The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, H.
Right arrow Articles by Shong, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, H.
Right arrow Articles by Shong, M.
The Journal of Immunology, 2003, 171: 616-627.
Copyright © 2003 by The American Association of Immunologists

Thyrotropin-Mediated Repression of Class II Trans-Activator Expression in Thyroid Cells: Involvement of STAT3 and Suppressor of Cytokine Signaling1

Ho Kim*, Jae Mi Suh*, Eun Suk Hwang*, Dong Wook Kim*, Hyo Kyun Chung*, Jung Hun Song*, Jung Hwan Hwang*, Ki Cheol Park*, Heung Kyu Ro*, Eun-Kyeong Jo{dagger}, Jong-Soo Chang{ddagger}, Tae-Hoon Lee2,§, Myung-Shik Lee, Leonard D. Kohn|| and Minho Shong3,*

* Laboratory of Endocrine Cell Biology, Department of Internal Medicine, {dagger} Department of Microbiology and Immunology, Chungnam National University School of Medicine, Daejon, Korea; {ddagger} Department of Biology, Daejin University, Kyeonggido, Korea; § Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea; Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; and || Department of Biomedical Sciences, Ohio University School of Osteopathic Medicine and Edison Biotechnology Institute, Athens, OH 45701


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has been suggested that class I and class II MHC are contributing factors for numerous diseases including autoimmune thyroid diseases, type 1 diabetes, rheumatoid arthritis, Alzheimer’s disease, and multiple sclerosis. The class II trans-activator (CIITA), which is a non-DNA-binding regulator of class II MHC transcription, regulates the constitutive and inducible expression of the class I and class II genes. FRTL-5 thyroid cells incubated in the presence of IFN-{gamma} have a significantly higher level of cell surface rat MHC class II RTI.B. However, the IFN-{gamma}-induced RT1.B expression was suppressed significantly in cells incubated in the presence of thyrotropin. Thyrotropin (TSH) represses IFN-{gamma}-induced CIITA expression by inhibiting type IV CIITA promoter activity through the suppression of STAT1 activation and IFN regulatory factor 1 induction. This study found that TSH induces transcriptional activation of the STAT3 gene through the phosphorylation of STAT3 and CREB activation. TSH induces SOCS-1 and SOCS-3, and TSH-mediated SOCS-3 induction was dependent on STAT3. The cell line stably expressing the wild-type STAT3 showed a higher CIITA induction in response to IFN-{gamma} and also exhibited TSH repression of the IFN-{gamma}-mediated induction of CIITA. However, TSH repression of the IFN-{gamma}-induced CIITA expression was not observed in FRTL-5 thyroid cells, which stably expresses the dominant negative forms of STAT3, STAT3-Y705F, and STAT3-S727A. This report suggests that TSH is also engaged in immunomodulation through signal cross-talk with the cytokines in thyroid cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Class I and class II MHC molecules play a critical role in T cell-dependent immunity and the inflammatory response by presenting the processed, endogenous, and exogenous Ags to the Th cells, respectively (1, 2, 3). Class I and class II MHC have been implicated as contributing factors for numerous diseases, including autoimmune thyroid diseases, type 1 diabetes, rheumatoid arthritis, Alzheimer’s disease, and multiple sclerosis (4, 5).

Constitutive and inducible expressions of class I and class II MHC are primarily regulated at the transcriptional level. The class II trans-activator (CIITA)4, a non-DNA-binding regulator of class II MHC transcription, regulates the constitutive and inducible expression of the class I and class II genes (6, 7, 8, 9). The CIITA expression level determines the inducibility and quantitative control of class II expression. In normal tissues the CIITA expression patterns are controlled by as many as four promoters that allow both the constitutive and IFN-{gamma}-inducible expressions of CIITA, which are constitutively expressed in dendritic cells and B lymphocytes by promoters I and III, respectively, and IFN-{gamma}-inducible expression in a melanoma cell line by promoter IV (10, 11). IFN-{gamma} regulates the type IV CIITA promoter through several cis-acting regulatory elements, including the IFN-{gamma} activation sequence (GAS), an E box, and an IFN regulatory factor (IRF) element (12, 13). The GAS and IRF elements bind the IFN-{gamma}-regulated transcription factors STAT1 and IRF-1, respectively. The E box is bound by the constitutively expressed upstream stimulating factor-1 (USF-1). All three of these cis-acting elements (GAS, E box, and IRF-1) are essential for IFN-{gamma}-induced activation of the type IV CIITA promoter (13). In addition, the cooperative interaction of IFN-{gamma}-activated STAT1{alpha} and constitutively expressed USF-1 is required for IFN-{gamma} activation of the type IV CIITA promoter (12).

Thyroid cells are one of the most common targets for organ-specific autoimmune diseases. Although the initial triggering molecular events have not been identified in the pathogenesis of autoimmune thyroid diseases, abnormal or aberrant class I and II MHC gene expression is consistently observed in follicular epithelial cells in autoimmune thyroid diseases (14, 15, 16). A recent study reported the importance of aberrant class II expression in the development of autoimmune thyroid diseases. Therefore, immunizing the mice with fibroblasts transfected with both the human thyrotropin (TSH) receptor and an MHC class II molecule, but not either alone, induces Graves-like disease (17). These results suggests that acquisition of the Ag-presenting ability on a thyroid cell, which is the result of aberrant class II expression, can activate the immune system for the development of autoimmune thyroid diseases (18). Understanding the mechanisms underlying the basis of the aberrant class II expression in thyroid cells appears to be important for understanding how autoimmune thyroid diseases might develop.

IFN-{gamma} can positively regulate class I and II MHC gene expression in thyroid cells and can mimic the changes in human thyrocytes observed in patients with autoimmune thyroid diseases. IFN-{gamma} induces the rapid and prolonged phosphorylation (Y701) of STAT1, which is critical for dimerization, nuclear translocation, and DNA binding in thyroid cells (19). This Janus kinase-1 (JAK)-mediated tyrosine phosphorylation of STAT1 is essential for inducing the IFN-{gamma}-responsive genes, such as ICAM-1 and CIITA in thyroid cells (19). In a series of recent discoveries, several regulators, a suppressor of cytokine signaling (SOCS), SH2-containing tyrosine phosphatases, and the protein inhibitors of activated STAT (PIAS), act as negative regulators of IFN-{gamma}-mediated JAK/STAT activation (20, 21, 22, 23, 24). However, the roles of these negative regulators of IFN-{gamma} signaling in thyroid cells have not been fully evaluated. Recent studies have shown the inhibitory effect of TSH on IFN-{gamma} signaling in thyroid cells and the inhibition of tyrosine phosphorylation on STAT1, JAK1, and IFN-{gamma} receptor. Furthermore, TSH was shown to induce SOCS-1 and SOCS-3 in thyroid cells (25).

This study found that TSH suppresses IFN-{gamma}-mediated class II gene expression through the inhibition of CIITA expression. These inhibitory effects were mainly caused by the TSH-mediated induction and activation of STAT3, which result in the expression of SOCSs in thyroid cells. The increased SOCS level suppresses the IFN-{gamma}-induced maximum levels of STAT1 and IRF-1 expression and activation, which are crucial for transcriptional regulation of the type IV promoter of CIITA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Highly purified bovine TSH was purchased from the Sigma-Aldrich (St. Louis, MO). The rat and mouse recombinant IFN-{gamma} was obtained from R&D Systems (Minneapolis MN). [{alpha}-32P]dCTP (3000 Ci/mmol) was purchased from DuPont-Merck (Wilmington, DE). Unless otherwise noted, all other materials were obtained from Sigma-Aldrich.

Cells

FRTL-5 rat thyroid cells (Interthyr Research Foundation, Baltimore, MD) were a fresh subclone (F1) that had all the properties previously detailed (26). Their doubling time with TSH was 36 ± 6 h, and they did not proliferate without TSH. After 6 days in the medium with no TSH, the addition of 1 x 10-10 M TSH stimulated thymidine incorporation into the DNA by at least 10-fold. The cells were diploid between their 5th and 20th passages. The cells were grown in six-hormone medium consisting of Coon’s modified F-12 supplemented with 5% calf serum, 1 mM nonessential amino acids, and a mixture of six hormones (6H): bovine TSH (1 µ/ml), insulin (10 µg/ml), cortisol (0.4 ng/ml), transferrin (5 µg/ml), glycyl-L-histidyl-L-lysine acetate (10 ng/ml), and somatostatin (10 ng/ml). Fresh medium was added to all the cells every 2 or 3 days, and the cells were passaged every 7–10 days. In individual experiments the cells were shifted to 5H medium with no TSH and 5% calf serum.

Mouse embryonic findroblasts (MEF) were prepared from the fetal mice on day 13.5 of gestation from IRF-1-/- and STAT1-/- mice (129S6/SvEv-Stattm1; Taconic Farms, Germantown, NY) (27, 28), where embryonic day 0.5 was the time when the vaginal plug was detected. The MEF were prepared from individual fetuses from each littermate, as previously described (29), with the genotype being determined by an evaluation of tail DNA from each fetal mouse. Each culture was derived from a separate fetal mouse. The cells (105/35-mm diameter dish) were seeded into 2 ml of the growth medium (10% (v/v) FBS in DMEM).

RNA isolation, Northern analysis, and RT-PCR

Total cellular RNA was isolated using the standard procedures, and Northern analysis was performed as previously described (25). The final washes were conducted at 65°C in 1x SSPE (150 mM NaCl, 10 mM NaH2PO4, and 1 mM EDTA, pH 7.4). The hybridization probes for STAT1, USF-1, IRF-1, SOCS-1, and SOCS-3 were the purified inserts of the expression vectors RcCMV-STAT1, pcDNA3-USF-1, pcDNA3-IRF-1, pEF-SOCS-1, and pEF-SOCS-3, respectively. All probes were radiolabeled using a random priming protocol (Amersham Pharmacia Biotech, Arlington Heights, IL). For RT-PCR, first-strand cDNA was synthesized using reverse transcriptase (Life Technologies, Grand Island, NY). PCR was performed using AmpliTaq DNA polymerase (Perkin-Elmer, Norwalk, CT) in a Perkin-Elmer 9700 thermocycler. The PCR conditions were: predenaturation at 94°C for 20 s, followed by 30 cycles of denaturation at 94°C for 15 s, annealing at 55°C for 45 s, and elongation at 72°C for 1 min. The primers used were: malic enzyme, 5'-GATGCATACAAAGAAAAGATGGC-3' (sense) and 5'-TTATGCTATATTAATTTATTACTAGTGTGGG-3' (antisense),; rat CIITA, 5'-GGACCTGGACTCACTTAGCGA-3' (sense) and 5'-ATCCAGCTGCTGCAGGTG-3' (antisense); mouse CIITA, 5'-GGACCTGGACTCACTTAGTGA-3' (sense) and 5'-ATCCAGCTGCTGCAGGTG-3' (antisense); and rat STAT3, 5'-CGGCCCTTAGTCATCAAGAC-3' (sense) and 5'-CCGCTCGAGTCACATCGGGGAGGTAGC-3' (antisense).

FACS analysis

FRTL-5 thyroid cells (1 x 106 cells) were stimulated for 48 h by adding directly TSH (1 mU/ml), forskolin (10 µM), and/or IFN-{gamma} (10 ng/ml). The cells were washed three times in an isotonic cold PBS buffer (supplemented with 0.5% BSA) after the trypsin/EDTA treatment, and then incubated for 30 min at 4°C with FITC-conjugated mouse anti-rat RT1.B mAbs (OX-6; BD PharMingen, San Diego, CA). Isotype mouse IgG1 (BD Biosciences, Heidelberg, Germany) was used as the control. Following this incubation, the unreacted anti-RT1.B Abs were removed by washing, and the cells were resuspended in 200 µl of a PBS buffer for the final flow cytometric analysis. The cells were analyzed using a FACScan (BD Biosciences).

CIITA and STAT3 promoter constructs

The sequence for the primers used to PCR amplify a 1703-bp DNA fragment of the type IV promoter of the human CIITA gene was derived from that reported previously (13). The sense primer was located at the 3' end of the type III promoter and has a sequence of GCCTGGCTCCACGCCCTGCTG. The antisense primer is located at the 3' end of the type IV promoter and has a sequence of CGCTGTTCCCCGGGCTCCCG. The designated name for this construct is hCIITAp1.7. The site-directed mutation constructs, hCIITA-GAS, hCIITA-E box, hCIITA-IRF, hCIITA-GAS+IRF, and hCIITA-GAS+E box, were supplied by Dr. Benveniste (13). The wild-type reporter and the mutant reporter containing a -478/-229 fragment of the STAT3 promoter in front of the minimal junB promoter-luciferase gene (p478/229-Luc) were used to determine TSH responsiveness (30, 31).

Transient transfection and luciferase assay

The hCIITA promoter and STAT3 promoter constructs were transfected in FRTL-5 thyroid cells by lipofection with a Lipofectamine regent (Invitrogen, San Diego, CA). For each well of the six-well plate, 4 µg of Lipofectamine were combined with 200 µl of OptiMEM (Life Technologies/BRL) and then added to 0.4 µg of the hCIITA promoter and STAT3 promoter construct. The cells were incubated overnight with a DNA/Lipofectamine mixture. 5H5% medium was then applied, and the cells were incubated for an additional 24 h before harvesting for the luciferase determinations. The cells were washed with PBS and lysed with 180 µl of a lysis buffer. The cells were cotransfected with 0.1 µg of the pRL-CMV plasmid containing the Renilla luciferase gene (Promega, Madison WI) according to the manufacturer’s protocol. The extracts were assayed in triplicate for their luciferase activity, and the light intensity was measured using a luminometer (Berthold, Bad Wildbad, Germany). The luciferase activity was integrated over a 10-s period. The firefly luciferase values were standardized to the Renilla values.

Development of stable cell lines

FRTL-5 cells (5 x 106) were electroporated in a 0.4-cm cuvette (Bio-Rad, Hercules, CA) at a voltage of 0.3 kV and a high capacitance of 950 µF (32, 33). The cells were transferred to a 10-cm plate and incubated with Coon’s modified Ham’s F-12 medium without G418, and the media was changed the next day. In the following 2 days, 0.4 mg/ml G418 was added, and the cells were subsequently maintained. Individual colonies appeared ~4 wk after transfection. The RcCMV-, STAT3-, STAT3-Y705F-, and STAT3-S727A-expressing clones were selected by Western blotting.

Preparation of small interfering RNA (siRNA)-STAT3 and transfection

The 21-nucleotide siRNA were synthesized and purified by a Silencer siRNA Construction Kit (Ambion, Houston, TX). The siRNA sequence targeting rat STAT3 (GenBank accession no. X91810) corresponded to the coding region 145–165. Desalted DNA of the sense (5'-AAAGAGTCACACGCCACTCTGCCTGTCTC-3') and antisense (5'-AACAGAGTGGCGTGTGACTCTCCTGTCTC-3') oligonucleotides were synthesized, and the eight nucleotides at the 3' end of both oligonucleotides have the sequence 5'-CCTGTCTC-3', which is complementary to the T7 promoter. To produce an efficient transcription template, the sense and antisense oligonucleotides for each siRNA must be converted to dsDNA with a T7 promoter in 37°C. The sense and antisense siRNA transcripts are transcribed for 2 h in separate reactions with T7 RNA polymerase. The reactions were then mixed, and the combined reaction was incubated overnight at 37°C for dsRNA. The 5' overhanging leader sequence and the DNA template were eliminated by a single-strand specific ribonuclease and DNase digestion, respectively. The resulting siRNA was recovered from the mixture of nucleotides, enzymes, short oligomers, and salts in the reaction by column purification. The FRTL-5 cells were transfected with siRNA by electroporation. For the electroporations, 2.5 µmol of dsRNA was added to prechilled 0.4-cm electrode gap cuvettes (Bio-Rad). FRTL-5 cells were resuspended to 3 x 107 cells/ml in cold PBS, added to the cuvettes, mixed, and pulsed once at 300 mV and 950 µF with a Gene Pulser electroporator II (Bio-Rad). The cells were plated into six-well culture plates containing 3 ml of complete medium and incubated at 37°C in a humidified 5% CO2 chamber. Cell viability immediately after electroporation was typically ~70%. The cells were treated and harvested 24 h later for the further assays.

Western blot analysis

Immunoblot analyses were performed using anti-STAT1 (Cell Signaling Technology, Beverly, MA), anti-IRF-1 (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-USF-1 (Upstate Biotechnology, Charlottesville, VA). The Abs against the phosphorylated forms of STAT1 (Y701) or CREB (S133) were affinity-purified rabbit polyclonal IgG (Cell Signaling Technology). For the Western blot, adherent FRTL-5 cells were stimulated with various agents for the indicated period of time at 37°C. The treated cells were then scraped, lysed by adding the SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 6% (w/v) SDS, 30% glycerol, 125 mM DTT, and 0.03% (w/v) bromophenol blue), and separated by 10% SDS-PAGE along with the biotinylated m.w. standards. The proteins were transferred to a nitrocellulose membrane by electrotransfer for 2 h. After soaking the membrane in a blocking buffer (1x TBS/0.1% Tween 20 with the blocking reagent in 5% milk), the membrane was incubated with the primary Abs overnight at 4°C. The blots were developed using the HRP-linked secondary Abs and a chemiluminescent detection system (Phototope-HRP Western Blot Detection Kit; New England Biolabs, Beverly, MA).

Other assays

The protein concentration was determined using the Bradford method (Bio-Rad), and recrystallized BSA was used as the standard.

Statistical significance

All experiments were repeated at least three times with different cells. The values are reported as the mean ± SD of these experiments. Significance between the experimental values was determined by two-way ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhibition of IFN-{gamma}-induced class II MHC and CIITA expression by TSH

To determine whether TSH affects the IFN-{gamma}-induced expression of class II MHC expression, the TSH-responsive FRTL-5 rat thyroid cells were examined for the expression of the rat class II MHC Ag RT1.B in response to IFN-{gamma} in the presence or the absence of TSH. The cells were cultured in the presence of IFN-{gamma} with or without TSH for 48 h and stained with fluorescein-labeled anti-rat RT1.B mAbs for flow cytometric analysis of RT1.B expression. TSH, insulin, and forskolin did not induce RT1.B expression in the FRTL-5 thyroid cells (data not shown). However, the cells incubated in the presence of IFN-{gamma} for 48 h had a significantly higher level of surface RTI.B (Fig. 1B). RT1.B expression was increased by IFN-{gamma} in a dose-dependent manner; ~70% of the cells exposed for 48 h with IFN-{gamma} (10 ng/ml) tested positive for RT1.B (Fig. 1A). The cells incubated in the presence of TSH and IFN-{gamma} showed a marked suppression of RT1.B expression (Fig. 1B, upper panel). In addition, forskolin did not induce RT1.B expression (data not shown), but it did inhibit IFN-{gamma}-mediated expression of RT1.B in FRTL-5 thyroid cells. In contrast, insulin did not inhibit the IFN-{gamma}-dependent up-regulation of RT1.B (data not shown).



View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 1. Flow cytometric analysis of TSH and forskolin effects on RT1.B in response to IFN-{gamma}. FRTL-5 thyroid cells were incubated with recombinant IFN-{gamma} (10 ng/ml) in the presence or the absence of TSH (1 mU/ml) or forskolin (10 µM) at 37°C and 5% CO2 for 48 h. The cells were washed three times in an isotonic cold PBS buffer (supplemented with 0.5% BSA) after trypsin/EDTA treatment and then were incubated for 30 min at 4°C with FITC-conjugated mouse anti-rat RT1.B mAbs (OX-6; BD PharMingen). Isotype mouse IgG1 (BD Biosciences) was used as a control. Following this incubation, the unreacted anti-RT1.B Abs were removed by washing, and the cells were resuspended in 200 µl of PBS buffer for the final flow cytometric analysis. The cells were analyzed using a FACScan (BD Biosciences).

 
These results suggest that TSH may inhibit certain steps of the IFN-{gamma}-induced expression of class II MHC in FRTL-5 thyroid cells. This study evaluated the effects of TSH and forskolin in IFN-{gamma}-mediated class II MHC (RT1.B{alpha}) RNA induction in FRTL-5 thyroid cells. FRTL-5 thyroid cells did not express RT.1B{alpha} RNA, but IFN-{gamma} markedly induced RT1.B{alpha} after 12 h of treatment (Fig. 2A, lane 1 vs lane 4). TSH and forskolin did not alter the basal RT.1B{alpha} RNA expression level. However, both TSH and forskolin resulted in a marked decrease in IFN-{gamma}-induced RT1.B{alpha} RNA expression (Fig. 2A, lane 4 vs lanes 5 and 6).



View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 2. A, Effects of TSH and forskolin on IFN-{gamma}-mediated RT1.B{alpha} gene expression. FRTL-5 thyroid cells were grown to near confluence in Coon’s modified Ham’s F-12 medium containing 5% (v/v) calf serum. The cells were maintained for 6 days with 5H medium not containing TSH and serum. The medium was replaced with fresh medium including the following additions as indicated: IFN-{gamma}, 10 ng/ml; TSH, 1 mU/ml; and forskolin, 10 µM. RNA was isolated 12 h after the final treatment and subjected to Northern analysis (20 µg/lane) using probes for RT1.B{alpha} and rat {beta}-actin. Representative Northern analyses are shown. B and C, Effects of TSH and forskolin on IFN-{gamma}-mediated CIITA gene expression. The total cellular RNA was isolated using standard procedures in FRTL-5 thyroid cells treated with TSH and/or IFN-{gamma}. First-strand cDNA was synthesized using reverse transcriptase (Life Technologies, Grand Island, NY); PCR was performed using AmpliTaq DNA polymerase (PerkinElmer, Norwalk, CT) in a PerkinElmer 9600 thermocycler. PCR conditions were: predenaturation at 94°C for 20 s, followed by 30 cycles of denaturation at 94°C for 15 s, annealing at 55°C for 45 s, and elongation at 72°C for 1 min. RT-PCR was performed at least three times, and representative analysis are shown.

 
The IFN-{gamma}-mediated induction of class II MHC requires the function of the transcriptional coactivator, CIITA, in class II MHC-negative cells such as thyroid cells (34). CIITA was expressed as a response to IFN-{gamma} and is an obligatory mediator of the IFN-{gamma}-inducible class II MHC expression. Therefore, CIITA is a critical component of the IFN-{gamma} signaling pathways that are necessary for class II MHC transcription. To investigate the regulation of CIITA expression, FRTL-5 thyroid cells cultured in TSH-depleted medium were incubated for 6, 12, and 24 h with IFN-{gamma} (10 ng/ml) in the presence or the absence of TSH (1 mU/ml). Total RNA was isolated, and RT-PCR was performed using the primers for CIITA, malic enzyme, and GAPDH. The results shown in Fig. 2, B and C, demonstrated that the FRTL-5 thyroid cells did not show any basal CIITA expression. However, IFN-{gamma} treatment induced CIITA expression after 2 h (Fig. 2C, lane 1 vs lane 2). The simultaneous treatment of TSH and IFN-{gamma} resulted in a significantly lower level of CIITA gene expression compared with the same time point of IFN-{gamma} treatment alone (Fig. 2B, lanes 2–4 vs lanes 6–8). In the same RNA samples, the action of TSH was demonstrated by a malic enzyme evaluation, which is found in the TSH/cAMP-induced genes in FRTL-5 thyroid cells (35). TSH increased the expression level of the malic enzyme transcript in the presence of IFN-{gamma} (Fig. 2B, lane 5 vs lanes 6–8), which suggests that the TSH-mediated action in FRTL-5 cells is functionally active even in the presence of IFN-{gamma}. Regardless of whether TSH pretreatment interferes with IFN-{gamma} action, IFN-{gamma} treatment was administered for relatively short periods of 2 and 4 h in FRTL-5 thyroid cells pretreated with TSH for 6 h (Fig. 2C). As shown in a previous experiment (Fig. 2B), the IFN-{gamma}-induced CIITA transcripts were reduced by TSH pretreatment. In addition, pretreatment with cycloheximide completely abolished the IFN-{gamma} effects on CIITA induction (Fig. 2C).

The IFN-{gamma} response elements in type IV CIITA promoter were targets for TSH-mediated repression

IFN-{gamma}-mediated transcriptional activation of the type IV promoter depends on cis-acting elements, which are composed of GAS (-133 to -142 bp), E box (-126 to -131 bp), and IRF (-55 to -66 bp) motifs. Two IFN-{gamma}-activated transcription factors, STAT1{alpha} and IRF-1, bind the GAS and IRF elements, respectively (12). To define the TSH-mediated repression of IFN-{gamma} action in the type IV CIITA promoter, the wild-type and mutant reporter constructs were used, in which a 1703-bp construct of the type IV CIITA 5'-flanking region was ligated into the pGL2-basic reporter construct. Fig. 3A illustrates the organization of the wild-type and mutant cis-acting elements, which are responsible IFN-{gamma}-mediated trans-activation in the type IV CIITA promoter containing 1703 bp. Each of these constructs was transiently transfected into FRTL-5 thyroid cells, and the cells were stimulated for 12 h with IFN-{gamma}. Luciferase activity was then measured, and the relative promoter activity was expressed as a multiple of the induction in the untreated control. A very low basal transcriptional activity of the full-length construct hCIITAp1.7(wild type (WT)) was detected, and a 6.3-fold induction of the CIITA promoter activity was observed after stimulation with IFN-{gamma} (Fig. 3B). TSH reduced the trans-activation of hCIITAp1.7(WT) by IFN-{gamma}. FRTL-5 thyroid cells stably transfected with the mutant constructs hCIITA-GAS, hCIITA-E-box, and hCIITA-GAS+E-box showed a lower, but significant, induction of CIITA promoter activity with IFN-{gamma} treatment. In addition, these constructs exhibited TSH-mediated repression of IFN-{gamma}-mediated trans-activation of CIITA promoter activity. However, the mutant constructs carrying the mutant IRF-1 sequence, hCIITA-IRF, and hCIITA-GAS+IRF did not show any significant induction with IFN-{gamma} treatment or TSH-mediated suppression of the promoter activities. This suggests that GAS, E-box, and IRF are critical elements for the trans-activation of CIITA by IFN-{gamma}. In addition, the TSH-mediated repression of IFN-{gamma} action involves the same regulatory motifs for IFN-{gamma} action.



View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 3. A and B, Effects of site-specific mutations in the human type IV CIITA promoter on TSH-mediated repression of IFN-{gamma}-mediated CIITA trans-activation. The cis-acting elements identified within the type IV promoter are indicated in the box. FRTL-5 thyroid cells were cotransfected with 0.4 µg of WT or mutated CIITA promoter constructs and 0.1 µg of the Renilla luciferase construct as indicated in Materials and Method. The cells were treated with IFN-{gamma} and/or TSH for 12 h. Firefly and Renilla luciferase activity were determined in triplicate, and the firefly promoter reporter activity was normalized to Renilla activity. The fold induction was calculated by dividing the luciferase activity of the IFN-{gamma}- or IFN-{gamma}/TSH-treated cells by the luciferase activity of the medium-treated cells. The results are represented as a mean ± SD from at least three experiments. C and D, Characterization of IFN-{gamma}-mediated CIITA induction in IRF-1 null cells. The total cellular RNA was isolated by standard procedures in MEFs. The first-strand cDNA was synthesized using reverse transcriptase (Life Technologies); PCR was performed using AmpliTaq DNA polymerase (PerkinElmer) in a PerkinElmer 9700 thermocycler. PCR conditions were: predenaturation at 94°C for 20 s, followed by 30 cycles of denaturation at 94°C for 15 s, annealing at 55°C for 45 s, and elongation at 72°C for 1 min. The primers used in this experiment are described in Materials and Method (C, upper panel). To detect STAT1 and IRF-1 in MEF cells, the whole cell lysates treated with IFN-{gamma} were resolved in 10% SDS-PAGE and further examined for immunoblot analyses with the anti-STAT1 Abs, anti-STAT1 tyrosine 701-phosphospecific and anti-IRF-1 Abs (C, lower panel). D, The hCIITA1.7(WT) reporter construct was transfected into IRF-1-/- and IRF-1+/- cells, and the cells were treated with mouse IFN-{gamma} (10 ng/ml) for 24 h. The fold induction was calculated by dividing the luciferase activity of the IFN-{gamma}-treated cells by the luciferase activity of the medium-treated cells. The results are represented as the mean ± SD from at least three experiments.

 
The binding motif for IRF-1 was absolute regulatory elements for IFN-{gamma}-mediated trans-activation of the type IV CIITA promoter. Hobart et al. (36) demonstrated the in vivo role of IRF-1 in CIITA mRNA induction using the IRF-1 knockout mouse. Whether IRF-1 is a critical regulatory factor in CIITA gene expression, the CIITA transcript was examined in IRF-1-null MEF after IFN-{gamma} treatment (Fig. 3C). MEFs from the IRF-1-/+ heterozygote mice exhibited CIITA expression with IFN-{gamma} treatment. However, MEFs from the IRF-/- null mice did not show CIITA gene expression following IFN-{gamma} treatment (Fig. 3C). IFN-{gamma} treatment rapidly induced the phosphorylation of 701 tyrosine residues of STAT1 in the MEFs from both IRF-1-/+ and IRF-1-/- mice (Fig. 3C), suggesting that the IFN-{gamma}-mediated STAT1 activation pathway is intact in IRF-1-deficient cells. Transfection of hCIITA1.7(WT) into IRF-1-deficient MEFs was performed to examine the IFN-{gamma}-mediated trans-activation of the type IV CIITA promoter. IFN-{gamma} failed to stimulate hCIITA1.7(WT) in IRF-1-deficient MEF cells. This suggests that the induction of the CIITA gene requires an adequate IRF-1 level with IFN-{gamma} treatment. In summary, these results suggest that IRF-1 is crucial for the IFN-{gamma}-mediated induction of CIITA in thyroid cells, and the nuclear factors that bind to GAS and the E box of the CIITA promoter might be additional factors for the maximal induction of CIITA in response to IFN-{gamma} in thyroid cells.

TSH represses IFN-{gamma}-mediated activation and induction of STAT1{alpha} and IRF-1, respectively

The regulatory elements, GAS, E-box, and IRF, are responsible for TSH-mediated repression of IFN-{gamma}-induced trans-activation of the type IV CIITA promoter. This study observed the effects of IFN-{gamma} and/or TSH on the activation and induction of the transcription factors, STAT1{alpha}, IRF-1, and USF-1, which are known to bind GAS, IRE, and the E box of the CIITA promoter, respectively, in FRTL-5 thyroid cells. IFN-{gamma} rapidly induced the maximum Y701-phosphorylated STAT1{alpha} levels within 1 h of treatment, and its level was persistently maintained for 48 h (Fig. 4A, lane 1 vs lane 2). STAT1/DNA complex formation also reached its maximum level within 1 h, which was maintained for 48 h (data not shown). In addition, IFN-{gamma} induced total STAT1 during the 6-h treatment, and its inducing effects were observed for 48 h (Fig. 4A, lane 6). IRF-1, the expression of which is dependent on STAT1{alpha}, was also induced by IFN-{gamma} treatment. IFN-{gamma}-mediated induction of IRF-1 became evident within 1 h, and its level persisted for 24 h. The USF-1 level did not change with IFN-{gamma} treatment in FRTL-5 thyroid cells (Fig. 4A). All these findings suggest that the initial activation of STAT1{alpha} phosphorylation by IFN-{gamma} is responsible for the sequential expression of IRF-1 and STAT1{alpha}.



View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 4. Effects of TSH on activation and induction of STAT1, IRF-1, and USF-1. A, FRTL-5 thyroid cells were grown to near confluence in Coon’s modified Ham’s F-12 medium containing 5% (v/v) calf serum. The cells were maintained for 6 days with 5H medium that did not contain TSH and serum. The medium was replaced with fresh medium including IFN-{gamma} (10 ng/ml), and whole cell lysates were resolved in 10% SDS-PAGE and further examined for immunoblot analyses with the anti-STAT1 Abs, anti-STAT1 tyrosine 701-phosphospecific, anti-IRF-1, and anti-USF-1 Abs. B, FRTL-5 thyroid cells were prepared as described above. FRTL-5 thyroid cells were treated with TSH (1 mU/ml) and forskolin (FSK; 10 µM) for 6 h, washed with PBS, and treated with IFN-{gamma} for an additional 30 min. The whole cell lysates were resolved in 10% SDS-PAGE and further examined for immunoblot analyses with anti-STAT1 tyrosine 701-phosphospecific Abs. A representative blot is presented. C, FRTL-5 thyroid cells were treated with IFN-{gamma} and TSH or forskolin for 2 h. The whole cell lysates were resolved in 10% SDS-PAGE and further examined for immunoblot analyses with the anti-STAT1 Abs, anti-STAT1 tyrosine 701-phosphospecific, anti-IRF-1, and anti-USF-1 Abs. D, Characterization of IFN-{gamma}-mediated IRF-1 induction in STAT1 null cells. To detect STAT1 and IRF-1 in MEF cells, whole cell lysates treated with IFN-{gamma} were resolved in 10% SDS-PAGE and further examined for immunoblot analyses with anti-STAT1 Abs, anti-STAT1 tyrosine 701-phosphospecific and anti-IRF-1 Abs.

 
The next experiment investigated the effects of TSH and forskolin on IFN-{gamma}-mediated activation and induction of STAT1 and IRF-1, respectively. Phosphorylation of the tyrosine 701(Y701) residue of STAT1{alpha} was monitored by phosphospecific Abs with IFN-{gamma} treatment. This study investigated whether pretreating FRTL-5 thyroid cells with either TSH or forskolin affects the IFN-{gamma}-induced Y701 phosphorylation of STAT1{alpha}. To accomplish this, FRTL-5 thyroid cells were treated for 6 h with either TSH or forskolin, and the cells were challenged with IFN-{gamma} and without TSH or forskolin (Fig. 4B). FRTL-5 thyroid cells pretreated with either TSH or forskolin showed a lower level of the Y701-phosphorylated form of STAT1 in response to IFN-{gamma} (Fig. 4B, lane 2 vs lanes 4 and 6). These observations suggest that the TSH-mediated induction of certain genes might be involved in the inhibition of the IFN-{gamma}/JAK signaling pathways, which activate STAT1.

To determine whether TSH and forskolin inhibit the IFN-{gamma}-induced activation or expression of STAT1, IRF-1, and USF-1, FRTL-5 cells were treated with TSH and forskolin with or without IFN-{gamma} for 2 h, and phosphorylated STAT1{alpha}, IRF-1, and USF-1 were analyzed by SDS-PAGE (Fig. 4C). Once more, the level of the Y701-phosphorylated form of STAT1{alpha} increased to a high level within 2 h of IFN-{gamma} addition (Fig. 4C, lane 1 vs lane 2). However, the simultaneous addition of IFN-{gamma} with TSH or FSK produced a significantly lower level of the Y701-phosphorylated forms of STAT1. In addition, the simultaneous addition of IFN-{gamma} with TSH and/or forskolin produced a relatively low IRF-1 level compared with the value resulting from IFN-{gamma} alone (Fig. 4C, lane 2 vs lanes 5 and 6). The level of USF-1 protein was not changed by IFN-{gamma}, TSH, and forskolin treatment.

The role of STAT1 in IRF-1 induction has been identified in STAT1 mutant cell lines, such as U3A and ME180 (37). STAT1+/- and STAT1-/- mouse MEFs were used to determine whether IRF-1 induction is dependent on STAT1 activation. MEF cells from STAT1+/- mice exhibited STAT1-Y701 phosphorylation and IRF-1 induction (Fig. 4D) in response to IFN-{gamma}. However, MEF cells obtained from the STAT1-/- mice did not show any STAT1 activation, and IRF-1 induction was not observed after the IFN-{gamma} treatment (Fig. 4D).

This study observed the mRNA level of the transcription factors IRF-1, USF-1, and STAT1{alpha} after the IFN-{gamma} treatment in the presence or the absence of TSH in FRTL-5 thyroid cells (Fig. 5). FRTL-5 thyroid cells that were maintained without TSH for 7 days were treated with IFN-{gamma} and IFN-{gamma} plus TSH. STAT1 RNA was induced for 6 h of IFN-{gamma} treatment and maintained at a high level until 48 h (Fig. 5A). The pattern of the time-dependent induction of STAT1{alpha} RNA with IFN-{gamma} treatment was similar to the results obtained by Western blot analysis (Fig. 4A). The onset of the rapid maximal induction of STAT1{alpha} RNA by IFN-{gamma} was noted 6 h after the treatment. This time point is relevant to the increase in STAT1{alpha} protein level. IRF-1 RNA was induced more rapidly than STAT1{alpha} with IFN-{gamma} treatment (Fig. 5A), and the time for IRF-1 RNA induction was also correlated with the increase in the IRF-1 protein level (Fig. 4A). USF-1 RNA was not changed by IFN-{gamma} treatment (Fig. 5A). Simultaneous treatment with IFN-{gamma} and TSH also increased IRF-1 and STAT1{alpha} levels compared with the untreated control values. However, the IRF-1 and STAT1{alpha} expression levels were lower than the IRF-1 and STAT1{alpha} levels with IFN-{gamma} alone (Fig. 5A). In addition, the maximum inducibilities of STAT1{alpha} and IRF-1 RNA were significantly blunted (Fig. 5, B and C). For the mRNA stability experiments, the cells were further cultured in the presence or the absence of actinomycin D (5 µg/ml) for the indicated times after TSH and/or IFN-{gamma} treatment before RNA was isolated. Five to 10 µg of the RNA was analyzed for IRF-1 mRNA expression by an RNase protection assay. This study did not find that TSH increases the level of IRF-1 RNA degradation in this experiment (data not shown).



View larger version (53K):
[in this window]
[in a new window]
  		FIGURE 5. Effect of TSH and on IFN-{gamma}-induced STAT1, IRF-1, and USF-1 RNA levels. A, After the FRTL-5 cells were grown to near confluence in complete 6H medium with 5% serum, the cells were maintained for 6 days with 5H medium that did not contain TSH. TSH-starved FRTL-5 cells were stimulated with TSH (1 mU/ml), IFN-{gamma} (10 ng/ml), or both. Total RNA was isolated and subjected to Northern analyses using probes for STAT1, USF-1, IRF-1, and {beta}-actin as described in Materials and Methods. The results shown are representative Northern analyses from one experiment with 20 µg RNA/lane. B and C, Results from A were quantified using an BAS 2000 image analyzer (Fuji, Tokyo, Japan.). Relative ratios between STAT1 and IRF-1 RNA and {beta}-actin RNA are presented as a multiple of the unstimulated control value. The results are represented as the mean ± SD of the values obtained from three separate experiments.

 
TSH induces SOCS-1 and -3 through the induction and activation of STAT3 in thyroid cells

The results showing that TSH represses IFN-{gamma} action, inhibits the Y701 phosphorylation of STAT1, and inhibits STAT1{alpha} and IRF-1 RNA induction suggest that TSH might be able to induce the negative regulators of IFN-{gamma} signaling pathways. Northern blot analysis was performed for SOCS-1, SOCS-2, SOCS-3, cytokine-inducible SH2 protein (CIS), PIAS-1, and PIAS-3. SOCS-2 RNA was not expressed in e FRTL-5 thyroid cells, and CIS, PIAS-1, and PIAS-3 RNA were not changed by either TSH or forskolin (data not shown). SOCS-1 and SOCS-3 RNA were induced by TSH addition in FRTL-5 cells; this was maintained in the absence of TSH for 7 days (Fig. 6A). The SOCS-1 and SOCS-3 RNA levels in the untreated cells were very low, but SOCS-1 and SOCS-3 RNA were induced within 2 h after TSH addition (Fig. 6A). The SOCS-1 RNA expression level reached a maximum around 2 h, but gradually decreased to the basal level 6 h after TSH treatment. SOCS-3 RNA was induced by TSH within 2 h and maintained its highest level until 12 h (Fig. 6A). IFN-{gamma} is a known SOCS-1 and SOCS-3 inducer by activating STATs, and this study found similar effects, as shown in Fig. 6A; IFN-{gamma} stimulated SOCS-1 and SOCS-3 RNA expression. The simultaneous addition of IFN-{gamma} with TSH resulted in a low SOCS-1 RNA expression level at 6 and 12 h compared with IFN-{gamma} alone (Fig. 6A, lanes 8 and 9 vs lanes 13 and 14). However, costimulation of IFN-{gamma} with TSH showed a higher SOCS-3 RNA expression level compared with IFN-{gamma} alone (Fig. 6A, lanes 4 and 5 vs lanes 14 and 15).



View larger version (55K):
[in this window]
[in a new window]
  		FIGURE 6. Effects of TSH and/or IFN-{gamma} on SOCS-1 and SOCS-3 RNA levels as well as the inhibitory roles of SOCS-1 and -3 on the CIITA promoter. A, After FRTL-5 cells were grown to near confluence in complete 6H medium with 5% serum, the cells were maintained for 6 days with 5H medium that did not contain TSH. The TSH-starved FRTL-5 cells were stimulated with TSH (1 mU/ml), IFN-{gamma}, and IFN-{gamma}/TSH. Total RNA was isolated and subjected to Northern analysis using probes for SOCS-1 and SOCS-3 described in Materials and Methods. The results shown are representative Northern analyses from four separate experiments with 20 µg RNA/lane. B, FRTL-5 thyroid cells were cotransfected with 0.4 µg of the WT CIITA promoter construct (hCIITA1.7(WT)) and 0.1 µg of the Renilla luciferase construct with or without the pEF-SOCS-1 and -3 expression plasmids. The cells were treated with IFN-{gamma} for 24 h. The firefly and Renilla luciferase activities were determined in triplicate, and the firefly promoter reporter activity was normalized to the Renilla activity. The fold induction was calculated by dividing the luciferase activity of the IFN-{gamma}-treated samples by the luciferase activity of the medium-treated sample. The results are represented as the mean ± SD from at least three experiments.

 
The hCIITA1.7(WT) constructs were transfected into FRTL-5 thyroid cells in the absence or the presence of SOCS-1 and -3 expression plasmids to observe the effects of SOCS-1 and -3 on IFN-{gamma}-induced up-regulation of the CIITA promoter. As shown in Fig. 6B, SOCS-1 and SOCS-3 expression caused inhibition of the IFN-{gamma}-induced increase in CIITA promoter activities. These results suggest that SOCS-1 and SOCS-3 are both active in repressing IFN-{gamma}-mediated CIITA induction in FRTL-5 thyroid cells.

The expression of SOCSs was dependent on STAT activation in various systems. The expression and post-translational modification of STATs were observed after TSH addition to determine whether the TSH-mediated induction of SOCS-1 and SOCS-3 requires STAT activation. TSH is known to activate the cAMP/protein kinase A (PKA) system, and the rapid S133 phosphorylation of CREB (Fig. 7A) was observed in thyroid cells. 133S phosphorylation was dependent on PKA because H89 (10 µM), a specific PKA inhibitor, completely abolished the TSH-induced S133 phosphorylation of CREB (data not shown). In this study the expression of STAT1 and STAT3 was observed by RT-PCR and Northern blot analysis after treatment with TSH (1 mU/ml). STAT1{alpha} RNA was not induced by TSH (data not shown), but we observed a significant induction of STAT3 RNA by TSH in FRTL-5 thyroid cells (Fig. 7, B and C). As a control experiment, TSH induced a malic enzyme transcript that is induced by the TSH-induced cAMP/PKA-dependent gene (35). The TSH-mediated induction of the malic enzyme transcript was reduced by pretreatment of H89 (Fig. 7C). In a similar experiment TSH-mediated induction of the STAT3 transcript was also decreased by H89 (Fig. 7C). TSH also increased STAT3 RNA expression (Fig. 7B). In addition, this study found that TSH treatment in FRTL-5 thyroid cells results in the Y705 and S727 phosphorylation of STAT3 (Fig. 7D), which is important for DNA binding and the maximal transcriptional activation of STAT3.



View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 7. Effects of TSH on the expression and post-translational modification of STAT3. A, FRTL-5 thyroid cells treated with TSH (1 mU/ml), and whole cell lysates were obtained and separated by 10% SDS-PAGE. The level of phosphorylated CREB S133 was measured by phosphospecific Abs. B, The total RNA was isolated and subjected to Northern analyses using the probes for STAT3 described in Materials and Methods. The results shown are representative Northern analyses from one experiment with 20 µg RNA/lane. C, Total cellular RNA was isolated by standard procedures in FRTL-5 thyroid cells treated with TSH and/or H89. First-strand cDNA was synthesized using reverse transcriptase (Life Technologies); PCRs for detecting STAT3 and malic enzyme were performed using AmpliTaq DNA polymerase (PerkinElmer) in a PerkinElmer 9600 thermocycler. D, FRTL-5 thyroid cells treated with TSH (1 mU/ml) and PMA (10 µM). Whole cell lysates were obtained and separated by 10% SDS-PAGE. The levels of phosphorylated STAT3-S727 and STAT3-Y705 were measured by phosphospecific Abs. E and F, The reporter containing the -478/-229 fragment of the STAT3 promoter in front of the minimal junB promoter-luciferase gene, (p478/229 Luc) was tested for TSH responsiveness. The three p478/229 Luc reporter constructs, the WT, that with a mutated STAT-binding element (mSBE), and that with a mutated CRE (mCRE), were tested for TSH responsiveness. Luciferase reporter plasmids containing the 2.2-kb STAT3 promoter (p2166 STAT3 Luc) and the three p478/229 Luc reporter constructs (WT, mCRE, and mSBE) were cotransfected with the Renilla luciferase constructs. The relative luciferase activities from three independent experiments are shown.

 
The next experiment observed the effects of TSH on the promoter activity of STAT3 in thyroid cells. The WT full-length STAT3 promoter (p2166 STAT3 Luc) and STAT3 promoter constructs containing the -478/-229 fragment of the STAT3 promoter were transfected in FRTL-5 thyroid cells with Lipofectamine (Fig. 7E). As shown in Fig. 7E, TSH increased full-length promoter (p2166 STAT3 Luc) activity by 4.5-fold and also increased the promoter activities of the p478/299 Luc constructs, which contains the canonical CRE and the STAT3-binding element by 6.5-fold (Fig. 7E). However, p478/299 Luc, which contains the mutant CRE and the mutant STAT-binding element, did not show an increase in promoter activity in response to TSH. Therefore, TSH-mediated activation of STAT3 involves STAT3 gene expression in cooperation with the CRE-binding protein. This regulatory mechanism is similar to that of the junB gene, which is activated by IL-6 through junB IL-6RE, which contains a low affinity STAT3-binding site and a CRE-like site (31).

After identifying that TSH is responsible for both activating and inducing STAT3 in the TSH-responsive FRTL-5 thyroid cells, two lines of experiments were performed to confirm that STAT3 is essential for TSH-mediated SOCS-3 induction. Recently, Tuschl et al. (38) demonstrated that RNA interference could be provoked in mammalian cell lines through the introduction of siRNA. The mediators of the sequence-specific mRNA degradation are the 21-23-nt siRNA duplexes that trigger the specific gene silencing in mammalian somatic cells without activating the unspecific IFN response. To determine whether STAT3 expression could be knocked down using siRNA, 21-nt siRNA duplexes specific to rat STAT3 were synthesized to transfect FRTL-5 thyroid cells. As shown in Fig. 8A, five different siRNAs for rat STAT3 were synthesized and tested for their effects by Western blot analysis. The siRNA, which targeted 145–165 bp of coding region, siRNA-STAT3–145, was effective in silencing the endogenous STAT3 (Fig. 8A). The silencing effect persisted for 72 h after the transfection of siRNA-STAT3–145. The USF-1 protein level was not altered by transfection with siRNA-STAT3–145. After the transfection of siRNA-STAT3–145, the siRNA-transfected cells were treated with TSH, and total RNA was obtained for Northern blot analysis of SOCS-3. The siRNA for STAT3 significantly reduced the maximum level of SOCS-3 in response to TSH compared with the control siRNA-transfected cells (Fig. 8B).



View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 8. Involvement of STAT3 in TSH-mediated SOCS-3 induction in FRTL-5 thyroid cells. A, Schematic of rat STAT3 mRNA and relative target locations (122–142, 145–165, 226–246, 476–496, and 791–811 bp) of the siRNAs for rat STAT3 used in this studies. Double-stranded RNA (2.5 µmol) was transfected in FRTL-5 cells by electroporation as described in Materials and Methods. The cells were harvested at 18 or 36 h to detect endogenous STAT3 by Western blot assays. B, Total RNA was isolated from the FRTL-5 cells transfected with siRNAs and subjected to Northern analyses using the probes for SOCS-3 described in Materials and Methods. The results shown are representative Northern analyses from one experiment with 20 µg RNA/lane. C, FRTL-5 cells stably transfected with RcCMV, RcCMV-STAT3-Y705F, and STAT3-S727A cultured in 6-cm dishes were transfected with Lipofectamine with the m67 reporter construct (100 ng) for 48 h. The cells were treated with TSH (1 mU/ml) or PMA (10 µM). The relative luciferase activities of the whole cell extracts are expressed on the y-axis as the mean ± SD. Experiments were performed in quadruplicate and were normalized to the Renilla luciferase activity. D, The total RNA was isolated from the stable cells and subjected to Northern analyses using the probes for SOCS-3 described in Materials and Methods. The results shown are representative Northern analyses from one experiment with 20 µg RNA/lane.

 
This study developed stable cell lines that carried the mutant STAT3, STAT3-Y705F, and STAT3-S727A, which have two mutated phosphorylation sites. To confirm that the mutant cell lines are defective in STAT3-dependent transcriptional activation, those cells were transfected with the m67 reporter construct, which has been reported to be a STAT3-responsive construct (39). The increase in m67 reporter activity produced by TSH or PMA was significantly lower in the STAT3-Y705F and STAT3-S727A stable cells compared with the RcCMV stable cells (Fig. 8C). Furthermore, the TSH induction of SOCS-3 was not observed in the STAT3-Y705F and STAT3-S727A stable cells (Fig. 8D). Overall, these observations suggest that the TSH-mediated induction of SOCS-3 requires endogenous STAT3 and its activation in FRTL-5 thyroid cells.

Role of STAT3 in TSH repression of IFN-{gamma}-mediated CIITA and MHC class II expression

The above results suggest that TSH-mediated STAT3 activation is involved in SOCS induction, and this resulted in a lower biological response to IFN-{gamma} in FRTL-5 thyroid cells. To confirm the role of STAT3 in the TSH-mediated suppression of IFN-{gamma}-induced CIITA gene expression, the stable cell lines that express the dominant negative mutant STAT3, RcCMV-STAT3-Y705F and RcCMV-STAT3-S727A, were established. TSH treatment resulted in the inhibition of IFN-{gamma}-induced phosphorylation of STAT1 and the induction of IRF-1 in the RcCMV stable cells (Fig. 9A, lane 2 vs lane 3). The stable cells expressing the STAT3 mutants showed rapid tyrosine phosphorylation of STAT1 and induction of IRF-1 in response to IFN-{gamma}. However, the TSH inhibition of STAT1 tyrosine phosphorylation and IRF-1 induction by IFN-{gamma} were not found in the stable cells expressing STAT3-Y705F and STAT3-S727A (Fig. 9A, lane 5 vs lane 6, lane 8 vs lane 9).



View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 9. Effects of STAT3 and dominant negative STAT3 mutant on TSH repression of IFN-{gamma}-induced CIITA and RT1.B expression. A, The FRTL-5 stable cell lines expressing RcCMV and STAT3 mutants were grown to near confluence in Coon’s modified Ham’s F-12 medium containing 5% (v/v) calf serum, and the cells were maintained for 6 days with 5H medium without TSH and serum. The medium was replaced with fresh medium including TSH (1 mU/ml) and/or IFN-{gamma} (10 ng/ml). The whole cell lysates were resolved in 10% SDS-PAGE and further examined for immunoblot analyses with the anti-STAT1 Abs, anti-STAT1 tyrosine 701 phosphospecific, anti-IRF-1, anti-USF-1, and anti-{beta}-actin Abs. B, The FRTL-5 stable cell lines expressing WT STAT3 and mutant STAT3-Y705F were treated with IFN-{gamma} in the presence or the absence of TSH. Total cellular RNA was isolated by standard procedures in FRTL-5 thyroid cells treated with TSH and/or IFN-{gamma}. The first-strand cDNA was synthesized using reverse transcriptase (Life Technologies); PCR with the rat CIITA primer (see Materials and Methods) was performed using AmpliTaq DNA polymerase (PerkinElmer) in a PerkinElmer 9700 thermocycler. To define the level of SOCS-3 expression under the above conditions, total RNA was isolated and subjected to Northern analysis using the probes for SOCS-3 described in Materials and Methods. The results shown are representative Northern blot analyses from four separate experiments with 20 µg RNA/lane. C, FRTL-5 stable cell lines expressing WT STAT3 and mutant STAT3-Y705F were treated with IFN-{gamma} in the presence or the absence of TSH. After the indicated time, the level of expression of RT1.B was analyzed by FACS as described in Fig. 1.

 
FRTL-5 thyroid cells expressing the dominant negative form of STAT3, RcCMV-STAT3-Y705F, showed IFN-{gamma}-induced CIITA expression (Fig. 9B, lane 5). However, TSH repression of IFN-{gamma}-induced CIITA expression was not observed (Fig. 9B, lane 6) in FRTL-5 thyroid cells stably expressing the dominant negative form of STAT3. As shown in Fig. 6A, SOCS-3 expression persisted for 24 h in TSH- or TSH/IFN-{gamma}-treated FRTL-5 thyroid cells. However, FRTL-5 thyroid cells expressing the dominant negative STAT3 exhibited a reduced SOCS-3 expression level in response to TSH (Fig. 8D) and TSH/IFN-{gamma} (Fig. 9B, lane 3 vs lane 6). To determine whether STAT3 is involved in the TSH repression of IFN-{gamma} induced expression of class II MHC expression, FRTL-5 rat thyroid cells stably expressing the dominant negative forms of STAT3 (RcCMV-STAT3-Y705F) were examined for their expression of the rat class II MHC Ag, RT1.B, in response to IFN-{gamma} in the presence or the absence of TSH (Fig. 9C). The cells were cultured in the presence of IFN-{gamma} with or without TSH for 48 h. The cells were washed and stained with fluorescein-labeled anti-rat RT1.B mAbs, and the cell surface RT1.B was quantified using a flow cytometry. The RT1.B expression level in FRTL-5 cells expressing RcCMV-STAT3-Y705F showed no changes in basal and IFN-{gamma}-induced RT1.B expression compared with WT cells (data not shown). However, the cells incubated in the presence of TSH and IFN-{gamma} did not show any suppression of RT1.B expression (Fig. 9C). These observations suggest STAT3/SOCS-3 are responsible for TSH repression of the biological responses elicited by IFN-{gamma} in thyroid cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The biological effects of IFN-{gamma} have long been studied in a number of cell types (40, 41). The signal transduction pathway initiated by the binding of IFN-{gamma} to its receptor leads to the sequential activation of the JAK1 and -2/STAT1 signaling pathways (42). The activation of this pathway results in the regulation of a number of IFN-{gamma}-responsive genes through transcriptional activation of the promoter, which has a GAS known as a binding site for the STAT1 homodimer (43). The negative regulation of IFN-{gamma}-activated JAK/STAT signaling has just begun to be investigated, and recently at least three families of proteins, SH2-containing tyrosine phosphatases, PIAS, and SOCS, were found to be involved in the negative regulation of IFN-{gamma} action (20, 21, 22, 23, 24). SOCS-1 and SOCS-3 have been demonstrated to inhibit the signaling induced by IFN-{gamma} by intervening at the proximal step in the signal cascade, inhibiting the JAK-mediated phosphorylation and homodimerization of STAT (44, 45, 46).

SOCSs are regulated by primarily by the STATs activated by cytokines and hormones. FRTL-5 cells have long been studied as TSH-responsive thyroid cell lines, and these cells express SOCS-1 and SOCS-3 in response to TSH and IFN-{gamma}. In previous experiments TSH has been shown to prevent the maximum induction of IFN-{gamma} target genes, such as ICAM-1, in response to IFN-{gamma} in thyroid cells (19). TSH repression of the IFN-{gamma}-induced ICAM-1 gene results from suppression of the maximum phosphorylation of the Y701 residue in STAT1 by IFN-{gamma} (19). The same phenomenon of TSH suppression of the IFN-{gamma}-induced Y701 phosphorylation of STAT1 was observed in this study. Because TSH suppression is evident in the presence of the tyrosine phosphatase inhibitors, this phenomenon may result from the inhibition of JAKs. This study showed that TSH was able to inhibit the tyrosine phosphorylation of JAK1, but not JAK2, in response to IFN-{gamma} in FRTL-5 thyroid cells. The interactions between the TSH-induced SOCSs and JAK1 may play role in the inactivation of JAK1 in thyroid cells (25).

If the above observations are valid, then TSH might inhibit the IFN-{gamma}-induced MHC class II expression in thyroid cells. As expected, TSH and forskolin were able to inhibit the maximum IFN-{gamma}-induced expression of RT1.B in FRTL-5 thyroid cells (Fig. 1). Insulin treatment did not mimic the actions of TSH and forskolin, and it was unable to inhibit the maximum IFN-{gamma}-induced expression of RT1.B in FRTL-5 thyroid cells (data not shown). This suggests that the TSH/cAMP signaling cascade might be involved in the suppression of IFN-{gamma}-induced expression of RT1.B in FRTL-5 thyroid cells. Because CIITA is an obligatory mediator of IFN-{gamma}-induced MHC class II expression, the effects of TSH on the IFN-{gamma}-induced expression of CIITA were examined by RT-PCR. The TSH repression of IFN-{gamma}-induced CIITA expression was observed 2 h after TSH addition, and marked repression was noted 6 h after TSH addition. It was reported that CIITA is phosphorylated and inactivated by PKA, and this phenomenon is believed to be a plausible mechanisms for the repression of MHC class II expression by cAMP and PGE2 (47). Because TSH activates cAMP/PKA signaling pathways through the binding of its G protein-coupled receptor in FRTL-5 thyroid cells, it is possible that inactivating the phosphorylation of CIITA by TSH-activated PKA may also be involved in the TSH repression of class II expression. To define the role of cAMP/PKA on CIITA function in thyroid cells, it may necessary to confirm the post-translational modifications, such as the phosphorylation of CIITA by the TSH stimulations.

Recently it was shown that CIITA gene expression is controlled by the alternative use of multiple distinct promoters; constitutive expression in dendritic cells and B lymphocytes by promoters I and II, respectively, and IFN-{gamma}-inducible expression by promoter IV (11). Sequence analysis of the type IV CIITA promoter demonstrated the sequences of numerous cis-acting elements, including GAS, an E box, and an IRF element within 154 bp from the translation initiation site (13). The last three cis-acting elements, GAS, E box, and IRF element, are involved in IFN-{gamma}-mediated CIITA transcription (Fig. 3) in thyroid cells. A deletion of the GAS and E box elements results in reduced CIITA promoter activity, but these mutant constructs still exhibited significant IFN-{gamma} responsiveness, which is mediated by the IRF element. Interestingly, the IRF-1 mutant construct showed a complete inability to induce CIITA promoter activities by IFN-{gamma}. This suggests that the IRF element plays a crucial role in IFN-{gamma} induction of the type IV CIITA promoter. These results suggest that the transcription factors that bind to the IRF elements are essential in mediating the IFN-{gamma} induction of CIITA and MHC class II expression in thyroid cells. These observations were further supported by the absence of IFN-{gamma} responsiveness in the induction of CIITA promoter activity in IRF-1-deficient MEF cells, which showed normal Y701 phosphorylation of STAT1 (Fig. 3, C and D). This study found that IRF-1 induction is entirely dependent on STAT1 activation by IFN-{gamma} in thyroid cells. TSH prevents induction of the IRF-1 protein and mRNA by inhibiting IFN-{gamma}-induced Y701 STAT1 phosphorylation.

TSH induces SOCS-1 and SOCS-3, but CIS and SOCS-2 were not induced by TSH in thyroid cells (25). The expression patterns of SOCS-1 and SOCS-3 by TSH are different. SOCS-1 showed the highest expression level ~2 h after TSH treatment; SOCS-3 showed a rapid induction, but its expression level gradually increased until 24 h. This study observed several time points of the combined TSH/IFN-{gamma} effects on SOCS-1 and SOCS-3 expression (Fig. 6). Although SOCS-1 expression was inhibited in the TSH plus IFN-{gamma} group compared with IFN-{gamma} alone group, SOCS-3 expression was actually higher in the cells treated with TSH and IFN-{gamma} compared with IFN-{gamma} alone (Fig. 6). These observations suggest that SOCS-3 may have a more potent and prolonged TSH-induced inhibitory effect on IFN-{gamma} signaling in thyroid cells.

This study suggests that TSH mediates SOCS induction through STAT3 induction and activation in thyroid cells. The STAT3 gene promoter has a functional STAT3 binding site and a CRE, which are functional elements for IL-6 induction of STAT3 (30). TSH can activate CREB through cAMP/PKA and can also phosphorylate the S727 residue of STAT3. These two pathways activated by TSH are involved in transcriptional activation of the STAT3 promoter. TSH has been shown to phosphorylate the tyrosine 705 residue in FRTL-5 thyroid cells. Several serine/threonine kinases have been identified as the serine kinases for the S727 residue of STAT3 (48). p38 and the extracellular signal-regulated kinases (ERKs) are not involved in the TSH-mediated S727 phosphorylation of STAT3 in FRTL-5 thyroid cells, because TSH did not activate p38 or the mitogen-activated protein kinases (ERK1/ERK2; data not shown), which are known to phosphorylate the S727 residue of STAT3. Recently, FKBP12-rapamycin-associated protein/mammalian target of rapamycin (FRAP/mTOR) was identified as a STAT3 serine kinase (49). TSH is able activate S6K1, which acts in the downstream kinase of FRAP/mTOR in FRTL-5 thyroid cells (J. M. Suh et al., unpublished observations). In addition, the results showing that rapamycin inhibits the TSH-induced S727 phosphorylation of STAT3 (data not shown) suggest that the TSH-mediated FRAP/mTOR pathways are important for regulating STAT3. TSH-mediated STAT3 activation and induction may play a role in the cellular proliferation of thyroid cells because STAT3 activation is usually related to the increase in the growth potential of the cells (50).

The critical role of STAT3 in the TSH repression of IFN-{gamma}-induced CIITA and MHC class II expression was further supported by the results with the FRTL-5 thyroid cell line carrying the dominant negative form of STAT3 and siRNA-STAT3. Lowering the endogenous STAT3 levels by siRNA-STAT3 significantly hampered SOCS-3 expression. Furthermore, TSH-induced SOCS-3 expression was lower in the STAT3 mutant cell lines. Finally, TSH repression of IFN-{gamma}-induced CIITA and RT1.B expression was not noted in the stable STAT3 mutant cell lines. In previous reports TSH was able to inhibit transcription of the MHC class I genes in FRTL-5 thyroid cells by inducing and activating several transcription factors. The MHC class I gene showed constitutive expression in the thyroid gland and was up-regulated in thyroid epithelial cells during the process of autoimmune thyroid diseases. This study did not observe the TSH effects on IFN-{gamma}-induced MHC class I expression in thyroid cells. Although CIITA is involved in MHC class I gene transcription (51), it has no essential roles, unlike in MHC class II gene transcription (52, 53). However, TSH intervenes in the proximal steps of the IFN-{gamma} signaling pathways; IFN-{gamma}-mediated MHC class I gene induction might also have been repressed by TSH in thyroid cells.

TSH is a major regulator of the proliferation and function of thyroid cells. Several TSH-responsive gene products, thyroperoxidase and thyroglobulin, are major autoantigens in autoimmune thyroid diseases. In previous studies TSH was reported to be involved in the repression of MHC class I through the suppression of transcription (54, 55, 56). These MHC class I results and this report suggest that TSH, which is a major regulator of thyrocyte function, is also engaged in immunomodulation through signal cross-talk with the cytokines in thyroid cells. It is suggested that the phosphoinositol 3-kinase-dependent FRAP/mTOR and cAMP/PKA pathways are closely related to the induction and activation of STAT3 in thyroid cells. One of effects in TSH-mediated STAT3 activation is that SOCS-1 and -3 induction plays a role in imparting the thyroid cells with resistance to IFN-{gamma} during the thyrocyte growth phase.


   Acknowledgments
 
We acknowledge Drs. Starr and Hilton for the expression vectors pEF-mSOCS-1 and pEF-mSOCS-3, Dr. Benveniste for the type IV CIITA promoter constructs, Dr. Bromberg for the expression vectors RcCMV-STAT3 and RcCMV-STAT3-Y705F, and Dr. Hirano for the STAT3 promoter constructs.


   Footnotes
 
1 This work was supported by the National Research Laboratory Program (M1-0104-00-0014), Ministry of Science and Technology, Korea. Back

2 Current address: Department of Oral Biochemistry, College of Dentistry, Dental Science Research Institute, Chonnam National University, 300 Yongbong-dong, Buk-ku, Kwangju 500-757, Korea. Back

3 Address correspondence and reprint requests to Dr. Minho Shong, Laboratory of Endocrine Cell Biology, Department of Internal Medicine, Chungnam National University School of Medicine, 640 Daesadong, Chungku, Taejon 301-721, Korea. E-mail address: minhos{at}cnu.ac.kr Back

4 Abbreviations used in this paper: CIITA, class II trans-activator; ERK, extracellular signal-regulated kinase; GAS, IFN-{gamma}-activated site; 6H, six hormone; IRF, IFN regulatory factor; JAK, Janus kinase; MEF, mouse embryonic findroblasts; PIAS, protein inhibitors of activated STAT; PKA, protein kinase A; SOCS, suppressor of cytokine signaling; siRNA, small interfering RNA; TSH, thyroid-stimulating hormone; USF, upstream stimulating factor-1; WT, wild type; CIS, cytokine-inducible SH2 protein; FRAP/mTOR, FKBP12-rapamycin-associated protein/mammalian target of rapamycin. Back

Received for publication June 17, 2002. Accepted for publication May 1, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Chapman, H. A.. 1998. Endosomal proteolysis and MHC class II function. Curr. Opin. Immunol. 10:93.[Medline]
  2. McDevitt, H. O.. 1998. The role of MHC class II molecules in susceptibility and resistance to autoimmunity. Curr. Opin. Immunol. 10:677.[Medline]
  3. Pieters, J.. 2000. MHC class II-restricted antigen processing and presentation. Adv. Immunol. 75:159.[Medline]
  4. Singer, D. S., E. Mozes, S. Kirshner, L. D. Kohn. 1997. Role of MHC class I molecules in autoimmune disease. Crit. Rev. Immunol. 17:463.[Medline]
  5. Mach, B., V. Steimle, E. Martinez-Soria, W. Reith. 1996. Regulation of MHC class II genes: lessons from a disease. Annu. Rev. Immunol. 14:301.[Medline]
  6. Waldburger, J. M., K. Masternak, A. Muhlethaler-Mottet, J. Villard, M. Peretti, S. Landmann, W. Reith. 2000. Lessons from the bare lymphocyte syndrome: molecular mechanisms regulating MHC class II expression. Immunol. Rev. 178:148.[Medline]
  7. Boss, J. M.. 1997. Regulation of transcription of MHC class II genes. Curr. Opin. Immunol. 9:107.[Medline]
  8. Harton, J. A., J. P. Ting. 2000. Class II transactivator: mastering the art of major histocompatibility complex expression. Mol. Cell. Biol. 20:6185.[Free Full Text]
  9. Collawn, J. F., E. N. Benveniste. 1999. Regulation of MHC class II expression in the central nervous system. Microbes Infect. 1:893.[Medline]
  10. Otten, L. A., V. Steimle, S. Bontron, B. Mach. 1998. Quantitative control of MHC class II expression by the transactivator CIITA. Eur. J. Immunol. 28:473.[Medline]
  11. Muhlethaler-Mottet, A., L. A. Otten, V. Steimle, B. Mach. 1997. Expression of MHC class II molecules in different cellular and functional compartments is controlled by differential usage of multiple promoters of the transactivator CIITA. EMBO J. 16:2851.[Medline]
  12. Muhlethaler-Mottet, A., W. Di Berardino, L. A. Otten, B. Mach. 1998. Activation of the MHC class II transactivator CIITA by interferon-{gamma} requires cooperative interaction between Stat1 and USF-1. Immunity 8:157.[Medline]
  13. Dong, Y., W. M. Rohn, E. N. Benveniste. 1999. IFN-{gamma} regulation of the type IV class II transactivator promoter in astrocytes. J. Immunol. 162:4731.[Abstract/Free Full Text]
  14. Kohn, L. D., H. Shimura, Y. Shimura, A. Hidaka, C. Giuliani, G. Napolitano, M. Ohmori, G. Laglia, M. Saji. 1995. The thyrotropin receptor. Vitam. Horm. 50:287.[Medline]
  15. Weetman, A. P., A. M. McGregor. 1994. Autoimmune thyroid disease: further developments in our understanding. Endocr. Rev. 15:788.[Abstract/Free Full Text]
  16. Tomer, Y., T. F. Davies. 1993. Infection, thyroid disease, and autoimmunity. Endocr. Rev. 14:107.[Abstract/Free Full Text]
  17. Shimojo, N., Y. Kohno, K. Yamaguchi, S. Kikuoka, A. Hoshioka, H. Niimi, A. Hirai, Y. Tamura, Y. Saito, L. D. Kohn, et al 1996. Induction of Graves-like disease in mice by immunization with fibroblasts transfected with the thyrotropin receptor and a class II molecule. Proc. Natl. Acad. Sci. USA 93:11074.[Abstract/Free Full Text]
  18. Shimojo, N., T. Arima, K. Yamaguchi, S. Kikuoka, L. D. Kohn, Y. Kohno. 2000. A novel mouse model of Graves’ disease: implications for a role of aberrant MHC class II expression in its pathogenesis. Int. Rev. Immunol. 19:619.[Medline]
  19. Chung, J., E. S. Park, D. Kim, J. M. Suh, H. K. Chung, J. Kim, H. Kim, S. J. Park, O. Y. Kwon, H. K. Ro, et al 2000. Thyrotropin modulates interferon-{gamma}-mediated intercellular adhesion molecule-1 gene expression by inhibiting Janus kinase-1 and signal transducer and activator of transcription-1 activation in thyroid cells. Endocrinology 141:2090.[Abstract/Free Full Text]
  20. Starr, R., T. A. Willson, E. M. Viney, L. J. Murray, J. R. Rayner, B. J. Jenkins, T. J. Gonda, W. S. Alexander, D. Metcalf, N. A. Nicola, et al 1997. A family of cytokine-inducible inhibitors of signalling. Nature 387:917.[Medline]
  21. Naka, T., M. Narazaki, M. Hirata, T. Matsumoto, S. Minamoto, A. Aono, N. Nishimoto, T. Kajita, T. Taga, K. Yoshizaki, et al 1997. Structure and function of a new STAT-induced STAT inhibitor. Nature 387:924.[Medline]
  22. Liu, B., J. Liao, X. Rao, S. A. Kushner, C. D. Chung, D. D. Chang, K. Shuai. 1998. Inhibition of Stat1-mediated gene activation by PIAS1. Proc. Natl. Acad. Sci. USA 95:10626.[Abstract/Free Full Text]
  23. You, M., D. H. Yu, G. S. Feng. 1999. Shp-2 tyrosine phosphatase functions as a negative regulator of the interferon-stimulated Jak/STAT pathway. Mol. Cell. Biol. 19:2416.[Abstract/Free Full Text]
  24. Endo, T. A., M. Masuhara, M. Yokouchi, R. Suzuki, H. Sakamoto, K. Mitsui, A. Matsumoto, S. Tanimura, M. Ohtsubo, H. Misawa, T. Miyazaki, et al 1997. A new protein containing an SH2 domain that inhibits JAK kinases. Nature 387:921.[Medline]
  25. Park, E. S., H. Kim, J. M. Suh, S. J. Park, O. Y. Kwon, Y. K. Kim, H. K. Ro, B. Y. Cho, J. Chung, M. Shong. 2000. Thyrotropin induces SOCS-1 (suppressor of cytokine signaling-1) and SOCS-3 in FRTL-5 thyroid cells. Mol. Endocrinol. 14:440.[Abstract/Free Full Text]
  26. Kohn, L. D., W. A. Valente, E. F. Grollman, S. M. Aloj, and P. Vitti. 1986. Clinical determination and/or quantification of thyrotropin and a variety of thyroid stimulatory or inhibitory factors performed in vitro with an improved cell line. U.S. Patent 4,609,622.
  27. Matsuyama, T., T. Kimura, M. Kitagawa, K. Pfeffer, T. Kawakami, N. Watanabe, T. M. Kundig, R. Amakawa, K. Kishihara, A. Wakeham, et al 1993. Targeted disruption of IRF-1 or IRF-2 results in abnormal type I IFN gene induction and aberrant lymphocyte development. Cell 75:83.[Medline]
  28. Meraz, M. A., J. M. White, K. C. Sheehan, E. A. Bach, S. J. Rodig, A. S. Dighe, D. H. Kaplan, J. K. Riley, A. C. Greenlund, D. Campbell, et al 1996. Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell 84:431.[Medline]
  29. Lowe, S. W., T. Jacks, D. E. Housman, H. E. Ruley. 1994. Abrogation of oncogene-associated apoptosis allows transformation of p53-deficient cells. Proc. Natl. Acad. Sci. USA 91:2026.[Abstract/Free Full Text]
  30. Ichiba, M., K. Nakajima, Y. Yamanaka, N. Kiuchi, T. Hirano. 1998. Autoregulation of the Stat3 gene through cooperation with a cAMP-responsive element-binding protein. J. Biol. Chem. 273:6132.[Abstract/Free Full Text]
  31. Hirano, T., K. Ishihara, M. Hibi. 2000. Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors. Oncogene 19:2548.[Medline]
  32. Kim, H., T. H. Lee, E. S. Park, J. M. Suh, S. J. Park, H. K. Chung, O. Y. Kwon, Y. K. Kim, H. K. Ro, M. Shong. 2000. Role of peroxiredoxins in regulating intracellular hydrogen peroxide and hydrogen peroxide-induced apoptosis in thyroid cells. J. Biol. Chem. 275:18266.[Abstract/Free Full Text]
  33. Park, E. S., H. Kim, J. M. Suh, S. J. Park, S. H. You, H. K. Chung, K. W. Lee, O. Y. Kwon, B. Y. Cho, Y. K. Kim, et al 2000. Involvement of JAK/STAT (Janus kinase/signal transducer and activator of transcription) in the thyrotropin signaling pathway. Mol. Endocrinol. 14:662.[Abstract/Free Full Text]
  34. Montani, V., S. I. Taniguchi, M. Shong, K. Suzuki, M. Ohmori, C. Giuliani, G. Napolitano, M. Saji, B. Fiorentino, A. M. Reimold, et al 1998. Major histocompatibility class II HLA-DR {alpha} gene expression in thyrocytes: counter regulation by the class II transactivator and the thyroid Y box protein. Endocrinology 139:280.[Abstract/Free Full Text]
  35. Kohn, A. D., J. Chan, D. Grieco, V. M. Nikodem, S. M. Aloj, L. D. Kohn. 1989. Thyrotropin increases malic enzyme messenger ribonucleic acid levels in rat FRTL-5 thyroid cells. Mol Endocrinol. 3:532.[Abstract/Free Full Text]
  36. Hobart, M., V. Ramassar, N. Goes, J. Urmson, P. F. Halloran. 1997. IFN regulatory factor-1 plays a central role in the regulation of the expression of class I and II MHC genes in vivo. J. Immunol. 158:4260.[Abstract]
  37. Darnell, J. E., Jr, I. M. Kerr, G. R. Stark. 1994. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264:1415.[Abstract/Free Full Text]
  38. Tuschl, T., P. D. Zamore, R. Lehmann, D. P. Bartel, P. A. Sharp. 1999. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev. 13:3191.[Abstract/Free Full Text]
  39. Bromberg, J. F., M. H. Wrzeszczynska, G. Devgan, Y. Zhao, R. G. Pestell, C. Albanese, J. E. Darnell, Jr. 1999. Stat3 as an oncogene. Cell. 98:295.[Medline]
  40. Bach, E. A., M. Aguet, R. D. Schreiber. 1997. The IFN{gamma} receptor: a paradigm for cytokine receptor signaling. Annu. Rev. Immunol. 15:563.[Medline]
  41. Young, H. A., K. J. Hardy. 1995. Role of interferon-{gamma} in immune cell regulation. J. Leukocyte Biol. 58:373.[Abstract]
  42. Darnell, J. E., Jr. 1997. STATs and gene regulation. Science 277:1630.[Abstract/Free Full Text]
  43. Decker, T., P. Kovarik, A. Meinke. 1997. GAS elements: a few nucleotides with a major impact on cytokine-induced gene expression. J. Interferon Cytokine Res. 17:121.[Medline]
  44. Greenhalgh, C. J., D. J. Hilton. 2001. Negative regulation of cytokine signaling. J. Leukocyte Biol. 70:348.[Abstract/Free Full Text]
  45. Chong, M. M., H. E. Thomas, T. W. Kay. 2001. {gamma}-Interferon signaling in pancreatic {beta}-cells is persistent but can be terminated by overexpression of suppressor of cytokine signaling-1. Diabetes 50:2744.[Abstract/Free Full Text]
  46. Karlsen, A. E., S. G. Ronn, K. Lindberg, J. Johannesen, E. D. Galsgaard, F. Pociot, J. H. Nielsen, T. Mandrup-Poulsen, J. Nerup, N. Billestrup. 2001. Suppressor of cytokine signaling 3 (SOCS-3) protects {beta}-cells against interleukin-1{beta}- and interferon-{gamma}-mediated toxicity. Proc. Natl. Acad. Sci. USA 98:12191.[Abstract/Free Full Text]
  47. Li, G., J. A. Harton, X. Zhu, J. P. Ting. 2001. Downregulation of CIITA function by protein kinase a (PKA)-mediated phosphorylation: mechanism of prostaglandin E, cyclic AMP, and PKA inhibition of class II major histocompatibility complex expression in monocytic lines. Mol. Cell. Biol. 21:4626.[Abstract/Free Full Text]
  48. Decker, T., P. Kovarik. 2000. Serine phosphorylation of STATs. Oncogene 19:2628.[Medline]
  49. Yokogami, K., S. Wakisaka, J. Avruch, S. A. Reeves. 2000. Serine phosphorylation and maximal activation of STAT3 during CNTF signaling is mediated by the rapamycin target mTOR. Curr. Biol. 10:47.[Medline]
  50. Bromberg, J., J. E. Darnell, Jr. 2000. The role of STATs in transcriptional control and their impact on cellular function. Oncogene 19:2468.[Medline]
  51. Martin, B. K., K. C. Chin, J. C. Olsen, C. A. Skinner, A. Dey, K. Ozato, J. P. Ting. 1997. Induction of MHC class I expression by the MHC class II transactivator CIITA. Immunity 6:591.[Medline]
  52. Chang, C. H., R. A. Flavell. 1995. Class II transactivator regulates the expression of multiple genes involved in antigen presentation. J. Exp. Med. 181:765.[Abstract/Free Full Text]
  53. Chang, C. H., J. D. Fontes, M. Peterlin, R. A. Flavell. 1994. Class II transactivator (CIITA) is sufficient for the inducible expression of major histocompatibility complex class II genes. J. Exp. Med. 180:1367.[Abstract/Free Full Text]
  54. Kirshner, S., L. Palmer, J. Bodor, M. Saji, L. D. Kohn, D. S. Singer. 2000. Major histocompatibility class I gene transcription in thyrocytes: a series of interacting regulatory DNA sequence elements mediate thyrotropin/cyclic adenosine 3',5'-monophosphate repression. Mol. Endocrinol. 14:82.[Abstract/Free Full Text]
  55. Saji, M., M. Shong, G. Napolitano, L. A. Palmer, S. I. Taniguchi, M. Ohmori, M. Ohta, K. Suzuki, S. L. Kirshner, C. Giuliani, et al 1997. Regulation of major histocompatibility complex class I gene expression in thyroid cells: role of the cAMP response element-like sequence. J. Biol. Chem. 272:20096.[Abstract/Free Full Text]
  56. Saji, M., J. Moriarty, T. Ban, L. D. Kohn, D. S. Singer. 1992. Hormonal regulation of major histocompatibility complex class I genes in rat thyroid FRTL-5 cells: thyroid-stimulating hormone induces a cAMP-mediated decrease in class I expression. Proc. Natl. Acad. Sci. USA 89:1944.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
S. A. Morshed, R. Latif, and T. F. Davies
Characterization of Thyrotropin Receptor Antibody-Induced Signaling Cascades
Endocrinology, January 1, 2009; 150(1): 519 - 529.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
Q. Zhang, H. Y. Wang, M. Marzec, P. N. Raghunath, T. Nagasawa, and M. A. Wasik
STAT3- and DNA methyltransferase 1-mediated epigenetic silencing of SHP-1 tyrosine phosphatase tumor suppressor gene in malignant T lymphocytes
PNAS, May 10, 2005; 102(19): 6948 - 6953.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
E. S. Hwang, D. W. Kim, J. H. Hwang, H. S. Jung, J. M. Suh, Y. J. Park, H. K. Chung, J. H. Song, K. C. Park, S. H. Park, et al.
Regulation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT1-Dependent Genes by RET/PTC (Rearranged in Transformation/Papillary Thyroid Carcinoma) Oncogenic Tyrosine Kinases
Mol. Endocrinol., November 1, 2004; 18(11): 2672 - 2684.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, H.
Right arrow Articles by Shong, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, H.
Right arrow Articles by Shong, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS