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Endocrinology Vol. 140, No. 10 4651-4658
Copyright © 1999 by The Endocrine Society

ARTICLES

Tumor Necrosis Factor-{alpha} Regulation of Thyroid Transcription Factor-1 and Pax-8 in Rat Thyroid FRTL-5 Cells

Masayuki Ohmori, Norikazu Harii, Toyoshi Endo and Toshimasa Onaya

Third Department of Internal Medicine, Yamanashi Medical University, Tamaho, Yamanashi 409-3898, Japan

Address all correspondence and requests for reprints to: Toshimasa Onaya, Professor and Chairman, Third Department of Internal Medicine, Yamanashi Medical University, 1110 Shimokato, Tamaho, Yamanashi 409-3898, Japan. E-mail: onayat{at}res.yamanashi-med.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor-{alpha} (TNF-{alpha}) is known to modulate the expression of thyroid-specific genes, such as thyroglobulin (TG), contributing to the pathogenesis of autoimmune thyroid disease. In the present study, we show that TNF-{alpha} suppresses DNA-binding activity of thyroid transcription factors, Pax-8 and thyroid transcription factor-1 (TTF-1), which is, in part, involved in TNF-{alpha}-induced decrease in TG gene expression. Transfected into rat thyroid FRTL-5 cells, the activity of reporter plasmid containing the rat TG promoter ligated to a luciferase gene was significantly suppressed in the presence of TNF-{alpha}. In gel mobility shift analyses, protein-DNA complexes formed by TTF-1 and Pax-8 were reduced when the nuclear extracts prepared from TNF-{alpha}-treated FRTL-5 cells were used. The suppressive effect of TNF-{alpha} on TTF-1-DNA complex formation is, in part, caused by suppression of TTF-1 gene transcription by TNF-{alpha}. Expressions of TTF-1 messenger RNA and protein, which were assessed by Northern blot and Western blot analyses, respectively, were decreased by TNF-{alpha} treatment of FRTL-5 cells. In contrast, TNF-{alpha} did not affect the expression of Pax-8 messenger RNA. Treatment of FRTL-5 cells with TNF-{alpha} caused a decrease in Pax-8 protein in nuclear extracts and accumulation of the protein in the cytoplasm, as assessed by Western blot analyses. Mutation of the TTF-1/Pax-8-binding site lost the TNF-{alpha}-induced decrease in TG promoter activity in a transfection experiment. These results indicate that TNF-{alpha} suppresses the activity of TTF-1 and Pax-8 by different mechanisms, which, in part, seem to be involved in TNF-{alpha}-induced decrease in TG gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TUMOR NECROSIS factor-{alpha} (TNF-{alpha}) is a cytokine produced by activated macrophages and monocytes in inflammatory sites, and it plays a number of important roles in the mechanism of defense against bacteria and viruses, such as T-cell stimulation, neutrophil activation, and antiviral activity (1, 2). TNF-{alpha}, as well as interleukin (IL)-1 and IL-6, have been demonstrated in thyroid tissues from patients with neoplastic or autoimmune diseases and can be produced by thyroid epithelial cells (3). It may contribute to the pathogenesis of thyroid diseases. For instance, subacute thyroiditis is caused by viral infections, which are, in general, accompanied by production of TNF-{alpha} in immunocompetent cells. It is assumed that TNF-{alpha}, as well as IL-1 and interferon-{gamma}, are also produced in chronic thyroiditis, particularly painless thyroiditis, in which macrophages and lymphocytes infiltrate the thyroid tissue, as in subacute thyroiditis (4).

TNF-{alpha} is involved in the modulation of thyroid growth and function. Administration of TNF-{alpha} to mice or rats decreases serum T3, T4, and TSH concentrations, iodide uptake, and TSH-induced T3 and T4 release (5, 6). In humans, administration of TNF-{alpha} results in a decrease in serum T3 concentrations similar to those seen in patients with euthyroid sick syndrome (7, 8). In cultured thyroid cells, TNF-{alpha} has a number of effects on thyroid growth and function. In human thyroid cells, TNF-{alpha} decreases TSH-induced 125I-incorporation and release of 125I-T3 and 125I-T4 into the medium (9). Rasmussen et al. reported that TNF-{alpha}, separately and added together with IL-1b, inhibit thyroglobulin (TG) production from cultured human thyroid cells (10). Also in rat thyroid FRTL-5 cells, TNF-{alpha} decreases TSH-induced iodide uptake and inhibits cell growth (11, 12). Tang et al. showed that TNF-{alpha} and interferon-{gamma}, in combination, have a marked inhibitory effect on 5'-deiodinase, TG, and thyroid peroxidase (TPO) gene expressions in FRTL-5 cells (13).

There are few reports on the molecular mechanism of the effect of cytokines on thyroid-specific genes, such as TG, TPO, and TSH receptor (TSHR). In the present study, we show that TNF-{alpha} decreases TG gene expression, in part, by reducing the DNA-binding activity of two thyroid-specific transcription factors, Pax-8 and thyroid transcription factor-1 (TTF-1), which are also involved in regulation of the TPO and TSHR genes (14, 15, 16, 17, 18, 19, 20, 21, 22, 23).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Bovine TSH is a highly purified preparation obtained from Sigma Chemical Co. (St. Louis, MO). Recombinant human TNF-{alpha} was a generous gift of Dainippon Pharmaceutical Co. (Osaka, Japan). [{alpha}-32P]deoxycycidine triphosphate (3000 Ci/mmol) and [{gamma}-32P]ATP (3000 Ci/mmol) were obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). The sources of other materials were detailed previously (24).

Cells
FRTL-5 rat thyroid cells [CRL 8305, American Type Culture Collection (ATCC), Rockville, MD] (25) were grown in Coon’s modified Ham’s F-12 supplemented with 5% calf serum (Life Technologies, Grand Island, NY). The medium includes a six-hormone mixture (6H medium), containing bovine TSH (10 mU/ml), insulin (1.3 x 10-6 M), cortisol (10-6 M), transferrin (6.3 x 10-11 M), glycyl-L-histidyl-L-lysine acetate (2.5 x 10-6 M), and somatostatin (6.1 x 10-9 M). Cells were passaged every 7–8 days and fed a fresh medium every 2–3 days. They were used before passage 20. FRT rat nonfunctioning thyroid cells (26) and Buffalo rat liver cells (BRL 3A, CRL 1442, ATCC) were in Coon’s modified Ham’s F-12 supplemented with 5% calf serum or 5% FCS, respectively.

Northern blot analysis
Total RNA from FRTL-5 cells was isolated by a PERFECT RNA Kit (5 Prime 3 Prime, Inc., Boulder, CO). Total RNA (15 µg/lane) was electrophoresed on a 1% agarose gel and transferred to a nitrocellulose membrane (Schleicher & Schuell, Inc., Keene, NH). Blots were prehybridized overnight at 42 C in 50% formamide, 5 x Denhardt’s solution (0.2% Ficoll, 0.2% polyvinilpyrolidone, and 0.2% BSA), 5 x SSPE (20 x SSPE = 3 M NaCl, 0.2 M sodium phosphate, and 20 mM EDTA, pH 7.4), 0.1% SDS, and 0.2 mg/ml heat-denatured salmon sperm DNA. Hybridization was performed at 42 C for 24 h with the radiolabeled probe in 50% formamide, 2.5 x Denhardt’s solution, 5 x SSPE, 0.1% SDS, 10% Dextran sulfate, and 0.1 mg/ml heat-denatured salmon sperm DNA. Filters were washed for 15 min at room temperature in a wash buffer A (6 x SSPE, 0.5% SDS), for 15 min at 37 C in a wash buffer B (1 x SSPE, 0.1% SDS), and for 15 min at 65 C in the wash buffer B. An imaging plate was exposed to the filters for overnight, and the results were analyzed with a Bas 2000 Image Analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan). The complementary DNAs (cDNAs) used in this report were as follows. The TG cDNA was provided by ATCC (CRL 57736). The rat TTF-1 probe was a fragment from +1 to +331 bp excised from the TTF-1 expression vector, RcCMV-THA; it was the kind gift of Dr. R. Di Lauro (Stazione Zoologica A. Dohrn, Naples, Italy) (15). The Pax-8 (17) and rat ß-actin cDNAs were kindly donated by Dr. L. D. Kohn (NIH, Bethesda, MD). All probes were radiolabeled with [{alpha}-32P] deoxycycidine triphosphate using a random primer labeling kit (Takara Shuzo Co., Tokyo, Japan).

Plasmids
Genomic sequences of TG gene from -688 to +75, and -168 to +36 bp were amplified by PCR, together with forward and reverse primers containing a 5'-adaptor sequence (KpnI or BglII), to facilitate directional cloning. PCR products were cloned into KpnI-BglII sites of plasmid pGL2-Basic (Promega Corp., Madison, WI); these luciferase (Luc) constructs are designated pTG Luc (-688) and pTG Luc (-168), respectively. Cloned inserts were sequenced in their entirety to ensure the PCR-generated misincorporations. To generate a chimera containing mutations in the sequence, pTG Luc (-168MT), objective promoter segments with mutation (see Fig. 6AGo) were generated by PCR using a forward primer that had the mutated sequence with KpnI site on the 5'-end and the reverse primer described above. All plasmids were prepared using a Plasmid Maxi Kit (QIAGEN, Chatsworth, CA).



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  		Figure 6. Effect of mutations in the TTF-1/Pax-8-binding site on the TG promoter activity in FRTL-5 cells. A, Mutations are denoted by comparison with the sequence of wild-type promoter. Dots denote mutated residues. The location of the TTF-1/Pax-8 site is presented. B, TG promoter activity was measured in the presence or absence of 100 ng/ml TNF-{alpha} and/or 10 mU/ml TSH. FRTL-5 cells were transfected with pTG Luc (-168) or pTG Luc (-168MT) containing mutations in the TTF-1/Pax-8 site. Twenty-four hours after transfection, the medium were shifted to 5H medium plus 0.2% calf serum, and 100 ng/ml TNF-{alpha} and/or 10 mU/ml TSH were added to the medium. The cells were cultivated for 48 h before harvest for luciferase assay. Promoter activities are presented as the mean ± SE of three separate experiments. One asterisk and two asterisks denote a statistically significant decrease induced by TNF-{alpha}, in the absence or presence of TSH, respectively.

 
Transient expression analysis
Transient transfection used FRTL-5 cells. Before transfection, FRTL-5 cells were grown to 80% confluency in 6H medium and then shifted to 5H medium (6H medium minus TSH) for 4 days. One day before transfection, they were returned to 6H medium. Transfection used an electroporation technique (Gene Pulser, Bio-Rad Laboratories, Inc., Richmond, CA), previously described (24). FRTL-5 cells were harvested, washed, and suspended, 1.5 x 107 cells/ml, in 0.8 ml PBS. Ten micrograms of the TG pro Tmoter-luciferase chimeric plasmid and 3 µg pCH110-ß-gal were added. The cells were pulsed (300 V; 960 microfarads), plated, and cultured in 6H medium. One day after transfection, the cells were washed with PBS, and cultures were continued for 48 h in 5H medium plus 0.2% calf serum in the absence or presence of 100 ng/ml TNF-{alpha} and 10 mU/ml TSH. The cells were harvested for both luciferase assay and ß-galactosidase assay. These assays were performed as described previously (27, 28, respectively). Luciferase activity was normalized by ß-galactosidase activity.

Nuclear and cytoplasmic extracts
Nuclear extracts were prepared basically as previously described (24). The basal 5H extracts from FRTL-5 cells were prepared using cells maintained in 5H medium plus 0.2% calf serum for 7 days after near confluency in 6H medium plus 5% calf serum was achieved. Cells were washed twice with PBS, pH 7.4, and resuspended in 5 pellet vol of 2% Tween-40 in buffer A [10 mM HEPES-KOH (pH 7.9) containing 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonylfluoride, 2 µg/ml leupeptin, and 2 µg/ml pepstatin-A]. After 10 min on ice, the lysate was centrifuged at 500 x g for 3 min. The supernatant was designated as the cytoplasmic fraction and was stored for Western blot analyses. The pellet was resuspended in (0.3 M sucrose and 2% Tween-40 in buffer A. After the cells were frozen in liquid nitrogen, thawed, and gently homogenized, the suspension was layered onto 1.5 M sucrose in buffer A and centrifuged at 25,000 x g in a swinging bucket rotor. Nuclei were washed with buffer A and lysed in 2.5 vol of buffer B [10 mM HEPES-KOH (pH 7.9), containing 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 10% glycerol, 0.5 mM DTT, 0.5 mM phenylmethylsulfonylfluoride, 2 µg/ml leupeptin, and 2 µg/ml pepstatin-A]. Lysed nuclei were centrifuged at 15,000 x g for 1 h and were used in electrophoretic mobility shift assays (EMSAs) or Western blot analyses.

EMSA
EMSAs were performed basically as previously described (24). Synthesized, double-stranded oligonucleotide, Oligo C, was labeled with [{gamma}-32P] ATP and T4 polynucleotide kinase, and then purified on an 8% native polyacrylamide gel. Two micrograms of nuclear extract were incubated in a 30-µl reaction vol for 20 min at room temperature in the following buffer: 10 mM Tris-HCl (pH 7.6), 50 mM KCl, 5 mM MgCl2, 1 mM DTT, 1 mM EDTA, 5% glycerol, 0.1% Triton X-100, and 0.5 µg poly (dI-dC). Labeled probe (50,000 cpm; ~0.5 ng DNA) was added, and incubation was continued for an additional 20 min at room temperature. DNA-protein complexes were separated on 4.5% native polyacrylamide gels. In experiments using antiserum to TTF-1 or Pax-8, nuclear extracts were incubated with the antiserum or its control counterpart in the same buffer for 30 min at room temperature before adding the labeled probe and processing as above.

Western blot analysis
Fifteen micrograms of each nuclear extract or 80 µg of each cytoplasmic protein, prepared as above, were analyzed on 0.1% SDS-10% PAGE, electrotransferred to nitrocellulose membrane (Schleicher & Schuell, Inc.), and immunodetected (28) by anti-TTF-1 (29) or anti-Pax-8 antiserum. Anti-Pax-8 antibodies were prepared as follows: a recombinant partial Pax-8 protein, corresponding to amino acids 1–157, was produced using the pET system (Novagen, Madison, WI), as previously described (30). Antisera were raised in rabbits by serial injections of emulsion of the protein (1 mg/ml) in Freund’s complete adjuvant (Wako, Osaka, Japan), and after 10 weeks, the sera were used in the experiments.

Other analyses
Protein concentration was determined by Bradford’s method (Bio-Rad Laboratories, Inc.), and recry stallized BSA was used as a standard. All experiments were repeated at least three times with different batches of cells. Where noted, values are the mean ± SE of these experiments; significance (P < 0.05) between experimental values was determined by Student’s paired t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TG messenger RNA (mRNA) levels are decreased by TNF-{alpha}
To show that TNF-{alpha} decreases the gene expression of TG, Northern blot analysis was performed (Fig. 1Go). TG mRNA was detected as a single band of approximately 8.5 kb long, the size being compatible with a previous report (31). Because TG mRNA transcript was barely detectable in FRTL-5 cells maintained in basal 5H medium plus 0.2% calf serum, we could not evaluate the inhibitory effect of TNF-{alpha} on TG gene expression in TSH-deprived cells [Fig. 1Go, TSH (-)]. To estimate the effect of TNF-{alpha} on TG mRNA levels, therefore, we challenged TNF-{alpha} and TSH, at the same time, into FRTL-5 cells maintained in basal 5H medium plus 0.2% calf serum [Fig. 1Go, TSH (+)]. Incubation of FRTL-5 cells with TNF-{alpha} for 24 h had little or no effect on TG gene expression (Fig. 1AGo, left), which is compatible with a previous report by Tang et al., where FRTL-5 cells were maintained in 5H medium plus 5% calf serum for 5 days and then incubated with TNF-{alpha} for 24 h (13). However, when cells were incubated for 48 h, 100 ng/ml TNF-{alpha} resulted in a significant decrease in TG mRNA levels (Fig. 1AGo, right). The molecular mechanisms, by which a longer time incubation with TNF-{alpha} decreases TG gene expression in FRTL-5 cells, are therefore characterized as follows.



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  		Figure 1. Effect of TNF-{alpha} on expression of TG mRNA transcripts in FRTL-5 cells. A, FRTL-5 cells were maintained for 7 days in 5H medium plus 0.2% calf serum and then incubated, with or without 100 ng/ml TNF-{alpha}, for 24 or 48 h in the presence or absence of 10 mU/ml TSH. Total RNA was prepared from the cells, and equal amounts (15 µg/lane) were subjected to sequential Northern blot analysis using the TG and ß-actin probes. The data represent a typical experiment that was repeated three times with different batches of cells on different days. B, The ratio of TG to ß-actin mRNA levels was calculated after quantitation by Bas 2000 image analyzer. The data are the mean ± SE from three separate experiments. A statistically significant decrease induced by TNF-{alpha} is noted by an asterisk.

 
TNF-{alpha} suppresses promoter activity of the TG: possible involvement of TTF-1 and Pax-8 in its action
To investigate the molecular mechanisms of the effect of TNF-{alpha} on TG gene expression, we transfected chimeric luciferase plasmid containing the TG promoter region from -688 to +75 bp, pTG Luc (-688), into FRTL-5 cells and measured luciferase activity expressed in the presence or absence of TNF-{alpha} and/or TSH. pTG Luc (-688) contains three TTF-1- and one Pax-8-binding elements (Fig. 2AGo) (17, 23). pTG Luc (-688) expressed significantly higher activity by comparison with the promoter-less control plasmid, pGL2-Basic, which expressed only weak activity (Fig. 2BGo). When 100 ng/ml TNF-{alpha} were added to the basal 5H medium, the promoter activity of pTG Luc (-688) was significantly suppressed (Fig. 2BGo, left). The suppression by TNF-{alpha} was also observed in the presence of TSH in the incubation medium. Thus, the addition of TSH significantly increased the luciferase activity of pTG Luc (-688), approximately 2- to 3-fold higher than in cells without TSH, as previously described (32). The simultaneous addition of TNF-{alpha} and TSH to the basal 5H medium significantly reduced the luciferase activity by comparison with the addition of TSH without TNF-{alpha} (Fig. 2BGo, left). TNF-{alpha} and TSH had no effect on the luciferase activity of the TG promoterless pGL2-Basic (Fig. 2BGo, right) or pGL2-Control, which contains an SV40 promoter and an SV40 enhancer inserted into the structure of the pGL2-Basic (data not shown). It has been reported that there exist three TTF-1-, one TTF-2-, and one Pax-8-binding sites within -688 bp of the rat TG and that both TTF-1 and Pax-8 activate transcription from TG promoter (23). TTF-2 represses transcriptional activation by both TTF-1 and Pax-8 (33). To evaluate the possible involvement of TTF-1 and/or Pax-8 in the TNF-{alpha}-induced suppression of TG promoter activity, we studied the following.



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  		Figure 2. TNF-{alpha} suppresses TG promoter activity. A, The schema of the TG promoter (23 ) and the location of the oligonucleotide C mimicking the sequence of the TG promoter and used in this report are diagrammatically presented. TG promoter-luciferase chimeric plasmid, pTG Luc (-688), contains three TTF-1- and one Pax-8-binding elements. TATA, TATA box-like motif, B, TG promoter activity was measured in the presence or absence of 100 ng/ml TNF-{alpha} and/or 10 mU/ml TSH. The pTG Luc (-688) was transfected into FRTL-5 cells. The plasmid, pGL2-Basic, was also transfected as a control. Twenty-four hours after transfection, the medium were shifted to 5H medium plus 0.2% calf serum, and 100 ng/ml TNF-{alpha} and/or 10 mU/ml TSH were added to the medium. The cells were cultivated for 48 h before harvest for luciferase assay. Promoter activities are presented as the mean ± SE of three separate experiments. One asterisk and two asterisks denote a statistically significant decrease induced by TNF-{alpha} in the absence or presence of TSH, respectively.

 
DNA-binding activities of both TTF-1 and Pax-8 are reduced in TNF-{alpha}-treated FRTL-5 cell nuclear extracts
To test whether TTF-1 and/or Pax-8 binding is altered in TNF-{alpha}-treated cells, we performed gel mobility shift assays using a radiolabeled synthetic oligonucleotide probe spanning -77 to -54 bp of the rat TG, Oligo C, which includes the TTF-1- and Pax-8-binding sites (Fig. 2AGo, and Ref. 17). Because the inhibitory effect of TNF-{alpha} on TG promoter activity could be demonstrated even in the absence of TSH (Fig. 2BGo), we hereafter studied the effect of TNF-{alpha} on TTF-1 and Pax-8 DNA-binding activities in TSH-deprived FRTL-5 cells. Nuclear extracts from FRTL-5 cells maintained in 5H medium plus 0.2% calf serum formed two protein-DNA complexes (Fig. 3AGo, arrows). The upper complex was FRTL-5 cell-specific. Thus, nuclear extracts from FRTL-5 cells, but not from FRT or BRL cells, formed the upper complex. The lower complex exists in FRT, as well as FRTL-5, cells. FRT cell nuclear extracts contain Pax-8 but no TTF-1 (20). The upper and lower complexes were, therefore, likely to be TTF-1 and Pax-8, respectively. These were confirmed by the ability of antibodies to TTF-1 (Fig. 3BGo, lane 3) and to Pax-8 (Fig. 3BGo, lane 5) to inhibit its formation and/or supershift the complex. Antibodies to TTF-1 (29) supershifted the upper complex. Anti-Pax-8 antibodies inhibited the formation of the lower complex. Each preimmune serum had no effect on the TTF-1 or Pax-8 complex (Fig. 3BGo, lanes 2 and 4, respectively). These results are consistent with the report by Di Lauro’s group that the upper band is the TTF-1/DNA complex and the lower is the Pax-8/DNA complex (17). It seems that both anti-TTF-1 and anti-Pax-8 antibodies used in this report do work in Western blot analyses (evidenced later) and in gel mobility shift analyses. Nuclear extracts prepared from TNF-{alpha}-treated FRTL-5 cells also formed the same protein-DNA complexes (Fig. 3CGo, lanes 2–4 vs. 1). However, the intensity of these bands was unequivocally decreased in a concentration-dependent manner (Fig. 3CGo, lanes 2–4 vs. 1). Thus, a significant decrease in both TTF-1 and Pax-8 binding was measured at 50 ng/ml, more than 50% inhibition occurred with 100 ng/ml, and maximal inhibition was achieved with 500 ng/ml TNF-{alpha} (Fig. 3CGo, lanes 2–4). TNF-{alpha} decreased, with similar intensity, TTF-1 and Pax-8 binding (Fig. 3CGo, lower panel). The TNF-{alpha} effect is time-dependent; the suppressive effect of 100 ng/ml TNF-{alpha} on both TTF-1 and Pax-8 binding is evident within 24 h, and maximal by 48 h, in FRTL-5 cells (Fig. 3DGo). These data support the presumption raised in the functional analyses, i.e. that altered DNA-binding activities of TTF-1 and/or Pax-8 are involved in the TNF-{alpha}-induced suppression of TG promoter activity.



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  		Figure 3. Formation of both TTF-1/DNA complex and Pax-8/DNA complex is decreased by TNF-{alpha} treatment. A, The radiolabeled synthetic oligonucleotide C was used as a probe and incubated with nuclear extracts from FRTL-5 cells, from FRT cells or from BRL cells. Gel mobility shift analysis was performed as described in Materials and Methods. The upper arrow denotes a protein/Oligo C complex, which exists in nuclear extracts from FRTL-5 cells but not from FRT or BRL cells. The lower arrow depicts a protein/Oligo C complex expressing in FRT, as well as FRTL-5, cells. B, The radiolabeled synthetic oligonucleotide C was used as a probe and incubated with nuclear extracts from FRTL-5 cells in the absence (-) or presence (+) of the noted rabbit antiserum against TTF-1 or Pax-8, or the corresponding preimmune serum. DNA-protein complexes were electrophoresed for a longer period than that of the experiments in A, C, and D. The upper arrow denotes a protein-DNA complex missing in FRTL-5 cell extracts and up-shifted (small arrow) after incubation with anti-TTF-1 antibodies but not with the corresponding preimmune serum. The lower arrow depicts a protein-DNA complex missing in FRTL-5 cell extracts after incubation with anti-Pax-8 antibodies but not with the corresponding preimmune serum. C, The radiolabeled synthetic oligonucleotide C was incubated with nuclear extracts from FRTL-5 cells. Nuclear extracts were prepared from the cells that were cultured in 5H medium plus 0.2% calf serum for 7 days and then treated for 48 h with the noted concentration of TNF-{alpha}. In lanes 5 and 6, the radiolabeled synthetic oligonucleotide C was incubated with nuclear extracts from the cells treated with 100 ng/ml TNF-{alpha} in the presence of the noted rabbit antiserum against TTF-1 or Pax-8. In the lower panel, radioactivities of the specific TTF-1-DNA complex and Pax-8-DNA complex were measured by a Bas 2000 Image Analyzer. The radioactivity of the band formed in the absence of TNF-{alpha} was set at unity, and the data are presented as the ratio of the activities. Data are the mean of three separate experiments. D, The radiolabeled synthetic oligonucleotide C was incubated with the noted nuclear extracts. Nuclear extracts were prepared from FRTL-5 cells that were cultured in 5H medium plus 0.2% calf serum for 7 days and then treated with 100 ng/ml TNF-{alpha} for the noted number of hours.

 
TTF-1 mRNA and protein levels are decreased by TNF-{alpha}
Reduction in TTF-1-DNA complex formation by nuclear extracts from TNF-{alpha}-treated FRTL-5 cells could be caused by suppression of TTF-1 gene transcription by TNF-{alpha}. We, therefore, examined the expression of TTF-1 mRNA by Northern blot analysis. Total RNA was prepared from FRTL-5 cells cultured for 48 h in 5H medium with or without TNF-{alpha}. Steady-state RNA levels of TTF-1 were decreased in a concentration-dependent manner, 48 h after TNF-{alpha} challenge (Fig. 4AGo, lanes 2–4 vs. 1), at the time when the TTF-1 binding to DNA was reduced and TG promoter activity was suppressed, as shown above (Figs. 3Go and 2Go, respectively). In addition, TTF-1 protein levels were assessed by Western blot analysis (Fig. 4BGo). A protein of approximately 46 kDa, which corresponds to TTF-1 (29), was detected in both TNF-{alpha}-treated and nontreated nuclear extracts from FRTL-5 cells. The amount of the TTF-1 protein in TNF-{alpha}-treated extracts was less than that in nontreated extracts (Fig. 4BGo, lanes 2–4 vs. 1). These results indicate that TNF-{alpha} decreases TTF-1 mRNA and protein levels, in part, responsible for TNF-{alpha}-induced reduction in DNA-binding activity of TTF-1.



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  		Figure 4. Effect of TNF-{alpha} on TTF-1 mRNA and protein levels in FRTL-5 cells. A, FRTL-5 cells were maintained for 7 days in 5H medium plus 0.2% calf serum and then challenged for 48 h with the noted concentration of TNF-{alpha}. Total RNA was prepared from the cells, and equal amounts (15 µg/lane) were subjected to sequential Northern blot analysis using the TTF-1 and ß-actin probes. The data represent a typical experiment that was repeated three times with different batches of cells on different days. B, FRTL-5 cells were maintained for 7 days in 5H medium plus 0.2% calf serum and then challenged for 48 h with the noted concentration of TNF-{alpha}. Nuclear extracts were prepared from the cells, and equal amounts (15 µg/lane) were subjected to sequential Western blot analysis using the anti-TTF-1 antibodies.

 
TNF-{alpha} decreases Pax-8 protein levels in nuclear extracts without altering expression of Pax-8 mRNA: involvement of cytoplasmic trapping of Pax-8 from the nucleus
To investigate whether the expression of Pax-8 mRNA is reduced by TNF-{alpha}, we performed Northern blot analysis. Total RNA was prepared from FRTL-5 cells cultured for 48 h in 5H medium with or without TNF-{alpha}. Steady-state RNA levels of Pax-8 did not change, even 48 h after TNF-{alpha} challenge (Fig. 5AGo), at the time when the Pax-8-DNA complex was reduced and TG promoter activity was suppressed, as shown above (Figs. 3Go and 2Go, respectively). These results indicate that TNF-{alpha} does not affect the transcription of Pax-8 gene. We, therefore, questioned whether the reduction in Pax-8-DNA complex formation induced by TNF-{alpha} (Fig. 3Go, C and D) might be caused by reduction in Pax-8 protein in nuclear extracts. To evaluate the possibility that TNF-{alpha} decreases Pax-8 protein in nuclear extracts, Western blot analysis was performed. A protein of approximately 60 kDa was detected in nuclear extracts from FRTL-5 cells with antiserum to the recombinant partial Pax-8 protein but not with the corresponding preimmune serum (Fig. 5BGo, lanes 2 vs. 1). The 60-kDa protein was not detected in rat liver BRL cells (data not shown). The speciality of the antiserum to Pax-8 was also confirmed by gel mobility shift analyses (Fig. 3BGo, lanes 5 vs. 4). Pax-8 protein levels in nuclear extracts were decreased by TNF-{alpha} treatment of FRTL-5 cells in a concentration-dependent manner (Fig. 5CGo, lanes 2–4 vs. 1). One possible explanation for this discrepancy between mRNA and protein might be that TNF-{alpha} induces cytoplasmic trapping and loss of Pax-8 protein from the nucleus, as in the case of phorbol ester and TTF-1 (34). To evaluate the possibility, Western blot analysis was performed using cytoplasmic proteins from FRTL-5 cells, treated or not with TNF-{alpha}. The same 60-kDa protein was detected in cytoplasmic extracts from FRTL-5 cells with antiserum to Pax-8 (Fig. 5DGo). Pax-8 protein levels in cytoplasmic extracts were increased by TNF-{alpha} treatment of the cells in a concentration-dependent manner (Fig. 5DGo, lanes 2 and 3 vs. 1). These results indicate that down-regulation of Pax-8 DNA-binding activity by TNF-{alpha}, in part, involves cytoplasmic trapping of Pax-8 protein from the nucleus.



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  		Figure 5. Effect of TNF-{alpha} on Pax-8 mRNA and protein levels in FRTL-5 cells. A, FRTL-5 cells were maintained in 5H medium plus 0.2% calf serum for 7 days and then exposed to the noted concentration of TNF-{alpha} for 48 h. Total RNA was prepared from the cells, and equal amounts (15 µg/lane) were subjected to sequential Northern blot analysis using the Pax-8 and ß-actin probes. The data represent a typical experiment that was repeated three times with different batches of cells on different days. B, FRTL-5 cells were maintained in 5H medium plus 0.2% calf serum for 7 days. Nuclear extracts were prepared from the cells, and equal amounts (15 µg/lane) were subjected to sequential Western blot analysis using a rabbit antiserum against Pax-8 or the corresponding preimmune serum. The arrow indicates the position of Pax-8 protein. Molecular size (kilodaltons) is indicated to the left. C, FRTL-5 cells were maintained in 5H medium plus 0.2% calf serum for 7 days and then exposed to the noted concentration of TNF-{alpha} for 48 h. Nuclear extracts were prepared from the cells, and equal amounts (15 µg/lane) were subjected to sequential Western blot analysis using anti-Pax-8 antibodies. D, FRTL-5 cells were maintained in 5H medium plus 0.2% calf serum for 7 days and then exposed to the noted concentration of TNF-{alpha} for 48 h. Cytoplasmic proteins were prepared from the cells, as described in Materials and Methods, and equal amounts (80 µg/lane) were subjected to sequential Western blot analysis using anti-Pax-8 antibodies.

 
Mutation data indicate the TTF-1- and Pax-8-binding sites are related to TNF-{alpha}-induced decrease in TG promoter activity
To determine the functional relevance of the TTF-1/Pax-8 element, we mutated in the pTG Luc (-168) chimeric construct having a minimal TG promoter (Fig. 6AGo). The mutation (MT) changes eight residues of the TTF-1/Pax-8-binding site and is identical to that reported by Civitareale et al. (14). Neither TTF-1 nor Pax-8 binds to the mutated sequence in gel mobility shift assays (17). When pTG Luc (-168) was transfected into FRTL-5 cells, it exhibited both TNF-{alpha}-induced decrease in promoter activity and TSH-responsiveness (Fig. 6BGo), as in the case of pTG Luc (-688) (Fig. 2BGo). In contrast, pTG Luc (-168MT) lost the basal promoter activity and the TNF-{alpha}-induced decrease in activity (Fig. 6BGo). These results indicate that the TTF-1/Pax-8-binding site is related to TNF-{alpha}-induced decrease, as well as the basal promoter activity of TG.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cytokines, including TNF-{alpha}, have been implicated in the pathogenesis of autoimmune thyroid diseases through various mechanisms (35). TNF-{alpha} is produced in the thyroid by intrathyroidal inflammatory cells, as well as by thyroid follicular cells themselves, and may act in a cascade to enhance the autoimmune processes (3, 35). It induces immunological changes in thyroid follicular cells, including enhancement of major histocompatibility complex (MHC) molecule expression (36). Apart from its immunoregulatory activities, TNF-{alpha} also has diverse effects on the growth and function of thyroid cells. Thus, it inhibits (solely or conjointly with other cytokines) the production of TG (10).

Schuppert et al. (37) analyzed the transcript levels of thyroid-specific transcription factors, TTF-1 and Pax-8, as well as thyroid-specific genes, TG, TPO, and TSHR, in various thyroid tissues (including those from patients with autoimmune thyroid diseases) and reported that Pax-8 expression is increased in Graves’ thyroids. In this report, we analyzed the DNA-binding activities, as well as transcript levels, of these thyroid-specific transcription factors in response to TNF-{alpha} (one of pathogenic cytokines in autoimmune thyroid diseases). This report focuses on TNF-{alpha} regulation of TTF-1 and Pax-8, in association with its regulation of TG gene expression.

In the present report, we show that TNF-{alpha} decreases TG mRNA levels and TG promoter activity in FRTL-5 cells. We show that TNF-{alpha} reduces DNA-binding activities of both TTF-1 and Pax-8. TNF-{alpha} decreases TTF-1 mRNA and protein levels, as well as TTF-1-DNA complex formation. In contrast, TNF-{alpha} does not alter Pax-8 mRNA levels. It causes a decrease in Pax-8 protein in nuclear extracts and accumulation of the protein in the cytoplasm. Moreover, we confirm the functional relevance of TTF-1/Pax-8 element in TNF-{alpha}-induced suppression of TG pr Homoter activity using mutant TG promoter chimera.

Pekary et al. recently demonstrated that TNF-{alpha} reduces Na+/I- symporter (NIS) mRNA levels in FRTL-5 cells (38). We have shown that TTF-1 activates transcription from NIS promoter (39). It is reasonable to consider that some common mechanisms could be involved in the suppressive effect of TNF-{alpha} on the expression of both TG and NIS genes. Thus, one of the possible explanations is that TNF-{alpha} suppresses both TG and NIS gene expressions by reducing TTF-1 activity. Further, because TTF-1 and/or Pax-8 interact with the promoters of not only TG but also TPO, TSHR, and NIS (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39), it seems to be reasonable to speculate that some common mechanisms could be involved in the regulation of the expression of these thyroid-specific genes by cytokines other than TNF-{alpha}.

There are several reports as to which mechanisms regulate TTF-1 binding activity. We reported that TSH, via its cAMP signal, decreases TTF-1 mRNA and protein levels in FRTL-5 cells (29). Avvedimento et al. (40) showed that TTF-1 could be phosphorylated by protein kinase A and that phosphorylation increased the binding of TTF-1 to its recognition sites in the rat TG gene. Kambe et al. (41) reported that the redox regulation of TTF-1 by TSH through thioredoxin is involved in its increased DNA-binding activity and in an increase in TG promoter activity. Interferon-{gamma} was reported to suppress the TSHR promoter activity, in part, by reducing the DNA-binding activity of TTF-1, by unknown mechanisms (42). TTF-1 exists also in lung and transactivates expression of the surfactant protein genes (43). In lung cells, cAMP and protein kinase A activation are associated with phosphorylation of TTF-1 and an increase in TTF-1 binding activity (44, 45). Phorbol ester down-regulates lung surfactant protein B gene expression by cytoplasmic trapping of TTF-1 (34). At present, there are few reports regarding the regulation of Pax-8. Van Renterghem et al. (46) showed that Pax-8 mRNA and protein levels are increased by cAMP in primary cultured dog thyrocyte. Kambe et al. (41) reported that the redox regulation of Pax-8 is involved in its increased DNA-binding activity and in an increase in TG promoter activity.

This report shows that TNF-{alpha} decreases TTF-1 mRNA, TTF-1 protein, and DNA-binding activity of TTF-1. The TNF-{alpha}-induced decrease in TTF-1 mRNA and protein levels seems to be less than that in TTF-1 binding activity (Figs. 4Go vs. 3). One possible explanation for this discrepancy is additional mechanisms induced by TNF-{alpha} treatment. One of the possible mechanisms might be the redox regulation. It has been demonstrated that oxidation of purified TTF-1 protein reduces the DNA-binding affinity by forming dimers or oligomers (47). Although TTF-1 protein may exist as either monomer or dimer, TTF-1 dimer shows remarkably reduced DNA-binding affinity, compared with monomer (47). It is not known whether TNF-{alpha} modifies the redox status in thyroid cells. The TNF-{alpha} effect on this issue and the significance of the TTF-1 function in pathological conditions, such as autoimmune thyroid diseases, require further investigation.

We previously hypothesized that autoimmune thyroid disease is a transcription factor disease (30). Thus, common transcription factors such as TTF-1 and Pax-8 coordinately regulate expression of TG, TPO, TSHR, and MHC genes. Abnormal expression of these common transcription factors results in increased expression of TG, TPO, and TSHR and MHC class I and class II genes. This results in a loss of immune tolerance and the development of autoimmune thyroid disease such as Graves’ disease. The present study shows that TNF-{alpha}, produced in subacute thyroiditis or chronic thyroiditis, decreases TG gene expression, in part, by reducing the activity of TTF-1 and Pax-8. There is a possibility that these transcription factors are important not only in Graves’ disease but also in cytokines-mediated thyroiditis. The roles of TTF-1 and Pax-8 in coordinate regulation of TPO, TSHR, NIS, and MHC genes, in response to TNF-{alpha} and/or other cytokines, require further investigation.

Received November 11, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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