no reduction was observed. The DNA-binding activity of both transcription factors was reduced in GSH-impoverished cells. Such reduced binding activity was accompanied by a reduction of TTF-1, but

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not Pax-8. However, the reduction of TTF-1 does not seem to be the only determinant of the failure of TTF-1 to activate transcription of specific target genes in BSO-treated, GSHimpoverished cells. Indeed, although overexpression of TTF-1 restores TPO natural minimal promoter transcriptional activity, it does not restore C5 or Tg natural minimal promoter transcriptional activities. Data reported by other groups (43) suggest that different regulatory mechanisms are involved in the control of Tg and TPO gene transcription; therefore, it seems conceivable that TTF-1 and Pax-8 play different roles in the transcriptional activation of Tg and TPO genes. Indeed, despite the fact that in human, rat, and bovine thyroid cells, Tg and TPO promoters and enhancers are targeted by the same thyroid-enriched transcription factors, TTF-1 and Pax-8 (23, 44 – 46), TTF-1 only weakly stimulates transcription of the TPO promoter, whereas it strongly stimulates transcription of the Tg promoter in nonthyroid cellular systems (44, 47, 48). Our data suggest that the decrease in GSH concentration in differentiated thyroid cells affects TTF-1 and Pax-8 functions by different posttranscriptional mechanisms. Our data and those reported by other groups on the TTF-1 DNA-binding ability in noncellular systems, suggest that the oxidative environment negatively influences its ability to bind specific recognition sequences. At the same time, Arnone et al. demonstrated that the oxidative environment improves TTF-1 homooligomerization via cystein residues in noncellular systems (14). A similar mechanism may be invoked to explain the inability of the overexpressed TTF-1 protein to restore the transcriptional activity of C5 and Tg promoters in BSO-treated cells. However, this mechanism cannot explain TTF-1 behavior on the TPO promoter. The overexpression of Pax-8 is able to overcome the BSO-dependent functional impairment and restore the BSAP as well as Tg and TPO natural minimal promoter transcriptional activities. This behavior of Pax-8 suggests the saturation of a BSO-dependent inactivating counterpart by the overexpressed protein. Interestingly, it has been recently reported that Pax-5, a transcription factor member of the Pax gene family with high structural homology with Pax-8, is able to heterodimerize with the underphosphorylate form of the retinoblastoma (Rb) gene product (49). In a variety of in vivo systems, oxidative stress has been shown to influence gene expression by hyper- or hypophosphorylating specific protein kinases and transcription factors (50 –52). In conclusion, our results support a differential role of GSH in the control of cell-specific gene expression in differentiated rat thyroid cells. Acknowledgments We thank Dr. Gianluca Tell and Mrs. Filomena Ciarfaglia for their technical help.

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