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Endocrinology Vol. 139, No. 4 1715-1722
Copyright © 1998 by The Endocrine Society

ARTICLES

Activation of Transcriptionally Active Nuclear Factor-{kappa}B by Tumor Necrosis Factor-{alpha} and Its Inhibition by Antioxidants in Rat Thyroid FRTL-5 Cells1

Toyone Kikumori, Fukushi Kambe, Takashi Nagaya, Tsuneo Imai, Hiroomi Funahashi and Hisao Seo

Department of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine (T.K., F.K., T.N., H.S.), Nagoya University, Nagoya 464–01; and Department of Surgery II, Nagoya University School of Medicine (T.K., T.I., H.F.), Nagoya 466, Japan

Address all correspondence and requests for reprints to: Fukushi Kambe, Department of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464–01, Japan. E-mail: kambe{at}riem.nagoya-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor-{alpha} (TNF-{alpha}) exerts pleiotropic effects on thyroid follicular cells. However, the intracellular signaling pathway for the TNF-{alpha} action has not been well elucidated. The present study examined the effects of TNF-{alpha} on the activation of nuclear factor-{kappa} B (NF-{kappa}B) and on the expression of interleukin (IL)-6 gene in rat thyroid FRTL-5 cells. The treatment of the cells with TNF-{alpha} resulted in the nuclear translocation of p65-p50 heterodimer as well as p50-p50 homodimer NF-{kappa}Bs. The treatment with the antioxidants 20 mM N-acetyl-L-cysteine (NAC) and 10 µM pyrrolidine dithiocarbamate (PDTC) inhibited the TNF-{alpha}-dependent activation of p65-p50 heterodimer but not the p50-p50 homodimer, indicating that generation of oxidants is required for the activation of the heterodimer NF-{kappa}B. When the plasmid containing the multimerized NF-{kappa}B sites upstream of a luciferase reporter gene was transfected into FRTL-5 cells, the treatment with NAC or PDTC prevented the TNF-{alpha}-dependent increase in the luciferase activities, indicating that the p65-p50 heterodimer is a transcriptionally active NF-{kappa}B. Accordingly, the TNF-{alpha}-dependent increase in IL-6 messenger RNA and in secretion of the protein was prevented by the treatment with NAC. These results strongly suggest that TNF-{alpha} increases the IL-6 gene expression through the activation of NF-{kappa}B in the thyroid cells, and that antioxidants suppress the TNF-{alpha}-dependent IL-6 expression by inhibiting the activation of the transcriptionally active NF-{kappa}B.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TUMOR NECROSIS FACTOR-{alpha} (TNF-{alpha}) exhibits multiple actions on thyroid follicular cells in vivo and in vitro. The TNF-{alpha} receptors have been identified in human thyroid follicular cells (1), human thyroid carcinoma cells (2, 3), and functional, nontransformed rat thyroid FRTL-5 cells (2). The effects of TNF-{alpha} on the thyroid follicular cells include an inhibition of proliferation (4, 5), a decrease in iodide uptake (6) and the expression of iodothyronine type I 5'-deiodinase (7, 8), and an increase in interferon-{gamma}-dependent expression of human leukocyte antigen class II antigens (9, 10) and the expression of intercellular adhesion molecule-1 (11, 12). Despite a number of effects of TNF-{alpha} on the thyroid follicular cells, the intracellular signaling pathway has not been investigated in detail.

It has been demonstrated in various cell types that the TNF-{alpha} exerts its effects through nuclear factor-{kappa} B (NF-{kappa}B), a transcription factor of dimeric complex. The prototypic NF-{kappa}B dimer consists of the p65 (RelA) and p50 subunits. At least three subunits, p52, c-Rel, and RelB, are identified in addition to p50 and p65 (13, 14). p50 is generated by a proteolytic cleavage of a precursor, p105 (15). In the inactive state, the NF-{kappa}B dimer is sequestrated in the cytoplasm bound to an inhibitory protein, I{kappa}B (16, 17). TNF-{alpha} induces the activation of NF-{kappa}B by promoting the dissociation of I{kappa}B and translocation of free NF-{kappa}B dimer into the nucleus (13, 14, 16, 17). The dissociation is caused by the phosphorylation of I{kappa}B and its subsequent proteolysis (14, 18, 19). Generation of reactive oxygen intermediates (ROI) such as hydrogen peroxide has been suggested to mediate the TNF-{alpha}-induced NF-{kappa}B activation, because antioxidants such as N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) inhibit the activation (20, 21, 22). The activated NF-{kappa}B dimer binds to the regulatory NF-{kappa}B elements in the target genes such as interleukin-6 (IL-6) and controls their transcription (14, 23, 24). Recently, it has been shown that phosphorylation of NF-{kappa}B increases its DNA binding activity (25).

TNF-{alpha} was shown to activate the NF-{kappa}B in human papillary thyroid carcinoma cells (26) and to increase the IL-6 production from thyrocytes prepared from the surgical specimens from patients with Graves’ disease and nonautoimmune thyroid nodule (27). However, no data are available about which subunits of NF-{kappa}B are activated by TNF-{alpha} in the thyroid cells. Also not known in thyroid cells is whether the activation of NF-{kappa}B mediates the TNF-{alpha}-dependent expression of the genes such as IL-6. We thus examined the effects of TNF-{alpha} on the activation of NF-{kappa}B and whether the activation of NF-{kappa}B leads to the expression of IL-6 and p105 genes using FRTL-5 cells. Our results will show that TNF-{alpha} induces the activation of p65-p50 heterodimer as well as p50-p50 homodimer NF-{kappa}Bs, and that the p65-p50 heterodimer is transcriptionally active, leading to the expression of IL-6 and p105 genes. It will be also shown that antioxidants inhibit the activation of p65-p50 heterodimer, thereby reducing their expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Recombinant human TNF-{alpha} (2.5 x 103 U/µg) was obtained from Asahi Chemical Industry Co. Ltd. (Tokyo, Japan). NAC and PDTC were purchased from Boehringer Mannheim (Mannheim, Germany) and Sigma Chemical Co. (St. Louis, MO), respectively. All other chemicals were obtained from Sigma Chemical Co. unless otherwise stated.

Cell culture
FRTL-5 cells (American Type Culture Collection, CRL8305, Rockville, MD) were cultured as previously described (28). When FRTL-5 cells were grown to near confluency, various doses of NAC or PDTC were added to the medium. After a 1-h incubation, TNF-{alpha} was added at a final concentration of 500 U/ml. The cells were incubated for 3 h and then harvested for preparation of nuclear extracts and extraction of total RNA. The culture protocol for luciferase assay and enzyme-linked immunosorbent assay will be described in the following sections.

Nuclear extract preparation
Nuclear extracts were prepared by the method of Schreiber et al. (29) with the following modifications. After washing twice with 10 ml PBS without Ca2+ and Mg2+ [PBS(-), Nissui Pharmaceutical Co., Tokyo, Japan], the cells, in a 75-cm2 culture flask (Falcon 3111; Becton Dickinson, Lincoln Park, NJ), were incubated on ice for 5 min in 10 ml of PBS(-) containing 2 mM EDTA. They were then harvested and pelleted by centrifugation at 1000 x g for 1 min at 4 C. The cell pellet was resuspended by gentle pipetting in 1.3 ml buffer A [10 mM HEPES-KOH, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 2 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM phenylmethanesulfonyl fluoride, and 1 µM pepstatin]. After centrifugation at 12,000 x g for 15 sec at 4 C, the cells were lysed by incubating on ice for 10 min in 100 µl buffer A containing 0.4% Nonidet P-40 and then centrifuged at 12,000 x g for 5 min. The pellet was washed with PBS(-), resuspended in 100 µl buffer C [20 mM HEPES-KOH, pH 7.9, 0.4 M KCl, 2 mM EDTA, 1 mM DTT, 1 mM phenylmethanesulfonyl fluoride, 1 µM pepstatin, and 20% (vol/vol) glycerol] and lysed by freezing and thawing. After centrifugation at 12,000 x g for 5 min at 4 C, the supernatant was used as a nuclear extract. The nuclear extract was aliquoted and stored at -80 C until analysis by electrophoretic mobility shift assay and Western blotting. Protein concentrations of the extracts were determined according to Bradford’s method (30) by the microassay kit (Bio-Rad, Richmond, CA) using the BSA as a standard.

Electrophoretic mobility shift assay (EMSA)
The detailed procedures were described previously (28). In brief, {kappa}Bwt oligonucleotide probe was prepared by annealing the sense and antisense oligonucleotides (5'-TCGAGCAGAGGGGACTTTCCGAG-AG-3' and 5'-TCGACTCTC GGAAAGTCCCCTCTGC-3') containing a canonical NF-{kappa}B binding site (underlined) in mouse Ig{kappa} enhancer (14) and by extending the nucleotides with Klenow enzyme in the presence of [{alpha}-32P]deoxycytidine triphosphate (dCTP) (3000 Ci/mmol; New England Nuclear, Boston, MA). The nuclear extract (10 µg protein) was incubated in a final volume of 20 µl containing 40 mM HEPES-KOH (pH 7.9), 75 mM KCl, 0.2 mM EDTA-NaOH (pH 8.0), 10% (vol/vol) glycerol, 0.5 mM DTT, 2 µg poly (dI-dC) (Pharmacia, Piscataway, NJ), and 0.04–0.06 pmol labeled {kappa}Bwt probe at 25 C for 15 min. The reaction mixture without dye was loaded directly onto a 4% polyacrylamide gel (acrylamide/bisacrylamide, 30:1) in 45 mM Tris-HCl (pH 8.0), 45 mM boric acid and 1 mM EDTA-NaOH (pH 8.0) and then electrophoresed at 160 V at 25 C. The gel was dried on a filter paper and exposed to XAR-5 film (Eastman Kodak, Rochester, NY) at -80 C with an intensifying screen.

The supershift analysis employed antibodies directed against p50, p52, p65, c-Rel and RelB (Santa Cruz Biotechnology, Santa Cruz, CA), and preimmune rabbit serum. Each antibody or preimmune serum (1 µl) was added to the binding reaction mixture before the addition of the labeled probe and incubated for 1 h at 25 C.

The displacement analysis was performed using unlabeled {kappa}Bwt oligonucleotide prepared by annealing the sense and antisense oligonucleotides followed by the extension. Another oligonucleotide {kappa}Bmu with a mutated NF-{kappa}B binding site was also used. The {kappa}Bmu oligonucleotide was prepared by annealing 5'-TCGAGCAGAG CTCACTTTCCGAGAG-3' and 5-TCGACTCTCGGAAAGTGAGCTCTGC-3' (mutated sequences are underlined) according to Edbrooke et al. (31). Each unlabeled oligonucleotide was added at 50-fold molar excess to the binding reaction mixture before the addition of the labeled probe.

Western blot analysis
The nuclear extracts containing 40 µg protein were mixed with 1 vol of a dissociation buffer containing 4% SDS and 10% mercaptoethanol. After denaturation in boiling water for 10 min, the extracts and molecular weight marker (Low; Bio-Rad) were fractionated on a SDS-10% polyacrylamide gel and electroblotted onto a membrane (Hybond-C, super, Amersham Life Sciences, Arlington Heights, IL) at 600 mA (4.0 mA/cm2 gel) for 15 min using Mill Blot-SDE system (Millipore Corp., Bedford, MA). A part of the membrane with the molecular weight marker blotted was stained with Coomassie brilliant blue R. The remaining membrane was soaked for 30 min in a blocking buffer [TBST solution (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween-20) containing 3% BSA]. Then, the membrane was incubated for 30 min with anti-p65 antibody (Santa Cruz Biotechnology) diluted at 1:3000 with TBST. After washing three times with TBST, the membrane was incubated for 30 min with antirabbit-IgG goat IgG conjugated with alkaline phosphatase (Zeimed, San Francisco, CA) diluted at 1:3000 with TBST. After washing three times, it was incubated in a color development solution [10 ml of AP buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2) containing 66 µl 50 mg/ml NBT (nitro blue tetrazolium in 70% dimethylformamide) and 33 µl 50 mg/ml BCIP (5-bromo-4-chloro-3-indolyl-phosphate in dimethylformamide)]. The color development was stopped by the incubation of the membrane in a stop solution (20 mM Tris-HCl, pH 8.0, 5 mM EDTA).

Luciferase assay
Annealing of the {kappa}Bwt sense and antisense oligonucleotides creates the ends, which are identical after dissection with XhoI (CTCGAG) and SalI (GTCGAC). Both 5' ends of the annealed oligonucleotide were phosphorylated by T4 polynucleotide kinase in the presence of 1 mM ATP and ligated with T4 DNA ligase. The multimerized oligonucleotides was digested with both XhoI and SalI to select the oligonucleotide with unidirectional alignment of NF-{kappa}B sites. An oligonucleotide having three NF-{kappa}B sites in tandem was purified by PAGE. The purified oligonucleotide was ligated into XhoI site of pGL3pro plasmid (pGL3 promoter; Promega, Madison, WI). The direction of the NF-{kappa}B sites was verified by DNA sequencing. This plasmid was named pGL3–3{kappa}Bpro. FRTL-5 cells (1 x 105 cells/well) were plated in six-well dishes (Falcon 3046) and were cultured for 3 days. The pGL3pro or pGL3–3{kappa}Bpro (2 µg DNA/well) was transfected into the cells using a LipofectAMINE reagent (GIBCO-BRL, Grand Island, NY). After a 16-h incubation of the cells with the liposome-DNA solution, 2 x fresh medium was added to the solution. After an additional 24-h incubation, various doses of NAC and PDTC were added to the medium. After a 1-h incubation, TNF-{alpha} (500 U/ml) was added, and the cells were incubated for 6 h. The cells were then harvested, and the luciferase activities in the cell lysates were determined by a luminometer (LB9501, Laboratorium Prof. Dr. Berthold GmbH & Co. KG, Bad Wildbad, Germany) and normalized with the protein contents of the cell lysates.

Northern blot analysis
Total RNA was prepared by the acid guanidinium thiocyanate-phenol-chloroform-extraction method (32). The detailed procedures for Northern blotting were described in our previous report (33). After fractionation of 15 µg total RNA on 0.8% agarose gel, the RNA was transferred onto GeneScreen Plus membrane (New England Nuclear). The membrane was hybridized with rat IL-6 and p105 complementary DNA (cDNA) probes labeled with [{alpha}-32P]dCTP (3000 Ci/mmol; New England Nuclear) using a random primed labeling kit (Boehringer Mannheim). Rat IL-6 cDNA was cloned by RT-PCR from the messenger RNA (mRNA) prepared from TNF-{alpha}-treated FRTL-5 cells. The primers for IL-6 were 5'-TTCCCTACTTCACAAGTCCG-3' and 5'-AGCCACTCCTTCTGTGACTC-3'. The amplified product was ligated to pGEM-T plasmid (TA cloning system, Promega). The authenticity of cDNA was verified by DNA sequencing. Rat p105 cDNA was provided by Dr. T. Okamoto (Nagoya City University School of Medicine, Nagoya, Japan). The mRNA levels were normalized by glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mRNA levels. The cloning of GAPDH cDNA was described in our previous report (34). After hybridization, the membrane was washed and exposed to XAR-5 film (Eastman Kodak) at -80 C with an intensifying screen. Quantitative analysis of the density of the specific bands were performed using BAS 2000 bioimage analyzing system (Fuji film Co., Tokyo, Japan).

RT-PCR
The detailed procedure of RT-PCR was previously described (35). In brief, RT was performed in 20 µl solution containing 10 µg total RNA and 100 pmol random d(N)6 primer (Takara Shuzo Co., Osaka, Japan). PCR was performed in 25 µl solution containing 1 µl RT-product, 5 pmol sense and antisense primers, and 2 µCi [{alpha}-32P]dCTP (3000 Ci/mmol, New England Nuclear). The primers for amplification of IL-6 were the same as those described above. The ß-actin primers were 5'-ACCTTCAACACCCCAGCCATG-3' and 5'-GGCCATCTCTTGCTCGAAG-TC-3'. Twenty five and thirty cycles were performed for ß-actin and IL-6 cDNA amplification, respectively; each cycle consisted of denaturation at 98 C for 15 sec, annealing at 55 C for 40 sec, and extension at 72 C for 45 sec. The numbers of cycles for each PCR were selected so that each product was within the exponential increase (35). Five microliters labeled PCR product was loaded onto a 4% polyacrylamide gel (acrylamide/bisacrylamide, 30:1) in 45 mM Tris-HCl (pH 8.0), 45 mM boric acid, and 1 mM EDTA-NaOH (pH 8.0) and electrophoresed at 100 V for 2 h at room temperature. The gel was dried on a filter paper and then exposed to XAR-5 film (Eastman Kodak) at -80 C with an intensifying screen. To confirm the authenticity of the amplified cDNAs, they were ligated to pGEM-T vector (Promega) and subjected to DNA sequencing.

Enzyme linked immunosorbent assay (ELISA)
FRTL-5 cells (1 x 105 cells/well) were plated in six-well dishes (Falcon 3046) and cultured in 1 ml medium for 3 days. NAC (20 mM) was added to the medium 1 h before the TNF-{alpha} addition. Then, TNF-{alpha} (500 U/ml) was added, and the cells were incubated for 6 h. The concentrations of IL-6 in the media were determined by a rat IL-6 ELISA kit (TOYOBO, Osaka, Japan). It was confirmed that the presence of 20 mM NAC did not affect the assay.

Statistical analysis
Results from different treatment groups were subjected to one-way ANOVA, and Student’s t test was applied to the evaluation of statistical significance. P values < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activation of NF-{kappa}B by TNF-{alpha} in FRTL-5 cells
Effects of TNF-{alpha} and the antioxidants NAC and PDTC on the activation of NF-{kappa}B in FRTL-5 cells were examined by EMSA. As shown in Fig. 1AGo, no apparent protein-{kappa}Bwt complex was observed when the nuclear extracts prepared from the untreated cells were examined. The treatment of the cells with NAC induced the activation of a single NF-{kappa}B-{kappa}Bwt complex. In contrast, TNF-{alpha} induced the activation of two distinct complexes: a fast-migrating complex, which exhibited the mobility similar to that induced by NAC, and an additional slow-migrating complex. The treatment with TNF-{alpha} together with NAC resulted in the activation of only the fast-migrating complex, indicating that NAC inhibited the TNF-{alpha}-dependent activation of the slow-migrating complex.



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  		Figure 1. Activation of NF-{kappa}B by TNF-{alpha} in FRTL-5 cells is modified by antioxidants. NAC (20 mM) (A) or PDTC (10 µM) (B) was added to culture medium 1 h before TNF-{alpha} addition. Then TNF-{alpha} (500 U/ml) was added, and cells were incubated for 3 h. Experiment was performed in duplicate. DNA binding activities of NF-{kappa}B in nuclear extracts prepared from cells untreated (lanes 1 and 2), treated with antioxidant alone (lanes 3 and 4), treated with TNF-{alpha} alone (lanes 5 and 6), and treated with TNF-{alpha} and antioxidant (lanes 7 and 8) were determined by EMSA using {kappa}Bwt oligonucleotide as a probe. Arrows indicate NF-{kappa}B/{kappa}Bwt complexes. Similar results were obtained from several separate experiments.

 
Another antioxidant, PDTC, had a similar effect on the TNF-{alpha}-dependent induction of NF-{kappa}Bs in FRTL-5 cells (Fig. 1BGo). PDTC also inhibited the TNF-{alpha}-dependent activation of the slow-migrating complex. In addition, a weak activation of the fast-migrating complex was also observed by the treatment with PDTC alone.

Dose effects of the antioxidants on the activation of NF-{kappa}B are summarized in Fig. 2Go. NAC or PDTC alone induced the activation of the fast-migrating complex in a dose-dependent manner. The minimum concentration required for the apparent induction of the fast-migrating complex by NAC was 2 mM and 10 µM by PDTC. As demonstrated in Fig. 1Go, NAC (10 mM and 20 mM) and PDTC (more than 10 µM) inhibited the TNF-{alpha}-induced activation of the slow-migrating complex. To our surprise, the amount of the fast-migrating complex was increased in the presence of NAC, whereas it was decreased in the presence of PDTC in a dose-dependent manner.



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  		Figure 2. Dose effects of antioxidants on activation of NF-{kappa}B by TNF-{alpha}. Various doses of NAC (A) or PDTC (B) were added to culture medium of FRTL-5 cells 1 h before TNF-{alpha} addition. Then TNF-{alpha} (500 U/ml) was added, and cells were incubated for 3 h. DNA binding activities of NF-{kappa}B in nuclear extracts prepared from cells that were untreated (lane 1), treated with increasing doses of antioxidant alone (lanes 2–4), treated with TNF-{alpha} alone (lane 5), and treated with increasing doses of antioxidant and TNF-{alpha} (lanes 6–8) were determined by EMSA using {kappa}Bwt oligonucleotide as a probe. Arrows indicate NF-{kappa}B/{kappa}Bwt complexes. Similar results were obtained from a separate experiment.

 
Characterization of NF-{kappa}B subunits activated by TNF-{alpha} and antioxidant
Supershift analysis using specific antibodies against each subunit of NF-{kappa}B was employed to characterize NF-{kappa}B subunits activated by TNF-{alpha} and NAC. As shown in Fig. 3AGo, NAC-induced, fast-migrating complex was supershifted only by anti-p50 antibody, indicating that the complex represents p50-p50 homodimer NF-{kappa}B. On the other hand, anti-p50 antibody supershifted both fast- and slow-migrating complexes induced by TNF-{alpha}, whereas anti-p65 antibody supershifted only slow-migrating complex (Fig. 3BGo). Thus, the slow- and fast-migrating complexes represent p65-p50 heterodimer and p50-p50 homodimer NF-{kappa}Bs, respectively. The fast-migrating complex observed in the nuclear preparation from the cells treated with both TNF-{alpha} and NAC was supershifted only by anti-p50 antibody (Fig. 3CGo), indicating that this complex represents p50-p50 homodimer NF-{kappa}B. The antibodies against p52, c-Rel and RelB, and preimmune rabbit serum did not affect the mobility of these complexes. Displacement analysis showed that the fast- and slow-migrating complexes disappeared in the presence of excess of the unlabeled {kappa}Bwt oligonucleotide, whereas the {kappa}Bmu oligonucleotide did not affect the complexes.



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  		Figure 3. Characterization of NF-{kappa}B subunits induced by NAC and TNF-{alpha}. Nuclear extracts of FRTL-5 cells treated with 20 mM NAC (A), 500 U/ml TNF-{alpha} (B), and both (C) were subjected to EMSA using a labeled {kappa}Bwt oligonucleotide as a probe. Subunits of NF-{kappa}Bs were characterized by supershift analysis employing antibodies directed against p50, p52, p65, c-Rel and RelB, and preimmune rabbit serum (pre). Displacement analysis was performed using 50-fold molar excess of unlabeled {kappa}Bwt and {kappa}Bmu oligonucleotides as competitors

 
To examine whether the changes in p65-p50 heterodimer binding by TNF-{alpha} and NAC are associated with the nuclear translocation of p65, Western blot analysis was performed using nuclear extracts. As shown in Fig. 4Go, a single band of approximately 65 kDa was detected in the TNF-{alpha}-treated nuclear extract, whereas the extract from the untreated cells showed no corresponding band, indicating that TNF-{alpha} induces the nuclear translocation of p65. Combined treatment with NAC and TNF-{alpha} resulted in a marked reduction of the band density, indicating that NAC inhibits the TNF-{alpha}-dependent translocation of p65 into the nucleus.



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  		Figure 4. NAC inhibits nuclear translocation of p65 induced by TNF-{alpha}. Nuclear extracts of FRTL-5 cells that were untreated (lane 1) and treated with 20 mM NAC (lane 2), 500 U/ml TNF-{alpha} (lane 3), and both (lane 4) were subjected to Western blot analysis using anti-p65 antibody. Positions of molecular weight markers, ovalbumin (45,000), BSA (66,000) and phosphorylase B (97,000), are indicated. Similar results were obtained from a separate experiment.

 
Taken together, these results demonstrate that TNF-{alpha} induces the activation of both p65-p50 heterodimer and p50-p50 homodimer NF-{kappa}Bs in FRTL-5 cells, and that the antioxidants inhibit the activation of p65-p50 heterodimer NF-{kappa}B.

p65-p50 heterodimer NF-{kappa}B-dependent transactivation of a luciferase reporter gene
The luciferase reporter plasmid, pGL3pro or pGL3–3{kappa}Bpro containing three NF-{kappa}B sites upstream of a luciferase reporter gene, was transfected into FRTL-5 cells, and the effects of TNF-{alpha} and antioxidants on the reporter gene expression were examined. As shown in Fig. 5Go, when the pGL3pro was transfected into the cells, no significant increase in the luciferase activities was observed by the addition of TNF-{alpha}, NAC, and PDTC, each alone or in combination. In contrast, when the pGL3–3{kappa}Bpro was transfected, TNF-{alpha} significantly increased the luciferase activities. The treatment with 2 mM NAC did not affect the TNF-{alpha}-dependent increase in the luciferase activities. However, 20 mM NAC markedly attenuated the increase. NAC alone did not significantly alter the luciferase activities. The treatment with 10 µM and 100 µM PDTC also markedly attenuated the TNF-{alpha}-dependent increase in the luciferase activities. PDTC alone did not significantly alter the luciferase activities.



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  		Figure 5. Antioxidants attenuate NF-{kappa}B-dependent increase in expression of luciferase reporter gene. Upper panel schematically depicts luciferase reporter gene constructs, pGL3–3{kappa}Bpro and pGL3pro. pGL3–3{kappa}Bpro (open column) or pGL3pro (hatched column) was transfected into FRTL-5 cells. Forty hours after transfection, NAC (2 mM and 20 mM) or PDTC (10 µM and 100 µM) was added to medium. After a 1-h incubation, TNF-{alpha} (500 U/ml) was added, and cells were incubated for 6 h. Luciferase activities in cell lysates were determined by a luminometer and normalized with protein contents of cell lysates. Experiment was carried out in triplicate. Values were expressed in arbitrary luciferase units relative to those of untreated cells and as mean ± SD. Similar results were obtained from a separate experiment.

 
Taken together with the EMSA studies, these results indicate that the attenuation of TNF-{alpha}-induced increase in the luciferase activities by the antioxidants is associated with a lack of p65-p50 heterodimer activation, and that NAC- or PDTC-induced activation of p50-p50 homodimer does not result in an increase in the reporter gene expression. Therefore, it is strongly suggested that TNF-{alpha} transactivates the luciferase gene only through the p65-p50 heterodimer NF-{kappa}B. It is thus likely that the TNF-{alpha}-dependent expression of endogenous NF-{kappa}B-responsive genes such as IL-6 and p105 is prevented by the antioxidants. This possibility was examined in the following experiments.

Inhibition of TNF-{alpha}-dependent IL-6 and p105 gene expression by antioxidant
The time course of TNF-{alpha}-dependent increase in the IL-6 mRNA level is shown in Fig. 6Go. The maximal increase in IL-6 mRNA was observed at 3 h after TNF-{alpha}. The levels of GAPDH mRNA were not changed by TNF-{alpha} (data not shown). Thus, in the following experiments, the duration of TNF-{alpha} treatment was fixed at 3 h. As shown in Fig. 7Go, the TNF-{alpha}-dependent increase in IL-6 mRNA was completely inhibited by the treatment with 10 mM and 20 mM NAC, whereas 2 mM NAC did not prevent the increase. In contrast, the ß-actin mRNA levels were not altered by NAC. This inhibitory effect of NAC correlated well with the absence of p65-p50 heterodimer binding presented in Fig. 2Go.



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  		Figure 6. Time course of IL-6 mRNA expression by TNF-{alpha}. Total RNA was extracted from FRTL-5 cells treated with TNF-{alpha} (500 U/ml) for 3, 6, and 12 h and subjected to Northern blot analysis using IL-6 and GAPDH cDNAs as probes. Experiment was performed in duplicate. Radioactivities of specific bands were quantitated by BAS 2000. IL-6 mRNA levels were normalized by GAPDH mRNA levels and expressed as mean ± range of two values in arbitrary units.

 


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  		Figure 7. Dose effects of NAC on TNF-{alpha}-dependent increase in IL-6 mRNA. Various doses of NAC were added to culture medium of FRTL-5 cells 1 h before TNF-{alpha} addition. Then TNF-{alpha} (500 U/ml) was added, and cells were incubated for 3 h. IL-6 and ß-actin mRNA levels were determined by RT-PCR. Experiment was performed in duplicate. Similar results were obtained from a separate experiment.

 
The effect of NAC on the IL-6 expression in FRTL-5 cells was studied using 20 mM NAC (Fig. 8Go). The treatment with NAC alone did not affect the basal level of IL-6 mRNA. TNF-{alpha} increased the mRNA level, and NAC inhibited this increase. Consistent with the alteration in the mRNA levels, IL-6 secretion from the FRTL-5 cells to the medium was markedly increased by TNF-{alpha}, and this increase was significantly attenuated by the treatment with NAC.



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  		Figure 8. NAC inhibits TNF-{alpha}-dependent increase in IL-6 expression. A, NAC (20 mM) was added to culture medium of FRTL-5 cells 1 h before TNF-{alpha} addition. Then TNF-{alpha} (500 U/ml) was added, and cells were incubated for 3 h. IL-6 and ß-actin mRNA levels in cells untreated (lanes 1 and 2), treated with NAC alone (lanes 3 and 4), treated with TNF-{alpha} alone (lanes 5 and 6), and treated with NAC and TNF-{alpha} (lanes 7 and 8) were determined by RT-PCR. Experiment was performed in duplicate. B, IL-6 concentrations in media obtained after a 6-h incubation with NAC and/or TNF-{alpha} were determined by ELISA. Experiment was performed in triplicate. Values were expressed as mean ± SD *, P < 0.05 vs. TNF-{alpha} alone. Similar results were obtained from a separate experiment.

 
When we examined the effects of NAC and TNF-{alpha} on the expression of another NF-{kappa}B-responsive gene, p105, results similar to those on IL-6 expression were obtained. As shown in Fig. 9Go, NAC alone did not affect the basal level of p105 mRNA. TNF-{alpha} increased the mRNA level and NAC markedly reduced the TNF-{alpha}-dependent increase in the p105 mRNA level. The GAPDH mRNA levels were not altered by NAC, TNF-{alpha}, or both.



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  		Figure 9. NAC inhibits TNF-{alpha}-dependent increase in p105 mRNA. After a 1-h incubation with NAC (20 mM), TNF-{alpha} (500 U/ml) was added, and FRTL-5 cells were incubated for 3 h. Aliquots of 15 µg total RNA were subjected to Northern blot analysis using p105 and GAPDH cDNAs as probes. Experiment was performed in duplicate. Size of each mRNA is indicated in right side. Similar results were obtained from a separate experiment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates for the first time that TNF-{alpha} induces the activation of two distinct types of NF-{kappa}Bs, p65-p50 heterodimer and p50-p50 homodimer, in the functional rat thyroid FRTL-5 cells. It is also shown that the p65-p50 heterodimer is transcriptionally active NF-{kappa}B, whereas the p50-p50 homodimer is unable to transactivate the reporter gene containing NF-{kappa}B sites and the IL-6 and p105 genes. These results are compatible with the previous findings that p65-p50 heterodimer functions as a potent activator of gene expression (36, 37), whereas p50-p50 homodimer cannot do, because p50 lacks an identifiable transactivation domain in the molecule (36, 38, 39).

In addition, it is strongly suggested that the IL-6 gene expression in the thyroid cells is increased by TNF-{alpha} through the activation of NF-{kappa}B. It is now established that TNF-{alpha} is secreted from the activated macrophages infiltrating into the thyroid gland of patients with autoimmune thyroid diseases (40, 41), and that IL-6 promotes B cell differentiation and T cell activation (42). It is thus possible that TNF-{alpha} increases the IL-6 production from the thyroid follicular cells and thereby stimulates the intrathyroidal autoimmune response in these patients.

It is also shown that an antioxidant, NAC, attenuated the TNF-{alpha}-dependent IL-6 and p105 gene expression by inhibiting the activation of p65-p50 heterodimer NF-{kappa}B in thyroid cells. NAC enters cells readily and scavenges ROI directly or indirectly by its conversion to L-cysteine and by increasing intracellular glutathione (43, 44). The inhibition of the heterodimer NF-{kappa}B activation by NAC could be due to this scavenging action, suggesting that generation of ROI mediates the TNF-{alpha}-dependent activation of the p65-p50 heterodimer NF-{kappa}B in the thyroid cells.

Although constitutive activation of p50-p50 homodimer has been shown in certain cell types (45, 46), the homodimer activation by antioxidant has not been reported. To our surprise, in FRTL-5 cells, NAC or PDTC alone activated the p50-p50 homodimer in a dose-dependent manner. The maximum dose of 20 mM NAC has been shown to be sufficient to inhibit the NF-{kappa}B activation in other cell types (47). PDTC at concentrations of more than 10 µM has also been shown to inhibit the NF-{kappa}B activation (47). Although further studies are required to clarify the mechanism of the activation of the homodimer by the antioxidants, it is possible that they may increase the degradation of p105 in FRTL-5 cells, resulting in the generation of p50 (15).

A striking difference in the activation of the p50-p50 homodimer by TNF-{alpha} was observed in the presence of either NAC or PDTC. NAC seemed to enhance the activation of the homodimer by TNF-{alpha}, whereas PDTC inhibited the activation. Because PDTC but not NAC possesses metal-chelating properties (48), the metal ion could be involved in the NAC-mediated enhancement of the p50-p50 homodimer activation by TNF-{alpha}.

Although the present study indicates that the p65-p50 heterodimer functions as an activator of gene expression in agreement with the previous reports (36, 37), the function of the p50-p50 homodimer activated by the antioxidants or by TNF-{alpha} is elusive. By employing transient transfection assays, p50-p50 homodimer has been shown to either activate (49, 50) or repress the transcription induced by NF-{kappa}B (39, 51). The basal expression of two endogenous genes, IL-6 and p105, in FRTL-5 cells was not increased nor decreased by the induction of the p50-p50 homodimer by NAC, indicating that the homodimer does not affect the basal transcription of both genes. However, we could not exclude the possibility that the homodimer has an inhibitory effect on the heterodimer function, because the simultaneous activation of both forms of NF-{kappa}B by TNF-{alpha} makes it impossible to determine the extent of the inhibitory effect of the homodimer.

In addition to a function of ROI as an intracellular mediator for signal transduction, it is well known that ROI directly modifies cellular lipids and proteins, and impairs their functions. Excess production of ROI is postulated to mediate the iodide-induced thyroid cell injury, which is followed by infiltration of immune cells and leads to the development of autoimmune thyroiditis. Recently, Bagchi et al. (52) showed that administration of antioxidant delay the onset and severity of autoimmune thyroiditis induced by iodine in obese strain (OS) chickens, which are genetically susceptible to spontaneous autoimmune thyroiditis. This effect of antioxidant may be attributable to its scavenging action of excess ROI, thereby reducing ROI-dependent cell injury. Our present findings may suggest another aspect of the antioxidant action. Inhibition of activation of transcriptionally active p65-p50 heterodimer by antioxidant may decrease the expression of the NF-{kappa}B-responsive genes such as cytokines and immunoreceptors (24, 37) in thyroid follicular cells, attenuating the intrathyroidal immune response.


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

Received August 13, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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