Increased binding of IFN regulating factor 1 mediates the synergistic induction of CIITA by IFN-{gamma} and tumor necrosis factor-{alpha} in human thyroid carcinoma cells

Michal A. Rahat, Inessa Chernichovski and Nitza Lahat

Immunology Research Unit, Carmel Medical Center, 7 Michal Street, Haifa 34362, and the Faculty of Medicine, Technion, Haifa, Israel

Correspondence to: M. A. Rahat


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Expression of MHC class II molecules is restricted to professional antigen-presenting immune cells, but it can be induced by IFN-{gamma} in other cell types. Thyroid cells have been shown to induce class II expression (mainly HLA-DR) following stimulation with IFN-{gamma} and addition of tumor necrosis factor (TNF)-{alpha} synergistically enhanced this expression. Class II transactivator (CIITA) has been implicated as the master regulator of MHC class II molecules and its transcription has been shown to be regulated from four different promoters, one of which is responsible for its induction by IFN-{gamma}. The aim of this study was to find whether CIITA is synergistically induced by IFN-{gamma} and TNF-{alpha} in the human thyroid MRO-87-1 cell line, and to investigate the molecular mechanisms responsible for this synergism. We have demonstrated that IFN-{gamma} and TNF-{alpha} synergistically induce HLA-DR{alpha} and CIITA mRNAs, but prolonged incubation resulted in the inhibition of CIITA mRNA accumulation. Several potential mechanisms that could explain the synergistic effect were explored. NF-{kappa}B did not bind the CIITA inducible promoter and addition of SN50, which inhibits NF-{kappa}B translocation to the nucleus, did not change the synergistic effect. Furthermore, IFN-{gamma} did not induce I{kappa}B{alpha} degradation. Synergistic activation of signal transducer and activator of transcription (STAT)-1 or IFN regulating factor (IRF)-1 was not observed, and STAT-1 did not bind the CIITA inducible promoter. IRF-1, although not synergistically induced or activated, bound synergistically to its specific cis element on the CIITA type IV promoter. Thus we propose that IRF-1 binding mediates the synergistic induction of HLA-DR{alpha} and CIITA in thyroid cells.

Keywords: class II transactivator, HLA-DR, IFN-{gamma}, IFN regulating factor-1, NF-{kappa}B, thyroid, tumor necrosis factor-{alpha}, signal transducer and activator of transcription-1, USF-1


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Expression of the MHC or the HLA class II molecules is critical for antigen presentation leading to the generation of an adaptive immune response. MHC class II expression is restricted to immune cells (e.g. B cells, monocytes/ macrophages, dendritic cells and activated T cells) and is tightly regulated, since changes in its concentration may determine both the type and magnitude of immune responses (13). However, MHC class II molecules may be expressed on non-immune tissue cells during an autoimmune disease (4) or in malignancy (5). Thyroid epithelial cells can be induced to express MHC class II following incubation with IFN-{gamma}, which is secreted in vivo by activated T cells, and thus partly render them capable of antigen presentation, promoting presentation of either tumor antigens or autoantigens (68). Tumor necrosis factor (TNF)-{alpha}, which is secreted mostly by activated macrophages during immune-mediated inflammatory responses, cooperates with IFN-{gamma} to regulate these processes. For example, IFN-{gamma} and TNF-{alpha} can synergistically enhance MHC class II gene expression in many cell types, including thyrocytes (8).

The expression of MHC class II molecules is regulated mainly at the transcriptional level. Three short conserved cis elements, termed W, X and Y boxes, located in all MHC class II promoters, mediate the coordinated and cooperative binding of the transcription factors RFX, NFY and CREB (reviewed in 913,14). An additional non-DNA binding transcription factor termed class II transactivator (CIITA) is necessary for both the constitutive and IFN-{gamma}-induced expression of MHC class II molecules (15,16), and has been suggested to be the `master regulator' responsible for the coordinated expression of their genes (17). CIITA interacts with RFX, NFY and CREB (18), and together they form the enhanceosome that governs MHC class II transcription (19). The expression of CIITA itself is regulated by multiple promoters. One promoter regulates its constitutive expression in dendritic cells, another is specific for the constitutive expression in B cells and a third promoter (termed type IV) controls the inducible expression of CIITA in multiple cell types (20). In a melanoma cell line, which expresses HLA-DR only after induction with IFN-{gamma}, expression of CIITA was derived only from promoter IV (20). Although the question of promoter utilization has not yet been addressed in thyroid cells, it is likely that promoter IV is responsible for CIITA induction, in a manner analogous to that of the melanoma cell line. CIITA promoter IV has been sequenced and several of its cis elements have been identified as critical for IFN-{gamma} induction, including the IFN-{gamma} activating sequence (GAS), the E-box and an IFN regulating factor (IRF) element (20). These elements bind the transcription factors signal transducer and activator of transcription (STAT)-1, USF-1 and IRF-1 respectively (21). In addition, the human CIITA promoter type IV includes a putative NF-{kappa}B site overlapping with an AP-2 site, as well as a NF-GMa site (22). Regulation of CIITA and MHC class II expression has been shown to involve yet another protein called IK, which functions as an inhibitor of IFN-{gamma}-induced expression of MHC class II molecules (23,24). IK transfection into B cells caused the disappearance of the constitutive expression of MHC class II as well as reduction in CIITA mRNA (23,24).

We have previously demonstrated that expression of HLA-DR on the surface of primary non-neoplastic thyroid cells and a malignant follicular thyroid cell line (MRO-87-1) could be induced by IFN-{gamma} in a dose-dependent way (25,26). Moreover, in both the primary and malignant thyroid cells, IFN-{gamma} induced HLA-DR{alpha} mRNA accumulation (27) and TNF-{alpha} synergistically enhanced this expression (25). In addition, IFN-{gamma} enhanced CIITA mRNA accumulation in untransformed thyrocytes (28). In this study we have shown that in the thyroid cell line this synergism was mediated by the accumulation of CIITA mRNA and that increased binding of the IRF-1 could have a role in mediating the synergistic effect. Other possible mechanisms have been ruled out, as no synergistic effect had been observed for the phosphorylation or expression of IRF-1 and STAT-1, and NF-{kappa}B did not bind the CIITA type IV promoter.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell line
The human thyroid carcinoma cell line MRO-87-1, derived from follicular carcinoma, was kindly provided by Dr G. J. F. Juillard (UCLA, Los Angeles, CA). The cell line was cultured in RPMI 1640 supplemented with 10% FCS and antibiotics. Between 3x106 and 5x106 cells were incubated for different time periods in the presence or absence of 100 U/ml IFN-{gamma} (Sigma, St Louis, MO), 20 ng/ml TNF-{alpha} (Sigma) or their combination. Cells were then harvested for RNA extraction, nuclear protein extraction or Western blot analysis. In addition, MRO-87-1 cells were incubated with several concentrations of the synthetic peptide SN50 (Calbiochem, San Diego, CA), which functions as a direct inhibitor of NF-{kappa}B translocation (29), 30 min before addition of IFN-{gamma} and TNF-{alpha}.

RNA extraction and Northern blot analysis
Total RNA was extracted using TriReagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. RNA samples were denatured at 65°C for 15 min in 2 M formaldehyde and 50% formamide, and then fractionated by electrophoresis (20–25 µg/lane) in a 1% agarose gel containing 0.66 M formaldehyde and MOPS buffer. After separation the RNA was transferred to a nylon blotting membrane (Sartolon; Sartorius, AG, Gottingen, Germany). HLA-DR and GAPDH mRNA were detected by hybridization to specific [{gamma}-32P]ATP 5'-end-labeled oligonucleotide probes. After stringent washes the membranes were autoradiographed. Steady-state mRNA was quantified using the BioImaging system (Dinco & Renium, Jerusalem, Israel) and TINA software (Raytest, Straubenhardt, Germany), and normalized to GAPDH.

Semi-quantitative RT-PCR analysis
RNA (1 µg) was reverse transcribed at 37°C for 1 h using 200 µM deoxynucleotides (Sigma), 5 µM random hexamers (Amersham Pharmacia Biotech, Piscataway, NJ), 20 U RNAguard (Amersham) and 20 U/µg of MMLV-RT (US Biochemicals, Cleveland, OH). The amplification reaction consisted of 0.5 µM of each primer, 100 µM of dNTPs (Sigma), 0.5 U of Taq polymerase (Roche Molecular Biochemicals, Mannheim, Germany), 1 µg of Flash anti-Taq (Chimerx, Milwaukee, WI) and 12–750 ng of the reversed-transcribed RNA, in a final volume of 10 µl. Amplification of CIITA was carried out as described before (24), with 32 cycles of 94°C for 30 s and 72°C for 1 min. Similarly, amplification of GAPDH was carried out for 30 cycles with the annealing temperature at 58°C for 30 s and amplification of IK was carried out for 37 cycles with the annealing temperature at 52°C for 30 s. The linear range of amplification was determined for each transcript (Fig. 2AGo–C) and amplification was performed within that linear range. To allow for relative comparison between the samples, the results were normalized for GAPDH expression and for the amounts of amplified RNA.





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Fig. 2. RT-PCR amplification of CIITA, IK and GAPDH (n = 7). Titration analysis was performed by making serial dilutions of total RNA before reverse transcription and amplification. MW, mol. wt marker (100-bp ladder); mix, negative control without RNA. Densitometric analysis was performed for the (A) CIITA mRNA (B), IK mRNA (C) and GAPDH mRNA, and the linear range of amplification for each transcript was determined.

 
Western blot analysis
Proteins were extracted at different time periods by using SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% w/v SDS, 10% glycerol and 0.1% w/v bromophenol blue). Equal amounts of protein extracts were loaded on a 7 or 10% SDS–PAGE. After electrophoretic separation, the proteins were transferred and fixed onto a cellulose nitrate membrane (Schleicher & Schuell, Dassel, Germany) in transfer buffer (25 mM Tris, 180 mM glycine and 20% methanol, pH 8.3). The membrane was probed with either rabbit polyclonal antibody directed against STAT-1 or with a rabbit polyclonal phospho-specific STAT-1, which recognized the phosphorylated tyrosine residue at location 701 (PhosphoPlus STAT-1 antibody kit; New England BioLabs, Beverly, MA). Likewise we have used the rabbit polyclonal antibody directed against I{kappa}B{alpha} and the rabbit polyclonal phospho-specific I{kappa}B{alpha}, which recognized the phosphorylated serine residue at location 32 (PhosphoPlus I{kappa}B{alpha} antibody kit; New England BioLabs). Alternatively we used rabbit polyclonal antibody directed against IRF-1 or the rabbit polyclonal antibody directed against RelA (p65) (Santa Cruz Biotechnologies, Santa Cruz, CA). The cellulose nitrate membrane was incubated for 1 h in blocking buffer (5% skimmed milk powder, 1% BSA, 0.01% Tween 20, 10 mM Tris, pH 8.0 and 150 mM NaCl) at room temperature. The primary antibodies were diluted 1:1000 in blocking buffer and incubated for 1 h at room temperature, and then washed 3 times in 1xTBST (10 mM Tris, pH 8.0, 150 mM NaCl and 0.5% Tween 20). The secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA), was diluted 1:5000 in blocking buffer, incubated for an additional 1 h at room temperature and then washed 3 times with 1xTBST. The enhanced chemiluminescence (ECL) system (Amersham) was used for detection.

Electrophoretic mobility shift assays (EMSA)
Nuclear extracts were prepared according to Schaffner (30). Cells (5x106) were washed with cold PBS, collected and centrifuged, and the pellet of cells was resuspended in 400 µl of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1 mM EDTA, 0.6% NP-40, 1 mM DTT, 1 mM PMSF, 10 µg/ml leupeptin and 50 µg/ml aprotinin). Cells were allowed to swell on ice for 15 min and then underwent lysis by vigorously shaking. The nuclei were centrifuged and the nuclear fractions were then resuspended in buffer C (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 10 µg/ml leupeptin and 50 µg/ml aprotinin). The nuclei were incubated on ice for an additional 15 min and pelleted. The amount of protein in the supernatants was determined by Bradford reagent (BioRad, Hercules, CA) and the nuclear extracts were kept in aliquots at –70°C. The inducible promoter of CIITA (type IV) was amplified from genomic DNA extracted from MRO-87-1 cells. The reaction consisted of 0.5 µM of each specific primer, 100 µM of dNTPs, 0.5 U Taq polymerase, 4 µl of [{alpha}-32P]dATP and 300 ng DNA. Amplification was performed for 40 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 30 s. The radioactively labeled PCR product (304 bp) was purified on 6% PAGE, eluted from the gel and then ethanol precipitated. Several oligonucleotides containing the binding sequence of NF-{kappa}B/AP-2, STAT-1/USF-1 and IRF-1 within the inducible promoter of CIITA were synthesized, and 5'-end-labeled with polynucleotide kinase and [{gamma}-32P]ATP. The labeled oligonucleotides were hybridized by boiling and slow cooling, and were then used for EMSA. The whole labeled promoter or the labeled boxes (~20,000 c.p.m., 2 ng) were incubated with 6 µg of the nuclear extracts in a binding reaction [15 mM HEPES, pH 7.9, 90 mM KCl, 6% glycerol, 5 µg poly(dI·dC) and 3mM DTT] for 30 min in room temperature. DNA–protein complexes were separated on native 4–6% PAGE, and the gel was dried and autoradiographed overnight. Alternatively, nuclear extracts were pre-incubated with specific antibodies for 30 min at 4°C before addition of the components of the binding reaction or with specific oligonucleotide competitors at 100-fold molar excess of unlabeled boxes.

Oligonucleotides
HLA-DR{alpha} probe: 5'-CCTCAGTTGAGGGCAGGAAGGGGAGATAGTGG; GAPDH probe: 5'-CGGAAGGCCA TGCCAGTGAGCTTCCCGT; CIITA primers (adopted from 24), sense: 5'-CAGGCTGTTGTGTGACATGGAAGGT, antisense: 5'-TGGAGAAAGGCATGGGAATCTGGTC; IK primers (adopted from 23), sense: 5'-ACCAAGACACCTCGGGACAA, antisense: 5'-CCAGCAGACCCAGCAAACTT; GAPDH primers, sense: 5'-ACCACAGTCCATGCCATCAC, antisense: 5'-TCCACCACCCTGTTGCTGTA; CIITA type IV promoter, sense: 5'-AGCTCCCTGCAACTCAGGACTTGC, antisense: 5'-TGGCAGCTCGTCCGCTGGTC; IRF-1 box, sense: 5'-TGCAGAAAGAAAGTGAAAGGGAAAAAGAAC, antisense: 5'-GTTCTTTTTCCCTTTCACTTTCTTTCTGCA; IRF-1 box mutated, sense: 5'-TGCAGAAATGGGGTGGGGAAGAAAAAGAAC, antisense: 5'-GTTCTTTTTCTTCCCCACCCCATTTCTGCA; NF-{kappa}B-AP-2 box, sense: 5'-GACCTCTTGGATGCCCCAGGCAGTTG, antisense: 5'-CAACTGCCTGGGGCATCCAAGAGGTC; NF-{kappa}B box mutated, sense: 5'-GACCTCTTTTGCTACCCAGGCAGTTG, antisense: 5'-CAACTGCCTGGGTAGCAAAAGAGGTC; AP-2 box mutated, sense: 5'-GACCTCTTGGATGCCTTGCCTAGTTG, antisense: 5'-CAACTAGGCAAGGCATCCAAGAGGTC; STAT-1–USF-1 box, sense: 5'-GCCACTTCTGATAAAGCACGTGGTGGCC, antisense: 5'-GGCCACCACGTGCTTTATCAGAAGTGGC; STAT-1 box mutated, sense: 5'-GCCACGGGAGATTAGGCACGTGGTGGCC, antisense: 5'-GGCCACCACGTGCCTAATCTCCCGTGGC; USF-1 box mutated, sense: 5'-GCCACTTCTGATAAAGTGTAACGTGGCC, antisense: 5'-GG-CCACGTTACACTTTATCAGAAGTGGC. Consensus sequences are underlined and mutated sequences are double underlined.

Statistical analyses
All data are presented as mean ± SE. Repeated measures analysis of variance was used for statistical analysis of the data. The Tukey–Kramer multiple comparison test was used to evaluate significance between experimental groups. P > 0.05 was not considered significant.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IFN-{gamma} and TNF-{alpha} synergistically enhance the accumulation of HLA-DR mRNA and CIITA mRNA, but not IK mRNA
We first examined the expression of mRNA coding for HLA-DR{alpha}, its master regulator CIITA and its inhibitor IK, to see if IFN-{gamma}, TNF-{alpha} or their combination exerted a synergistic effect. TNF-{alpha} alone did not induce the accumulation of HLA-DR{alpha} mRNA (Fig. 2Go). In contrast, IFN-{gamma} alone increased the expression of HLA-DR{alpha} mRNA after 18 h of incubation by 5.4 ± 1.3-fold (P < 0.05) and after 48 h by 6.7 ± 1.4-fold (P < 0.01) compared to non-stimulated cells. Incubation with both stimulators synergistically induced HLA-DR{alpha} mRNA accumulation already after 6 h by 4.1 ± 1.7-fold (which did not yet reach significance compared to the non-stimulated cells) and after 18 h by 9.3 ± 2.7-fold (P < 0.001) compared to the non-stimulated cells. However, after prolonged incubation (48 h) with both stimulators the synergism was no longer evident and HLA-DR{alpha} mRNA levels were similar to those induced by IFN-{gamma} alone (5.9 ± 0.75-fold compared to the non-stimulated cells).

Similarly, TNF-{alpha} did not induce accumulation of CIITA mRNA (Fig. 3AGo), whereas IFN-{gamma} alone induced CIITA mRNA by 8.7 ± 2.1-fold after 6 h and by 41.3 ± 8.5-fold after 24 h (P < 0.001 compared to non-stimulated cells). The combination of stimulators showed marked synergy and induced CIITA mRNA by 38.7 ± 8.9-fold (P < 0.001 compared to non-stimulated cells) already after 6 h, demonstrating the ability of the combined stimulation to enhance induction compared to that observed by IFN-{gamma} alone. However, after 24 h of incubation with the combined stimulation, CIITA mRNA was induced only by 18.3 ± 4.6-fold (P < 0.05) as compared to the 41.3 ± 8.5-fold induction by IFN-{gamma} alone. Thus, IFN-{gamma} alone induced both HLA-DR{alpha} and CIITA mRNAs, while TNF-{alpha} together with IFN-{gamma} resulted in their synergistic expression. However, no synergistic effect was observed after prolonged incubation and TNF-{alpha} even inhibited the long-term effects of IFN-{gamma} alone on the accumulation of CIITA mRNA.




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Fig. 3. CIITA mRNA accumulation. MRO-87-1 thyroid cells were incubated with 100 U/ml IFN-{gamma}, 20 ng/ml TNF-{alpha} or their combination for 6 and 24 h (n = 6). Total RNA was extracted and subjected to RT-PCR for the amplification of CIITA mRNA (A) or IK mRNA (B). TNF-{alpha} did not induce expression of CIITA mRNA, addition of IFN-{gamma} alone significantly increased and addition of the combined stimulation synergistically increased CIITA mRNA at 6 h (*P < 0.001 compared to non-stimulated cells). In contrast, addition of TNF-{alpha}, IFN-{gamma} or their combination did not significantly change the accumulation of IK mRNA.

 
In contrast to the enhancing effect on HLA-DR and CIITA mRNA accumulation, 24 h of incubation with TNF-{alpha}, IFN-{gamma} and their combination increased the accumulation of IK mRNA by 1.6 ± 0.5-, 1.9 ± to 0.3- and 2.05 ± 0.4-fold respectively (Fig. 3BGo). This small increase in IK mRNA accumulation did not reach significance and the combined stimulation did not produce a synergistic effect.

Induction and activation of transcription factors by IFN-{gamma} and TNF-{alpha} is not synergistic
We next examined whether the synergistic effects of IFN-{gamma} and TNF-{alpha} observed at the HLA-DR{alpha} and CIITA mRNA accumulation resulted from a synergistic activation of their respective transcription factors. Addition of TNF-{alpha} alone did not cause the phosphorylation of the transcription factor STAT-1 (Fig. 4Go). IFN-{gamma} alone rapidly caused the tyrosine phosphorylation of STAT-1 after 10 min (4.5 ± 1.3-fold, P < 0.05 compared to non-stimulated cells), an effect that lasted after 2 h (4.3 ± 0.9-fold, P < 0.05 compared to non-stimulated cells), but was reduced thereafter. Addition of both stimulators resulted in a similar rapid phosphorylation of STAT-1 after 10 min (5.1 ± 1.6-fold, P < 0.01 compared to non-stimulated cells), that was gradually reduced afterwards, so that activation of STAT-1 exhibited no synergistic effect. The expression of STAT-1 was essentially unchanged by the stimulators and only after 18 h in the presence of IFN-{gamma} alone was this expression slightly elevated (1.7 ± 0.26-fold, P < 0.05 compared to non-stimulated cells).



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Fig. 4. Activation of the transcription factor STAT-1. MRO-87-1 thyroid cells were incubated with 100 U/ml IFN-{gamma}, 20 ng/ml TNF-{alpha} or their combination for the indicated periods of time (n = 6). Cellular extracts were separated on a 10% SDS–PAGE, and the phosphorylation and expression of STAT-1 were evaluated by Western blot analysis as described in Methods. IFN-{gamma}, but not TNF-{alpha}, induced rapid and significant phosphorylation of STAT-1 (*P < 0.05 compared to non-stimulated cells). The combined stimulation did not result in a synergistic effect (**P < 0.01 compared to non-stimulated cells). Expression of STAT-1 was not changed, except after 18 h of stimulation with IFN-{gamma} (*P < 0.05).

 
IRF-1 was already present in the non-stimulated MRO-87-1 cells, but addition of TNF-{alpha}, IFN-{gamma} or their combination caused a modification in the protein that was manifested by the appearance of a heavier and specific band (as judged by the use of a blocking peptide) after 2 h of stimulation. We assume that this modification is most likely a phosphorylation of the transcription factor (31). TNF-{alpha} alone increased IRF-1 phosphorylation by 1.9 ± 0.3-fold after 2 h and this effect lasted after 18 h of incubation, although it did not reach significance compared to non-stimulated cells (Fig. 5Go). IFN-{gamma} alone or IFN-{gamma} and TNF-{alpha} increased phosphorylation of IRF-1 to a similar extent after 2 h of incubation (2.47 ± 0.18-fold, P < 0.05 and 2.67 ± 0.26-fold, P < 0.01 compared to non-stimulated cells) and this effect lasted after 18 h of incubation. TNF-{alpha} had a similar effect on the expression of IRF-1, which was increased by 1.6 ± 0.2 after 2 h, lasted after 18 h but did not reach significance, compared to non-stimulated cells. Expression of IRF-1 was also similarly increased after 2 h of incubation by IFN-{gamma} alone (2.5 ± 0.2-fold, P < 0.01 compared to non-stimulated cells) or by IFN-{gamma} and TNF-{alpha} (2.3 ± 0.2-fold, P < 0.05 compared to non-stimulated cells) and lasted after 18 h. Although TNF-{alpha} alone caused an increase in the activation and expression of IRF-1, the combined stimulation did not increase it synergistically.



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Fig. 5. Activation of the transcription factor IRF-1. MRO-87-1 thyroid cells were incubated with 100 U/ml IFN-{gamma}, 20 ng/ml TNF-{alpha} or their combination for the indicated periods of time (n = 6). Cellular extracts were separated on a 7% SDS–PAGE and the expression of IRF-1 was evaluated by Western blot analysis as described in Methods. Following 2 h of incubation, TNF-{alpha}, but to a greater extent IFN-{gamma}, exerted a modification in IRF-1, which was probably phosphorylation of the protein. Thus, expression and phosphorylation of IRF-1 were significantly enhanced by IFN-{gamma} or IFN-{gamma} together with TNF-{alpha} (*P < 0.05, **P < 0.01 compared to non-stimulated cells), but the effect of addition of TNF-{alpha} alone did not reach significance. No synergistic effect was observed in the activation or expression of IRF-1 when both stimuli were added.

 
In addition, we examined the phosphorylation and extent of degradation of I{kappa}B{alpha} by addition of TNF-{alpha}, IFN-{gamma} and their combination, but we could find no synergistic effect, nor did IFN-{gamma} alone induce degradation of I{kappa}B{alpha} (data not shown). Furthermore, analysis of the translocation of the p65 subunit of NF-{kappa}B by Western blot analysis, which was performed on nuclear extracts, revealed that translocation of NF-{kappa}B was similarly increased by TNF-{alpha} alone or the combined stimulation and no synergism was evident, whereas IFN-{gamma} alone had no effect (data not shown).

Binding of transcription factors to the CIITA promoter
We next tried to determine whether a synergistic effect could be revealed in the binding of the transcription factors to their respective consensus sequences. We have amplified the sequence of the proximal promoter as described in Methods and examined the binding of transcription factors to the whole promoter (Fig. 6BGo). Two bands with different mobilities were observed. The slow migrating band (complex a) was constitutive and was clearly evident without any stimulation. Addition of IFN-{gamma}, TNF-{alpha} or their combination gradually and similarly decreased the intensity of the binding of complex a, and after 24 h of stimulation it reached 0.7 ± 0.08-fold compared to the non-stimulated cells; however, this trend did not reach significance. In contrast, a fast migrating band (complex b) was observed only after stimulation of the cells with IFN-{gamma} or with the combined stimulation. TNF-{alpha} alone or IFN-{gamma} alone did not change the binding of complex b, but after 2 and 6 h of incubation with the combined stimulation this binding was increased by 2 ± 0.7- and 2.3 ± 0.5-fold respectively (P < 0.05 compared to non-stimulated cells), demonstrating a synergistic effect. To reveal which transcription factors bind the promoter we pre-incubated the nuclear extracts with several specific antibodies (Fig. 6CGo). Antibodies directed against AP-2, p50, p65 and STAT-1 did not cause super-shifting or inhibition of the binding of either one of the complexes. In contrast, anti-USF-1 super-shifted the slow migrating complex (complex a) and anti-IRF-1 super-shifted the fast migrating complex (complex b), thus implicating the involvement of these two proteins in the transcription of CIITA in MRO-87-1 cells.





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Fig. 6. Binding of transcription factor to the CIITA type IV promoter. (A) The CIITA type IV promoter is depicted. The primers used for amplification of the promoter are underlined, the cis elements for the binding of transcription factors are boxed and the transcription start site is indicated by an arrow. (B) EMSA analysis was performed to evaluate the binding of transcription factors to the promoter (n = 4). MRO-87-1 cells were incubated with 100 U/ml IFN-{gamma}, 20 ng/ml TNF-{alpha} or their combination for the indicated periods of time, and nuclear extracts were prepared as described in Methods. The nuclear extracts were incubated with the CIITA type IV promoter, which was radioactively labeled during its amplification (see Methods). Two complexes were observed: the slow migrating complex (complex a) was constitutive and the intensity of its binding did not change, while the fast migrating complex (complex b) was inducible and its binding was increased by the combined stimulation. (C) In order to identify the specific transcription factors comprising complex a and b, the nuclear extracts from MRO-87-1 cells that were stimulated with TNF-{alpha} and IFN-{gamma} for 6 h were pre-incubated with specific antibodies. Anti-IRF-1 caused super-shifting of complex b, while anti-USF-1 caused super-shifting of complex a. However, the other antibodies used did not change the binding of complex a or b.

 
We next examined the binding of each protein to its respective isolated box, by incubating the nuclear extract with an oligonucleotide containing its consensus sequence or a mutated sequence. Several bands were observed binding the IRF-1 box; however, their intensity was not changed (Fig. 7AGo) and they were not chased by addition of unlabeled oligonucleotide or the mutated box (Fig. 7BGo), and therefore represented non-specific binding. In contrast, a specific band (marked with an arrow in Fig. 7Go) was observed after incubation with each of the stimulators (1.7 ± 0.3-fold from non-stimulated cells for IFN-{gamma} and 1.3 ± 0.13-fold from non-stimulated cells for TNF-{alpha}). A synergistic increase in the binding intensity was observed with the combined stimulation at 2 h (6.2 ± to 0.5-fold from non-stimulated cells; Fig. 7AGo) and at 6 h (4.1 ± 0.36-fold from non-stimulated cells; Fig. 7BGo). This binding was competed away by a molar excess of the unlabeled box and the binding was abrogated when the mutated oligonucleotide was used (Fig. 7BGo), demonstrating the specificity of the binding of IRF-1 to its box.




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Fig. 7. Binding of nuclear extracts to the IRF-1 box. (A) EMSA analysis was performed to evaluate the binding of IRF-1 to its box (n = 3). MRO-87-1 cells were incubated with 100 U/ml IFN-{gamma}, 20 ng/ml TNF-{alpha} or their combination for the indicated periods of time, and nuclear extracts were prepared as described in Methods. The nuclear extracts were incubated with the 5'-end-labeled oligonucleotide containing the IRF-1 box. Several non-specific bands were observed, but a specific band (marked with an arrow) was increased after addition of the combined stimulation for 2 and 6 h. (B) In order to prove the specificity of the binding, nuclear extracts from MRO-87-1 cells that were stimulated with TNF-{alpha} and IFN-{gamma} for 6 h were allowed to bind the 5'-end-labeled box in the presence of a 100-fold molar excess of unlabeled box (c) or a 100-fold molar excess of the mutated box (Im).

 
Since the STAT-1 and USF-1 boxes are so close to each other (Fig. 6AGo) we have used one oligonucleotide to assay their binding. A constitutive binding of a complex to the STAT-1–USF-1 box was observed, that did not change with the addition of each of the stimulators or their combination, and was evident even without any stimulation (Fig. 8AGo). This complex was competed away by a molar excess of the unlabeled box (Fig. 8BGo). Competition with mutations that were introduced to the STAT-1 box caused complete abrogation of the binding of the complex, whereas mutations that were introduced to the USF-1 box did not change this binding (Fig. 8BGo), indicating that USF-1 was the protein binding the sequence and that STAT-1 was not involved.




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Fig. 8. Binding of nuclear extracts to the STAT-1–USF-1 box. (A) EMSA analysis was performed to evaluate the binding of either STAT-1 or USF-1 to their respective cis elements (n = 3). MRO-87-1 cells were incubated with 100 U/ml IFN-{gamma}, 20 ng/ml TNF-{alpha} or their combination for the indicated periods of time, and nuclear extracts were prepared as described in Methods. The nuclear extracts were incubated with the 5'-end-labeled oligonucleotide containing both the STAT-1 and the USF-1 boxes. Several non-specific bands were observed, but the binding of a specific band (marked with an arrow) was unchanged throughout the experimental protocol. (B) In order to identify which of the transcription factors is involved in the complex, nuclear extracts from MRO-87-1 cells that were stimulated with TNF-{alpha} and IFN-{gamma} for 6 h were allowed to bind the 5'-end-labeled STAT-1–USF-1 box in the presence of a 100-fold molar excess of unlabeled oligonucleotide (c), a 100-fold molar excess of the oligonucleotide with a mutation in the STAT-1 binding site (Sm) or the oligonucleotide with a mutation in the USF-1 binding site (Um).

 
In addition, incubation of the nuclear extracts with an oligonucleotide containing the NF-{kappa}B/AP-2 site (as specified in the methods) showed no binding of any protein complex (data not shown).

The NF-{kappa}B inhibitor SN50 has no effect on CIITA mRNA accumulation. To further investigate whether NF-{kappa}B is involved in the transcription of CIITA, we incubated MRO-87-1 cells with an increasing dose of the synthetic peptide SN50, that inhibits translocation of NF-{kappa}B to the nucleus (29). Without the presence of SN50 CIITA was induced by the combined stimulation at 6 h (22.9 ± 4.8-fold, P < 0.01compared to the non-stimulated cells) and to a lesser extent at 24 h (5.2 ± 1.6-fold). Since increasing amounts of SN50 did not significantly change the accumulation of CIITA mRNA at these time periods (Fig. 9Go), NF-{kappa}B was not involved in CIITA transcription in the MRO-87-1 thyroid cells.



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Fig. 9. The NF-{kappa}B inhibitor peptide SN50 has no effect on CIITA mRNA accumulation. MRO-87-1 cells were incubated with 100 U/ml IFN-{gamma} and 20 ng/ml TNF-{alpha} for 6 or 24 h with increasing amounts of the synthetic peptide SN50. Total RNA was extracted and the CIITA mRNA was amplified as described before. The synthetic SN50 peptide contains the nuclear localization signal sequence of the p50 subunit of NF-{kappa}B, and thus competes with it and inhibits NF-{kappa}B translocation to the nucleus. Addition of SN50 did not significantly change the accumulation of CIITA mRNA, indicating that NF-{kappa}B did not take part in the regulation of CIITA type IV promoter transcription.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we have demonstrated the synergistic induction of HLA-DR{alpha} and its master regulator CIITA by IFN-{gamma} and TNF-{alpha} in human thyroid cells, and we have investigated the molecular mechanisms responsible for this synergistic effect. We demonstrate that TNF-{alpha} exerts a synergistic effect by increasing the binding of the transcription factor IRF-1 to its element on the CIITA inducible type IV promoter. Other possible mechanisms that could explain the synergistic effect, such as a synergistic activation of STAT-1 via its tyrosine phosphorylation, a synergistic induction of IRF-1 and a synergistic activation of NF-{kappa}B via IFN-{gamma}-induced I{kappa}B{alpha} degradation, were demonstrated not to occur in this thyroid cell line. Moreover, the induction of CIITA did not require the putative element for NF-{kappa}B or the binding of STAT-1 to its GAS element.

TNF-{alpha} does not always act in synergism with IFN-{gamma} to induce MHC class II and CIITA expression in tissue cells, and depending on the cell type and state of differentiation (32), it may even repress their expression. For example, in the fibrosarcoma HT1080 cell line TNF-{alpha} blocked IFN-{gamma}-induced CIITA mRNA accumulation by increasing its degradation (33). Additionally, although a synergistic effect of TNF-{alpha} and IFN-{gamma} on MHC class II induction was found in rat astrocytes, CIITA induction was not synergistic, suggesting that the enhancing effect of TNF-{alpha} occurred down-stream of CIITA induction (22,34). However, many studies, as well as our own, demonstrated the enhancing effect of TNF-{alpha} on IFN-{gamma}-induced MHC class II expression in thyrocytes (reviewed in 8). Here we show that TNF-{alpha} and IFN-{gamma} synergistically increase the accumulation of HLA-DR{alpha} and CIITA mRNAs after short periods of incubation. CIITA mRNA accumulation was detected prior to that of HLA-DR{alpha} mRNA, in accordance with the role of CIITA as the master regulator of HLA-DR expression in these cells. However, prolonged incubation with TNF-{alpha} and IFN-{gamma} inhibited CIITA mRNA accumulation, suggesting that TNF-{alpha} may have a two-phase effect, possibly mediated by different mechanisms, as was demonstrated by the increased CIITA mRNA degradation in the HT1080 cell line (33). Induction of CIITA was much larger than that of HLA-DR, suggesting that its higher expression may be necessary to overcome the inhibitory effects of other transcription factors that may bind to HLA-DR promoter. Accumulation of IK mRNA, a gene product that functions as an inhibitor of IFN-{gamma}-induced expression of CIITA, was not changed with addition of IFN-{gamma}, TNF-{alpha} or their combination over time, suggesting that expression of IK is irrelevant to this system.

Several molecular mechanisms regulating the synergism between IFN-{gamma} and TNF-{alpha} had been proposed. The main mechanism involves interactions between the transcription factors NF-{kappa}B, which is activated by TNF-{alpha}, and IRF-1, which is induced by IFN-{gamma}, when binding to their respective elements on gene promoters. Other possible mechanisms include activation of NF-{kappa}B by IFN-{gamma}-induced degradation of I{kappa}B (3537), and induction of IRF-1 by TNF-{alpha} via the NF-{kappa}B site located on its promoter (35,38). Since CIITA type IV promoter includes NF-{kappa}B, STAT-1 and IRF-1 sites (21,22), increased activation of these transcription factors may lead to the synergistic effect observed.

We first investigated whether the putative site for NF-{kappa}B/AP-2 is involved in the transcription of CIITA. However, we could not see super-shifting of complex a or complex b when anti-AP-2, anti-p65 or anti-p50 were used in an EMSA assay with the CIITA type IV promoter. In addition, incubation of nuclear extracts with the isolated NF-{kappa}B/AP-2 box exhibited no binding (data not shown) and inhibition of NF-{kappa}B translocation into the nucleus with increasing dosage of SN50 did not change the transcription of CIITA mRNA. Furthermore, IFN-{gamma} alone did not induce I{kappa}B{alpha} degradation and thereby NF-{kappa}B activation in this thyroid cell line (data not shown). Thus, neither NF-{kappa}B nor AP-2 participated in the transcription of CIITA from its inducible promoter and the possibility that the binding of NF-{kappa}B to its element mediated the synergistic effect was ruled out.

We next looked for a synergistic effect on the activation of STAT-1 and IRF-1. Although STAT-1 was rapidly phosphorylated after stimulation with IFN-{gamma}, TNF-{alpha} did not exert any synergistic effect on phosphorylation. Similarly, IRF-1 expression, which was observed even without any stimulation, was increased after 2 h of stimulation by IFN-{gamma}, TNF-{alpha} or their combination, but no synergistic effect was observed. This is in agreement with the previous finding that TNF-{alpha} and IFN-{gamma} did not increase IRF-1 mRNA levels synergistically in the FRTL-5 thyroid cells (39). In addition, following 2 h of incubation with either stimulator or their combination, a modification of the IRF-1 protein, probably its phosphorylation (31), was observed, but no synergistic activation could be measured.

We then investigated whether the binding of STAT-1 or IRF-1 was synergistically affected by the stimuli. Two protein complexes were found to bind to the CIITA type IV promoter. Complex a showed a constitutive binding which was not affected by the stimulation with either cytokines. This binding was super-shifted by anti-USF-1, but not by anti-STAT-1, and was competed away by the molar excess of the USF-1 box, but not of STAT-1 box. This suggests that USF-1 is the binding protein in complex a, and that STAT-1 is not involved in the binding and transactivation of the CIITA type IV promoter in this thyroid cell line. In contrast, complex b was synergistically induced by the combined stimulation. This binding was super-shifted by anti-IRF-1 and was competed away by molar excess of IRF-1 box, but not by its mutated sequence, establishing the specificity of IRF-1 binding. Unlike the other possible mechanisms we investigated, IRF-1 binding was synergistically elevated by TNF-{alpha} and IFN-{gamma}, suggesting its involvement in the synergistic induction of CIITA mRNA in this thyroid cell line. We suggest that IRF-1 synergistically increased its binding to the promoter following the combined stimulation, although it was not synergistically induced or phosphorylated. A similar mechanism of increased DNA binding activity of a transcription factor without a corresponding induction of its mRNA expression was reported for the binding of AP-1 to its sequence after hypoxia (40).

The characteristics of the binding of complex a are in contrast to melanoma cells (21), where a cooperative interaction between IFN-{gamma}-activated STAT-1, that binds the GAS element, and the constitutively expressed USF-1, that binds the E-box, is required for induction of transcription of the CIITA type IV promoter. Conflicting data in rat astrocytes reveal either similar binding of STAT-1 and interaction with USF-1 (22) or, similar to our findings, the lack of such binding (34). These confusing data may indicate a cell-specific role for STAT-1 binding in the induction of CIITA.

In conclusion, we have demonstrated that in the MRO-87-1 human thyroid cells both HLA-DR{alpha} and CIITA are synergistically expressed after stimulation with IFN-{gamma} and TNF-{alpha}, and that this synergism is mediated, at least partially, by the increased binding of IRF-1 to its box in the CIITA type IV promoter.



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Fig. 1. HLA-DR{alpha} mRNA accumulation. MRO-87-1 thyroid cells were incubated without any stimulation (white bars), with 100 U/ml IFN-{gamma} (dotted bars), with 20 ng/ml TNF-{alpha} (hatched bars) or with their combination (black bars) for the indicated periods of time (n = 6). HLA-DR{alpha} mRNA accumulation was assayed in a Northern blot and normalized to that of GAPDH as described in Methods. TNF-{alpha} had no effect on HLA-DR{alpha} mRNA accumulation, but IFN-{gamma} significantly increased it after 18 and 48 h (*P < 0.05; **P < 0.01 compared to the non-stimulated cells). However, the combined stimulation synergistically increased HLA-DR{alpha} accumulation even earlier, reaching significance after 18 h stimulation (***P < 0.0001).

 

    Acknowledgments
 
This work was supported by research grant 3674 from the Chief Scientist Office of the Israeli Ministry of Health.


    Abbreviations
 
CIITA class II transactivator
GAS IFN-{gamma} activating sequence
IRF IFN regulating factor
STAT signal transducer and activator of transcription
TNF tumor necrosis factor

    Notes
 
Transmitting editor: I. Pecht

Received 13 April 2001, accepted 10 August 2001.


    References
 Top
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 Introduction
 Methods
 Results
 Discussion
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