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Endocrinology Vol. 140, No. 9 4214-4220
Copyright © 1999 by The Endocrine Society

ARTICLES

Tumor Necrosis Factor-{alpha} and Interferon-{gamma} Suppress Both Gene Expression and Deoxyribonucleic Acid-Binding of TTF-2 in FRTL-5 Cells

Asako Miyazaki, Hiroki Shimura, Toyoshi Endo, Kazutaka Haraguchi 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 M.D., Ph.D., Third Department of Internal Medicine, Yamanashi Medical University, 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}) and interferon-{gamma} (IFN-{gamma}) are cytokines that can individually or additively suppress thyroid cell function and the expression of thyroid-specific genes, such as thyroglobulin (TG) and thyroperoxidase (TPO). Thyroid transcription factor-2 (TTF-2) is a DNA-binding protein that modulates the expression of TG and TPO genes. In the present study, we examine the effects of TNF-{alpha} and IFN-{gamma} on TTF-2 gene expression, as well as the DNA-binding activity of TTF-2. FRTL-5 cells were maintained in 5H medium containing 0.2% calf serum for 7 days, then incubated with TNF-{alpha}, IFN-{gamma}, or TNF-{alpha} plus IFN-{gamma}. Total RNA was isolated and Northern blotted. TNF-{alpha} (50 ng/ml) only slightly suppressed (61 ± 2% compared with control), whereas IFN-{gamma} (100 U/ml) modestly decreased TTF-2 messenger RNA (mRNA) levels (34 ± 4%). TNF-{alpha} and IFN-{gamma} simultaneously caused a marked decrease in TTF-2 mRNA levels (13 ± 2%). The suppressive effects of TNF-{alpha} and IFN-{gamma} on TTF-2 mRNA levels were concentration dependent and maximal at 50 ng/ml TNF-{alpha} with 100 U/ml IFN-{gamma}. The suppressive effect was also time dependent, reaching a maximum 12 h after exposure. Moreover, the suppressive effects of TNF-{alpha} and IFN-{gamma} upon rat TG and TTF-2 mRNA levels were similar. To test whether TNF-{alpha} and IFN-{gamma} alter TTF-2-binding to DNA, we performed electrophoretic mobility shift assays using a TTF-2-binding element in the rat TG gene as a probe. Formation of the TTF-2/DNA complex was decreased by TNF-{alpha} and/or IFN-{gamma}. Our results demonstrate that TNF-{alpha} and IFN-{gamma} additively reduce the gene expression and DNA-binding of TTF-2. These data suggest that TTF-2 is involved in the TNF-{alpha} and IFN-{gamma}-induced suppression of thyroid-specific gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEVERAL CYTOKINES INVOLVED in the pathogenesis of autoimmune thyroid disease, such as tumor necrosis factor-{alpha} (TNF-{alpha}), and interferon-{gamma} (IFN-{gamma}), affect the growth, differentiation, and function of thyroid cells. For example, IFN-{gamma} secreted by activated T lymphocytes alters the growth of thyroid cells in association with inducing the aberrant expression of major histocompatibility complex (MHC) class II antigen (HLA-DR) (1, 2, 3, 4, 5, 6) and increasing the expression of MHC class I antigen (6) on thyroid cell surfaces. Furthermore, the IFN-{gamma}-induced expression of MHC class II antigen is enhanced by TNF-{alpha} (7). Expression of these antigen-presenting molecules must be important in the initiation and/or perpetuation of autoimmune thyroid disease.

In addition, cytokines, individually or additively, can affect thyroid cell function. Both TNF-{alpha} and IFN-{gamma} inhibit 125I organification and thyroid hormone release (8). Both TNF-{alpha} and IFN-{gamma} also inhibit the expression of thyroid-specific genes, such as thyroid peroxidase (TPO) (9, 10, 11, 12) and thyroglobulin (TG) (12, 13). Some mechanisms in common could be involved in the suppressive effect of TNF-{alpha} and IFN-{gamma} on the expression of these thyroid-specific genes. The mechanism regulating TG and TPO promoter activities has been studied extensively during the last decade. The tissue-specific transcription factors, TTF-1, TTF-2, and Pax-8, bind to both TG and TPO promoters (14). However, the mechanisms involved in the suppressive effect of TNF-{alpha} and IFN-{gamma} on these genes are not well understood. IFN-{gamma} suppresses TSH receptor gene expression in part, by reducing the DNA-binding affinity of TTF-1 (15).

TTF-2 was identified as a thyroid-specific DNA-binding protein that can recognize a single binding site in the minimal promoters of rat TG (16) and TPO (17), both of which are exclusively expressed in the thyroid. Mutagenesis in the TG promoter has revealed that TTF-2 is important for TG and TPO promoter activity in thyroid cells (18). Furthermore TSH and insulin exert a positive effect not only on TG gene expression, but also on that of TTF-2 (19). Cloning of TTF-2 cDNA has recently revealed that TTF-2 is a member of the forkhead family of transcription factors (20), which bind to DNA as monomers and contain a common 100-amino acid DNA-binding domain (21). Furthermore, Titf-2-null mutant mice have either a sublingual or a completely absent thyroid gland (22). A report also describes two human siblings with thyroid agenesis who were homozygous for a missense mutation within its forkhead domain (23). These facts suggest that TTF-2 plays important roles in the development of the thyroid as well as the regulation of TG gene expression.

In this study, we examined the effect of cytokines on TTF-2. We investigated the effects of TNF-{alpha} and IFN-{gamma} on levels of TTF-2 messenger RNA (mRNA) and its transcription rate. We also examined the effect of TNF-{alpha} and IFN-{gamma} on the DNA-binding activity of TTF-2. Furthermore, we discuss the possibility that TTF-2 is involved in TNF-{alpha}- or/and IFN-{gamma}-induced suppression of thyroid specific gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant human TNF-{alpha} was obtained from Prepro Tech, Inc. (Rocky Hill, NJ). Recombinant rat interferon-{gamma}, recombinant human TGF-ß, IL-1, and IL-6 were obtained from Genzyme Transgenics Corp. (Cambridge, MA). Materials used for cell culture were previously described (24). All chemicals and reagents were obtained from commercial sources and were of reagent or molecular biology grade.

Cell culture
FRTL-5 cells were cultured in Coon’s modified Ham’s F-12 medium supplemented with 5% calf serum containing bovine TSH (1 mU/ml), bovine insulin (10 µg/ml), human transferrin (5 µg/ml), glycyl-L-histidyl-L-lysine (10 ng/ml), somatostatin (10 ng/ml), and hydrocortisone (0.36 ng/ml) (6H). After reaching confluence, the cells were cultured for 7 days in medium without TSH (5H) and 0.2% calf serum. It has been reported that aged FRTL-5 cells are more susceptible to the cytotoxic effect of TNF-{alpha} (25), and this effect of TNF-{alpha} is enhanced by IFN-{gamma} (26). Therefore, we used this cell lines in early passage (<20 passages).

Northern blot analysis
Total RNA extracted from cells using guanidine isothiocyanate (27). For Northern blots, 20 µg of total RNA was resolved by electrophoresis on 1.2% agarose gels containing 0.66 M formaldehyde, then blotted onto nitrocellulose membranes. A rat TTF-2 cDNA (base 1598–2137) (20) was obtained by RT-PCR (27) from the mRNA of FRTL-5 cells. The PCR product was subcloned into the pCR2 vector (Invitrogen, San Diego, CA). Rat ß-actin cDNA was donated by Dr. L. D. Kohn (NIH, Bethesda, MD).

Nuclear run-on transcription analysis
FRTL-5 cells were maintained in 5H medium with 0.2% calf serum for 7 days, then incubated with 50 ng/ml TNF-{alpha} or/and 100 U/ml IFN-{gamma}. Nuclei were isolated from each sample as described (27). For transcription, nuclei were incubated in a 0.1 ml reaction mixture consisting of 90 mM NH4Cl, 2.5 mM MgCl2, 0.5 mM MnCl2, 1 mM dithiothreitol, 4 mM each of ATP, GTP, and CTP, and 200 µCi [{alpha}-32P] uridine 5'-triphosphate for 45 min at 26 C. The reaction was stopped by the adding of 100 µl of a stop mixture consisting of 200 µg/ml ribonuclease (RNase)-free deoxyribonuclease I, 200 µg/ml proteinase K, 200 µg/ml yeast transfer RNA, 20 mM HEPES-KOH (pH 8.0), and 20 mM CaCl2. Radiolabeled RNA was purified and hybridized to 4 µg DNA immobilized on nitrocellulose membranes. For micrograms each of rat TTF-2 cDNA, rat ß-actin cDNA, and pGEM7Zf(+) (Promega Corp., Madison, WI) plasmid DNA (to control for nonspecific binding) were immobilized on the nitrocellulose membranes. Prehybridization (3 h at 45 C) and hybridization (2 days at 45 C) proceeded in hybridization solution consisting of 50% formamide, 5 x SSPE, 10 x Denhardt’s solution, 0.1% SDS, 0.25 mg/ml heat-denatured salmon sperm DNA, 50 µg/ml poly (A), and 25 µg/ml transfer RNA. After hybridization, the membranes were washed three times in 2 x SSC-0.1%SDS for 30 min at room temperature, then three times in 0.1% SSC-0.1%SDS for 30 min at 45 C. The membranes were next washed in 2 x SSC with 10 µg/ml RNase A at 37 C, then 0.2 x SSC-0.1% SDS twice at room temperature and visualized by autoradiography.

Nuclear extracts
FRTL-5 cells were maintained in 5H medium with 0.2% calf serum for 7 days, then incubated with or without 50 ng/ml TNF-{alpha} or/and 100 U/ml IFN-{gamma}. Nuclear extracts were prepared as described (27).

Electrophoretic mobility shift analyses
Electrophoretic mobility shift analyses were performed using a double-stranded oligonucleotide, spanning -106 to -83 bp on the TG promoter (Oligo K) labeled with {gamma}-32P]ATP and T4 polynucleotide kinase. Nuclear extract (3 µg) was incubated with 50,000 cpm of the labeled probe ([approx]0.5 ng DNA) in a 15-µl reaction volume for 20 min at room temperature, and in the following buffer: 10 mM Tris-HCl, pH 7.6, 200 mM KCl, 5 mM MgCl2, 1 mM EDTA, 12.5% glycerol, 0.1% Triton X-100, 1 mM dithiothreitol, and 1 µg/15 µl poly (dl-dC). DNA-protein complexes were separated on 5% native polyacrylamide gels at 4 C and visualized by autoradiography.

Measurement of 125I uptake by FRTL-5 cells
Uptake of 125I by FRTL-5 cells was measured as previously described (27). Briefly, the cells were washed twice with 1 ml modified HBSS (137 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl2, 0.4 mM MgSO4, 0.5 mM MgCl2, 0.4 mM Na2HPO4, 0.44 mM KH2PO4, and 5.55 mM glucose with 10 mM HEPES buffer, pH 7.3) and incubated for 40 min at 37 C in 200 µl modified HBSS containing about 0.02 µCi carrier-free Na125I and 1.0 µM NaI, with a final specific activity of 10–20 mCi/mmol. When incubations were terminated, the cells were washed twice with ice-cold HBSS. Four hundred microliters of 95% ethanol were added to each well for 30 min, total of the well contents were then transferred into vials for counting with a {gamma}-counter.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of TNF-{alpha} and/or IFN-{gamma} on TTF-2 mRNA levels
We first examined the effects of TNF-{alpha} or IFN-{gamma} at various concentrations on TTF-2 mRNA levels. FRTL-5 cells were maintained in 5H medium with 0.2% calf serum for 7 days, then incubated with various concentrations of TNF-{alpha} or IFN-{gamma} for 12 h. Total RNA was isolated and Northern blotted using 32P-labeled rat TTF-2 cDNA (Fig. 1Go). Two mRNAs (2.8 and 2.3 kb) appeared, because of alternative use of the two polyadenylation signals identified in the 3'-untranslated region of the cDNA (20). As shown in Fig. 1Go, both TNF-{alpha} and IFN-{gamma} reduced TTF-2 mRNA levels. This effect was concentration dependent, and maximal at 50 ng/ml of TNF-{alpha} (61 ± 2% compared with control), or 100 U/ml of IFN-{gamma} (34 ± 4% compared with control). The maximal effect of IFN-{gamma} was significantly (P < 0.01) higher than that of TNF-{alpha}. In contrast, neither TNF-{alpha} nor IFN-{gamma} altered the rat ß-actin mRNA and 28S rRNA levels as shown in previous studies (12). Because normalization of the cytokine effects on TTF-2 mRNA level with both ß-actin mRNA and 28S rRNA resulted in no significant difference, we normalized the amounts of mRNA with rat ß-actin mRNA content in the following studies.



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  		Figure 1. Effect of TNF-{alpha} or IFN-{gamma} on TTF-2 mRNA levels and TG mRNA levels in FRTL-5 cells. FRTL-5 cells were maintained in 5H medium containing 0.2% calf serum for 7 days, then incubated with TNF-{alpha} or IFN-{gamma} for 12 h. Total RNA (20 µg) was Northern blotted using 32P-labeled rat TTF-2 cDNA, rat TG cDNA, and rat ß-actin cDNA as probes. As evidenced by ethidium bromide staining of the gel, total RNA used for analysis was the same amount and not degraded. B, Quantitation was using a BAS 2000 image analyzer (Fuji Photo Film Co., Ltd.). TTF-2 mRNA/ß-actin mRNA or 28S rRNA ratios and TG mRNA/ß-actin mRNA ratios is expressed as a percent of the control. Data are expressed as means ± SEM of values obtained from three separate experiments.

 
TNF-{alpha} and IFN-{gamma} suppress the expression of thyroid-specific genes, such as TG and TPO (12). We compared the effects of TNF-{alpha} and IFN-{gamma} on TG and TTF-2 mRNA levels by Northern blot analysis with the radiolabeled rat TG cDNA probe and the same filters. Figure 1Go shows that the dose-dependent suppressive effects of TNF-{alpha} and IFN-{gamma} upon rat TG and TTF-2 mRNA levels were similar.

We evaluated the time-dependent effect of TNF-{alpha} on TTF-2 mRNA levels in FRTL-5 cells that were maintained in 5H medium with 0.2% calf serum for 7 days, then in 50 ng/ml TNF-{alpha} for various periods. TNF-{alpha} suppressed the TTF-2 mRNA levels in a time-dependent manner (Fig. 2Go), reaching a maximum after 12 h.



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  		Figure 2. Time-dependent effect of TNF-{alpha} on TTF-2 mRNA levels in FRTL-5 cells. FRTL-5 cells were maintained in 5H medium containing 0.2% calf serum for 7 days, then incubated with 50 ng/ml TNF-{alpha}. Total RNA was isolated at various times thereafter. Total RNA (20 µg) was Northern blotted using 32P-labeled rat TTF-2 cDNA and rat ß-actin cDNA as probes. B, TTF-2 mRNA/ß-actin mRNA ratio is expressed as a percent of the control. Data are expressed as means ± SEM of values obtained from three separate experiments.

 
We also investigated the time-dependent effect of IFN-{gamma} on TTF-2 mRNA levels in FRTL-5 cells (Fig. 3Go). FRTL-5 cells were maintained in 5H medium containing 0.2% calf serum for 7 days, then incubated with 100 U/ml IFN-{gamma} for various periods. IFN-{gamma} time dependently decreased TTF-2 mRNA levels in a similar manner to that elicited by TNF-{alpha}. The effect was also maximal after 12 h.



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  		Figure 3. Time-dependent effect of IFN-{gamma} on TTF-2 mRNA levels in FRTL-5 cells. FRTL-5 cells were maintained in 5H medium containing 0.2% calf serum for 7 days, then incubated with 100 U/ml IFN-{gamma}. Total RNA was isolated at various times. Total RNA (20 µg) was Northern blotted using 32P-labeled rat TTF-2 cDNA and rat ß-actin cDNA as probes. B, TTF-2 mRNA/ß-actin mRNA ratio is expressed as a percent of the control. Data are expressed as means ± SEM of values obtained from three separate experiments.

 
It is reported that TNF-{alpha} and IFN-{gamma} added to FRTL-5 cells together synergistically inhibit functions and the expression of thyroid specific genes (6, 8, 28, 29). Thus, we investigated the combined effect of TNF-{alpha} and IFN-{gamma} upon the TTF-2 mRNA levels. Simultaneous exposure to TNF-{alpha} and IFN-{gamma} time dependently decreased TTF-2 mRNA levels, also reaching a maximum after 12 h. At each time point, TNF-{alpha} plus IFN-{gamma} exhibited additive suppression (13 ± 2% at 12 h) (Fig. 4Go). Our results show that TNF-{alpha} and IFN-{gamma} additively reduced TTF-2 mRNA levels in a time-dependent manner.



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  		Figure 4. Additive effect of TNF-{alpha} and IFN-{gamma} on TTF-2 mRNA levels in FRTL-5 cells. FRTL-5 cells were maintained in 5H medium containing 0.2% calf serum for 7 days, then incubated with 50 ng/ml TNF-{alpha} and 100 U/ml IFN-{gamma}. Total RNA was isolated at various times. Total RNA (20 µg) was Northern blotted using 32P-labeled rat TTF-2 cDNA and rat ß-actin cDNA as probes. B, Radioactivity level of TTF-2 mRNA/ß-actin mRNA ratio is expressed as a percent of the control. Data are expressed as means ± SEM of values obtained from three separate experiments.

 
TNF-{alpha} and/or IFN-{gamma} decrease levels of TTF-2 gene transcription
To determine whether TNF-{alpha} and/or IFN-{gamma} decrease levels of TTF-2 mRNA synthesis, we performed run-on assays with nuclei isolated from cells maintained in 5H plus 0.2% calf serum or exposed for 12 h to 50 ng/ml TNF-{alpha} and/or 100 U/ml IFN-{gamma} (Fig. 5Go). Both TNF-{alpha} (50 ng/ml) and IFN-{gamma} individually suppressed newly transcribed TTF-2 mRNA (58 and 42% compared with control, respectively). When TNF-{alpha} and IFN-{gamma} were added together, the transcription rate was markedly decreased (21%). Transcription of the ß-actin gene was not significantly affected by the two cytokines. These results suggested that the TNF-{alpha} and/or IFN-{gamma}-induced decrease in TTF-2 mRNA occurred at the level of transcription.



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  		Figure 5. Effect of TNF-{alpha}, IFN-{gamma}, or TNF-{alpha} plus IFN-{gamma} on transcription rate of TTF-2 gene in FRTL-5 cells. Nuclei were isolated from FRTL-5 cells incubated in 5H medium containing 0.2% calf serum (control) and from cells exposed to TNF-{alpha} (50 ng/ml), IFN-{gamma} (100 U/m), or TNF-{alpha} plus IFN-{gamma} for 12 h. RNA transcripts labeled with [32P] UTP and isolated from nuclei were added to hybridization reactions with either rat TTF-2 cDNA, ß-actin cDNA, or control empty vector pGEM7Z plasmid DNA as indicated. B, quantitation using a BAS 2000 image analyzer (Fuji Photo Film Co., Ltd.). The radioactivity level of nascent RNA/cDNA complexes from control nuclei was set at 100%. Data are expressed as means of values obtained from two separate experiments.

 
Binding of TTF-2 is reduced in TNF-{alpha} or/and IFN-{gamma}-treated FRTL-5 cells
To directly test whether or not TTF-2-binding to DNA is altered in TNF-{alpha} or/and IFN-{gamma}-treated cells, we performed electrophoretic mobility shift assays using a radiolabeled synthetic oligonucleotide probe, spanning -106 to -83 bp in the rat TG promoter (Oligo K), which includes the TTF-2-binding site. Nuclear extracts were prepared from FRTL-5 cells incubated with or without 50 ng/ml TNF-{alpha} or/and 100 U/ml IFN-{gamma} for 12 h. Nuclear extracts from FRTL-5 cells maintained in medium without the two cytokines formed a protein-DNA complex (Fig. 6Go). Because this complex was not formed with nuclear extracts from nonthyroid Buffalo rat liver cells, this band consisted of a TTF-2/DNA complex (data not shown). Nuclear extracts prepared from cells exposed to TNF-{alpha} and/or IFN-{gamma} also formed this protein-DNA complex, but to a decreased intensity. Both TNF-{alpha} and IFN-{gamma} individually inhibited formation of this complex up to 43 ± 8% and 32 ± 8%, respectively, compared with control. Like TTF-2 mRNA levels and transcription rates, TNF-{alpha} and IFN-{gamma} together decreased formation of the TTF-2/DNA complex (17 ± 7% compared with control).



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  		Figure 6. Effect of TNF-{alpha}, IFN-{gamma}, or TNF-{alpha} plus IFN-{gamma} treatment of FRTL-5 cells on TTF-2/thyroglobulin DNA complex formation. Double-stranded oligonucleotides containing the TTF-2-binding site in the TG promoter (oligo K) were radiolabeled and incubated with equal amount of nuclear extracts (2 µg) from FRTL-5 cells maintained in 5H medium containing 0.2% calf serum (control), and from cells exposed to TNF-{alpha} (50 ng/ml), IFN-{gamma} (100 U/m), or TNF-{alpha} plus IFN-{gamma} for 12 h. B, Radioactivity level of protein/DNA complexes measured using the BAS 2000 image analyzer (Fuji Photo Film Co., Ltd.) is represented as percent of the control. Data are expressed as means ± SEM of values obtained from three separate experiments.

 
Suppression of TTF-2 gene expression in TNF-{alpha} and IFN-{gamma}-specific
Because cytokines function through specific receptors on the cell surface and signal transduction, factors other than TNF-{alpha} and IFN-{gamma} may induce different effects. We therefore investigated the effects of other cytokines on TTF-2 mRNA levels. FRTL-5 cells were incubated with TGF-ß, IL-1, or IL-6 for 12. Total RNA was harvested and Northern blotted. None of TGF-ß, IL-1, or IL-6 decreased TTF-2 mRNA levels. In addition, iodide uptake was determined to test the activity of TGF-ß, IL-1, and IL-6 in FRTL-5 cells because it was reported that these cytokines caused approximately 30% decrease of sodium iodide symporter mRNA levels in FRTL-5 cells (30). As shown in Fig. 7Go, iodide uptake were decreased to 80% compared with control by treatment of these cytokines, showing that the cytokines used in this experiment were active against FRTL-5 cells. These results indicated that the effect of TNF-{alpha} and IFN-{gamma} on the TTF-2 mRNA levels in rat FRTL-5 cells is specific.



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  		Figure 7. Specificity of TNF-{alpha}- and IFN-{gamma}-induced effect on TTF-2 mRNA levels in FRTL-5 cells. FRTL-5 cells were maintained in 5H medium containing 0.2% calf serum for 7 days, then incubated with TNF-{alpha}, IFN-{gamma}, TGF-ß, IL-1, or IL-6 for 12 h. Total RNA (20 µg) was Northern blotted using 32P-labeled rat TTF-2 cDNA and rat ß-actin cDNA as probes. Furthermore, iodide uptake was determined after incubation with TGF-ß, IL-1 or IL-6. B, TTF-2 mRNA/ß-actin mRNA ratios and iodide uptake are expressed as percent of the control. Data are expressed as means ± SEM of values obtained from three separate experiments. The SEM of values obtained from iodide uptake were so small (TGF-ß, 0.017; IL-1, 0.018; IL-6, 0.019). *, P < 0.05 vs. control in iodide uptake.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several cytokines, including TNF-{alpha} and IFN-{gamma}, have been implicated in the pathogenesis of autoimmune thyroid disease. TNF-{alpha} and IFN-{gamma} induce the aberrant expression of MHC class II antigen (1, 2, 3, 4, 5, 6) and increase the expression of MHC class I antigen (29, 31) on thyroid cell surfaces. IFN-{gamma}-induced HLA-DR expression requires the activation of STAT1 and is associated with increased HLA-DR mRNA levels (32). Apart from their immunoregulatory activities, cytokines also have diverse effects on the function and growth of thyroid cells (5, 6, 8, 31). However, the mechanisms of these effects have remained unclear. TNF-{alpha} and IFN-{gamma} inhibit, solely or conjointly, the mRNA expression of TPO (9, 10, 11, 12), TG (12, 13) and TSHR (15, 33). Therefore, some common mechanisms could be involved in the suppressive effect of TNF-{alpha} and IFN-{gamma} on the expression of these thyroid-specific genes. IFN-{gamma} reportedly suppresses TSHR promoter activity in part, by reducing the DNA-binding affinity of TTF-1 (15), an important transcription factor for the TSHR gene. In addition, TNF-{alpha} appears to diminish the mRNA levels of TTF-1 and DNA-binding activities of TTF-1 and Pax-8 (34). The present study demonstrates the potent suppression of TTF-2 mRNA levels, as well as the DNA-binding activity of TTF-2, by TNF-{alpha} and IFN-{gamma} in FRTL-5 cells.

TNF-{alpha} and IFN-{gamma} inhibited TTF-2 mRNA levels in a dose- and time-dependent manner. Furthermore, run-on assays indicated that the decreases induced by TNF-{alpha} and IFN-{gamma} in mRNA levels were accompanied by a decrease in the transcription rate. More importantly, when TNF-{alpha} and IFN-{gamma} were added to the incubation medium together, the time-dependent decrease in TTF-2 gene expression was additive. Published studies report synergistic effects of TNF-{alpha} and IFN-{gamma} upon the enhanced expression of class II MHC antigens (6, 29) as well as suppressed 125I organification and thyroid hormone release (8). We therefore speculate that the synergistic suppression of TG and TPO gene expression by TNF-{alpha} and IFN-{gamma} is involved in decreased thyroid function. We suggest that the suppression of TTF-2 gene expression by combined TNF-{alpha} and IFN-{gamma} results at least in part, in the synergistic effects on TG and TPO, because TTF-2 is one of the most important factors regulating the expression of these genes.

IL-1, IL-6, and TGF-ß also inhibit thyroid-specific gene expression (24, 35, 36, 37), but their mechanisms are not clearly understood. We confirmed that the TNF-{alpha} and IFN-{gamma} actions were specific because other cytokines did not alter the TTF-2 mRNA levels. In addition, other IFNs, such as IFN-{alpha} and IFN-ß, did not decrease the TTF-2 mRNA levels (data not shown). These findings were consistent with a report showing that IFN-{alpha} and IFN-ß does not alter the expression of thyroid-specific genes (38).

Consistent with TTF-2 gene expression, TNF-{alpha} and IFN-{gamma} also decreased levels of TTF-2/DNA complexes. Decreased DNA binding activity would explain the observed decreases in Tg expression. However, there is still a possibility that the protein/DNA complex found with FRTL-5 nuclear extract is a part of this complex. Although a supershift experiment would document this point, TTF-2 antibodies are not available at the present time.

TTF-2 was isolated as a thyroid-specific DNA-binding protein that recognized a single binding site on the minimal promoters of the rat TG (16), and rat TPO genes (17). The recently cloned TTF-2 cDNA encodes a new member of the forkhead family of transcription factors. TTF-2 is transiently expressed in the thyroid and anterior pituitary bud and its mRNA is down-regulated immediately before the onset of thyroid differentiation. In adult thyroid tissue, TTF-2 mRNA is again detectable (20). So far, it is suggested that TTF-2 at least in adult thyroid tissue, functions as a transcriptional activator. Thus, mutagenesis in TG or TPO promoters has shown that TTF-2 interaction with the K or Z region of the TG or TPO promoter, respectively, is indispensable for full promoter activity (18). Second, TSH and insulin-induced up-regulation of TTF-2 gene expression parallels the effect of these factors on TG gene expression (19). Concatemerized TTF-2-binding elements activate the ß-globin promoter in FRTL-5 cells (23, 39). The present study shows that TTF-2 mRNA, like TG mRNA, levels are down-regulated by TNF-{alpha} and IFN-{gamma}. These findings are consistent with the important role of TTF-2 as a mediator of the transcriptional activation of thyroid-specific genes, such as TG.

Received December 3, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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