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Endocrinology Vol. 141, No. 6 2090-2097
Copyright © 2000 by The Endocrine Society

ARTICLES

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 Cells1

Jongkyeong Chung, Eun Shin Park, Dohoon Kim, Jae Mi Suh, Hyo Kyun Chung, Jeonghoon Kim, Ho Kim, Soo Jung Park, O-Yu Kwon, Heung Kyu Ro and Minho Shong

Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305–701; and Departments of Internal Medicine (E.S.P., H.K., S.J.P., H.K.R., M.S.) and Anatomy (O-Y.K.), Chungnam National University, Taejon 301–040, Korea

Address all correspondence and requests for reprints to: Dr. Minho Shong, Department of Internal Medicine, Chungnam National University School of Medicine, 640 Daesadong Chungku Taejon 301–040, Korea. E-mail: minhos{at}hanbat.chungnam.ac.kr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TSH is known as an important hormone that plays the major role not only in the maintenance of normal physiology but also in the regulation of immunomodulatory gene expression in thyrocytes. The adhesion molecule intercellular adhesion molecule-1 (ICAM-1) was identified as one of the proteins that are abnormally expressed in the thyroid gland during autoimmune thyroid diseases. In this study we found that TSH inhibits interferon-{gamma} (IFN{gamma})-mediated expression of the ICAM-1 gene, and we investigated the involved mechanisms in rat FRTL-5 thyroid cells. After exposure to IFN{gamma}, ICAM-1 expression is positively regulated at the level of transcription. This effect occurs via the IFN{gamma}-activated site (GAS) element in the ICAM-1 promoter as a consequence of the activation of STAT1 (signal transducer and activator of transcription-1), but not of STAT3. On the other hand, after exposure to TSH plus IFN{gamma}, ICAM-1 transcription is negatively modulated. We found that this inhibitory effect of TSH also occurs via the GAS element. Electrophoretic mobility shift assays confirmed that the IFN{gamma}-induced DNA-binding activities of STAT1 were reduced by TSH. Furthermore, our results showed that the inhibitory effect of TSH on IFN{gamma} signaling is caused by inhibition of tyrosine phosphorylation on STAT1, Janus kinase-1 (Jak1), and IFN{gamma} receptor {alpha}, but not Jak2. In conclusion, we have identified a novel mechanism in which TSH modulates the IFN{gamma}-mediated Jak/STAT signaling pathway through the inhibition of Jak1 and STAT1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN AN AUTOIMMUNE disease, lymphocytes infiltrate specific target organs and produce high concentrations of cytokines, which are involved in abnormal gene regulation (1, 2, 3) and functional impairment in target cells (2, 3). Although a number of cytokines are implicated in the pathogenesis of thyroid autoimmune disease (1, 2, 3), interferon-{gamma} (IFN{gamma}) has been studied intensively as the major cytokine involved (2). IFN{gamma} is a pleiotropic cytokine with antiproliferative and immunomodulatory activities that are crucial for the regulation of immune responses (4, 5, 6). IFN{gamma} signaling is initiated when IFN{gamma} binds to its receptor, thereby inducing its dimerization (5). Upon this event, the receptor-associated Janus family tyrosine kinases, Jak1 and Jak2, transphosphorylate each other, resulting in their activation (4, 5, 6). The cytoplasmic domains of the receptors then become tyrosine phosphorylated by the Jaks, enabling the recruitment of STAT1 (7, 8, 9). The activated Jaks phosphorylate a tyrosine residue of STAT1, causing the phosphorylated STAT1 to dimerize by reciprocal SH2 phosphotyrosine interaction and enter the nucleus, where it activates transcription of IFN{gamma}-responsive genes by binding to the IFN{gamma}-activated site (GAS) element in their promoters (7, 8, 9).

IFN{gamma} induces the gene expression of intracellular adhesion molecule-1 (ICAM-1), rendering the cells expressing it immunocompetent (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). ICAM-1 is expressed in thyroid cells and plays an important role in inflammatory processes and in the T cell-mediated host defense system (10). It functions as a costimulatory molecule on antigen-presenting cells to activate major histocompatibility complex (MHC) class II-restricted T cells and on other cell types in association with MHC class I to activate cytotoxic T cells (10). Also, ICAM-1 in the endothelium plays an important role in stimulating the migration of leukocytes to sites of inflammation (10). Abnormal ICAM-1 expression probably contributes to the clinical manifestations of a variety of diseases, such as malignancies (e.g. melanomas and lymphomas) (23, 24), atherosclerosis (25), ischemia (26), certain neurological disorders (27), and many inflammatory disorders (e.g. asthma and autoimmune disorders) (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 28).

The expression of ICAM-1 can be induced in a cell-specific manner by several cytokines, including tumor necrosis factor-{alpha} (29, 30), interleukin-1 (31), and IFN{gamma} (32, 33), and can be inhibited by glucocorticoids (34, 35). ICAM-1 expression is mainly regulated at the level of transcription through a promoter containing several enhancer elements, among them a {kappa}B element that mediates the effects of 12-O-tetradecanoylphorbol-13-acetate, interleukin-1, lipopolysaccharide, tumor necrosis factor-{alpha}, and glucocorticoids (29, 30, 34, 35). In addition, the expression of ICAM-1 depends on an IFN{gamma} signal transduction pathway in which the STAT1 transcription factor is a critical intermediate (32, 33). Recently, it has been shown that a putative GAS element, a cis-element required for STAT-dependent transcription, is indeed present in the ICAM-1 promoter, located at -115 bp from the translation initiation site (22, 29, 30, 32, 33, 34, 36, 37). Others have also shown that STAT1 is required for the expression of ICAM-1 by examining the functions of wild-type or dominant negative forms of STAT1 in a STAT1-deficient cell line (38). Transfection of the wild-type STAT1 expression plasmid restored STAT1 expression as well as the IFN{gamma}-dependent activation of cotransfected ICAM-1 promoter constructs and endogenous ICAM-1 gene expression. However, mutations of STAT1 at tyrosine 701 (Jak phosphorylation site), glutamic acid 428/429 (putative DNA-binding site), histidine 713 (splice site resulting in STAT1ß formation), or serine 727 (mitogen-activated protein kinase phosphorylation site) all decreased the capacity of STAT1 to activate the ICAM-1 promoter (38). Collectively, these results strongly indicate that the STAT1-dependent signaling pathway plays the main role in regulating the expression of ICAM-1.

The glycoprotein hormone TSH is the major regulator of growth and differentiation in the thyroid gland and acts by binding to its receptor at the basolateral membrane of thyroid follicular cells (39, 40, 41). The TSH receptor belongs to the large superfamily of G protein-coupled receptors (39, 40, 41). In the human thyroid, the binding of ligand to TSH receptor leads to stimulation of adenylyl cyclase and phospholipase C by interacting with Gs and Gq/11 (42). The cAMP regulatory cascade controls growth and differentiated function (thyroid hormone secretion and iodide trapping), whereas Ca2+ and diacylglycerol stimulate iodination and thyroid hormone synthesis (39).

Previous studies have shown that in the thyroid cell, exposure to TSH strongly suppresses the expression of many immunomodulatory genes induced by IFN{gamma}, including MHC class I (43, 44) and ICAM-1 (22). This hormonal suppression thus contributes to preventing autoimmunity (2, 44), and therefore it is imperative to understand the molecular basis of TSH-mediated suppression of IFN{gamma}-induced gene expression in the thyroid cell. Although the TSH signaling pathway and the IFN{gamma} signaling pathway are each well known, the molecular mechanism of this suppression is not fully understood. Thus, in the present study we have studied the molecular mechanisms of how TSH modulates IFN{gamma} signaling in thyroid cells, using ICAM-1 gene expression as a model system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The highly purified bovine TSH used in the experiments was obtained from the Hormone Distribution Program of the NIDDK, NIH (NIDDK-bTSH I-1; 30 U/mg), or prepared as previously described (43, 44) (26 ± 3 U/mg). Rat recombinant IFN{gamma} was purchased from Life Technologies, Inc. (Grand Island, NY). [{alpha}-32P]Deoxy-CTP (3000 Ci/mmol) and [{alpha}-32P]ATP were obtained from NEN Life Science Products (Boston, MA). The source of all other materials was Sigma (St. Louis, MO) unless otherwise noted.

Cell culture
FRTL-5 rat thyroid cells (Interthyr Research Foundation, Baltimore, MD) were a fresh subclone (F1) that had all of the properties previously described (46, 47). Their doubling time with TSH was 36 ± 6 h; without TSH, they did not proliferate. Cells were diploid and between their 5th and 20th passage. Cells were grown in 6H medium consisting of Coon’s modified F-12 supplemented with 5% calf serum, 1 mM nonessential amino acids, and a mixture of six hormones: bovine TSH (1 x 10-10 M), 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 cells every 2 or 3 days, and cells were passaged every 7 or 10 days. To perform the experiments, we starved 70% confluent cells from TSH in 5H medium for 6 days, then stimulated them with 1 x 10-10 M TSH and/or IFN{gamma} (100 U/ml).

Northern blot analysis
Total cellular RNA was isolated using standard procedures (43), and Northern blot analyses were performed as previously described (43). Final washes were carried out at 65 C in 1 x SSPE (150 mM NaCl, 10 mM NaH2PO4, and 1 mM EDTA, pH 7.4). The rat ICAM-1 probe used in the experiment was the whole complementary DNA sequence, obtained by PCR using the published rat sequence (48) and cloned in the EcoRI site of pUC19 plasmid. Rat ß-actin probe was provided by Dr. B. Paterson (NCI, NIH). All probes were radiolabeled using the random priming method (Amersham Pharmacia Biotech, Arlington Heights, IL).

Construction of ICAM-1 promoter/luciferase chimeras and deletion mutants
Construction of the chimera pCAM-1822, containing 1822 bp of the 5'-flanking region of the rat ICAM-1 gene, was performed using high fidelity PCR (22). To generate chimeras containing additional 5'-deletions of the promoter region, objective promoter segments were amplified with Pfu polymerase using appropriate forward primers with the BglII site on the 5'-end and reverse primers with the HindIII site on the 3'-end. Mutations of promoter sequences were generated by PCR using primers that had the mutated sequence (44). Amplified fragments were ligated into the pGL2-basic vector containing a luciferase reporter gene and were sequenced to ensure nucleotide fidelity (49). We made several 5'-deletion mutants: pCAM-1822, pCAM-1822 GAS(m), pCAM-431, pCAM-431 GAS(m), pCAM-175, pCAM-175 GAS(m), and pCAM-95 (diagrammatically presented in Fig. 2AGo). They contained the sequence between nucleotides at the numbered 5'-end and +1 bp, the start of translation. All plasmid preparations were purified twice by CsCl gradient centrifugation (44).



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  		Figure 2. Effects of IFN{gamma} and/or TSH on the promoter activity of rat ICAM-1 in FRTL-5 thyroid cells. A, Schematic representation of the ICAM-1 reporter genes used in the experiments. Nucleotide numbering is relative to the translational start site, which is designated +1 (22 ). The figure shows the cloned subfragments of the ICAM-1 promoter region located upstream of a promoterless firefly luciferase complementary DNA in pGL2-basic vector (see Materials and Methods). B, Inhibitory effects of TSH on IFN{gamma}-induced ICAM-1 promoter activity. The FRTL-5 cells that stably express various reporter plasmids, as indicated, were cultured near confluence, then stimulated with TSH (1 x 10-10 M), IFN{gamma} (100 U/ml), or both for 24 h. All of the cell lysates were prepared at the same time, and their luciferase assays were performed as described in Materials and Methods. The luciferase activity from untreated pCAM-1822 was the control for comparison of promoter activities in all other treated cells. The wild-type palindromic GAS sequence (5'-TTTCCGGAAA-3') was mutated to a nonpalindromic GAS sequence (5'-TTaCCGGtAc-3') in pCAM-1822 GAS(m), pCAM-431 GAS(m), and pCAM-175 GAS(m) as described in Materials and Methods. All experiments were repeated at least three times. Data were normalized for transfection efficiency and are presented as the mean ± SE of the experiments. P < 0.005, determined by two-way ANOVA.

 
Transfections and selection of stable transfectants
Transient transfections were conducted with FRTL-5 cells at 80% confluence. Twenty micrograms of pCAM-1822, equivalent molar amounts of the deletion mutants, or pGL2-basic (negative control) plasmids were cotransfected with 5 µg pSV-GH by electroporation as described previously (44) and further cultured for 48 h. The medium was saved for RIA of human GH as an internal control (Nichols Institute Diagnostics, San Juan Capistrano, CA) and cells were harvested for luciferase assay. Luciferase assays were performed as previously described (50). We also stably transfected FRTL-5 cells with pCAM-1822, pCAM-1822 GAS(m), pCAM-431, pCAM-431 GAS(m), pCAM-175, pCAM-175 GAS(m), pCAM-95, or pGL2-basic. Briefly, near-confluent FRTL-5 cells in 6H medium were cotransfected with 20 µg of these plasmid DNAs and 10 µg pRc-Neo containing a neomycin resistance marker. Seven days after transfection, 400 µg/ml G418 (Life Technologies, Inc.) was added to the medium to select stable transfectants.

Nuclear extracts
FRTL-5 cells were grown in the presence of complete 6H medium until reaching nearly 80% confluence, then maintained in 5H medium to starve TSH for the time periods as noted. After cells were exposed to IFN{gamma}, nuclear extracts were prepared from the FRTL-5 cells as previously described (44), except that they were washed with Dulbecco’s modified PBS without Mg2+ and Ca2+. After centrifugation at 500 x g, cells were resuspended in 5 pellet vol 0.3 M sucrose and 2% Tween 40 in buffer A [10 mM HEPES-potassium hydroxide (KOH); pH 7.9, containing 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonylfluoride (PMSF), 2 µg/ml leupeptin, and 2 µg/ml pepstatin A] and lysed by serial freezing, thawing, and gentle homogenization. Nuclei were isolated by centrifuging at 25,000 x g on buffer A containing a 1.5-M sucrose cushion and were further lysed in buffer B [10 mM HEPES-KOH (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EGTA, 10% glycerol, 0.5 mM DTT, 0.5 mM PMSF, 2 µg/ml leupeptin, and 2 µg/ml pepstatin A]. Nuclear lysates were centrifuged at 100,000 x g for 1 h, and the supernatant was dialyzed against a buffer containing 10 mM Tris (pH 7.9), 1 mM MgCl2, 1 mM DTT, 1 mM EDTA, and 5% glycerol and saved for gel shift analyses.

Electrophoretic mobility shift assay (EMSA)
Oligonucleotides derived from the 5'-flanking region (5'-CGAGGTTTCCGGGAAAGTG GCCCC-3'; a core sequence of palindromic GAS is underlined) of the rat ICAM-1 gene (22) were used for EMSA. Gel-purified oligonucleotides were labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase and purified on an 8% native polyacrylamide gel (44). EMSA was performed as previously described (44). Binding reactions were carried out in a volume of 20 µl for 30 min at room temperature. The reaction mixtures contained 1.5 fmol [32P]DNA, 2 µg nuclear extract, and 0.5 or 1 µg poly(dI-dC) in 10 mM Tris-Cl (pH 7.9), 5 mM MgCl2, 50 mM KCl, 1 mM DTT, 1 mM EDTA, 0.1% Triton X-100, and 12.5% glycerol. After incubation, reaction mixtures were subjected to electrophoresis on 4–4.5% native polyacrylamide gels in 0.5 x TBE buffer (44.5 mM Tris; 44.5 mM borate; 1 mM EDTA, pH 8.3). Gels were dried and autoradiographed. For supershift assays, extracts were incubated in the same buffer containing antibodies or control rabbit IgG at 20 C for 20 min before being processed as described above.

Immunoprecipitation and Western blot analysis
Cells were washed with ice-cold STE [NaCl 150 mM, 50 mM Tris (pH 7.4), and 1 mM EDTA] and lysed in lysis buffer [20 mM Tris (pH 7.4), 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, 50 mM ß-glycerophosphate, 2 mM DTT, 1 mM Na3VO4, 1 mM PMSF, and 4 µg/ml aprotinin]. Cell lysates were incubated with antibodies against Jak1 (Transduction Laboratories, Inc., Lexington, KY), Jak2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and IFN{gamma} receptor1 (Santa Cruz Biotechnology, Inc.) at 4 C for 2 h, and immunecomplexes were precipitated by protein A or G. They were washed twice in lysis buffer with 500 mM NaCl and once with 50 mM Tris (pH 7.4) and 150 mM NaCl. Precipitated proteins were eluted from protein A beads by boiling in the presence of 1 x SDS sample buffer and were separated in SDS-PAGE. Immunoblot analyses were performed using an antiphosphotyrosine antibody, 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY).

Statistical significance
All experiments were repeated at least three times with different batches of cells. Values are the mean ± SE of these experiments. Significance between experimental values was determined by two-way ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TSH inhibits IFN{gamma}-stimulated ICAM-1 gene expression
To determine whether the TSH-induced signaling pathway modulates IFN{gamma}-induced ICAM-1 expression in vivo, FRTL-5 cells were treated with TSH and/or IFN{gamma} for 24 h. Total RNA was isolated, and ICAM-1 expression was determined by Northern blot analyses (Fig. 1Go). ICAM-1 expression in FRTL-5 cells was strongly induced by IFN{gamma} compared with the ß-actin control (Fig. 1AGo, lane 3). Basal level expression and IFN{gamma}-induced expression of ICAM-1 were inhibited by TSH (Fig. 1Go, A and B, lane 1 vs. lane 2; Fig. 1Go, A and B, lane 3 vs. lane 4, respectively). This inhibition was evident at 6 h after the addition of TSH. However, the expression of ß-actin was not affected by either TSH or IFN{gamma}. This inhibition of IFN{gamma}-induced ICAM-1 expression by TSH suggests that TSH may interfere with the downstream signaling cascade and/or transcriptional activities that are triggered by IFN{gamma}. To identify the mechanisms involved in the inhibition of IFN{gamma}-induced ICAM-1 expression by TSH, we analyzed the regulatory properties of ICAM-1 gene by using reporter plasmids that carry the 5'-flanking region of the rat ICAM-1 gene (22).



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  		Figure 1. Effects of TSH and IFN{gamma} on ICAM-1 mRNA levels in rat FRTL-5 cells. A, After FRTL-5 cells were grown to near confluence in complete 6H medium with 5% serum, cells were maintained for 6 days with 5H medium without TSH. TSH-starved FRTL-5 cells were stimulated with TSH (1 x 10-10 M), IFN{gamma} (100 U/ml), or both for 24 h. Total RNA was isolated and subjected to Northern analyses using probes for rat ICAM-1 and ß-actin as described in Materials and Methods. The results shown are representative Northern analyses from one experiment with 20 µg RNA/lane. B, Results from A were quantitated using a BAS 2000 image analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan). Relative ratios between ICAM-1 mRNA and ß-actin mRNA were presented as a percentage of the unstimulated control value. Data are the mean ± SEM of values obtained from three separate experiments.

 
Regulation of ICAM-1 promoter activities by TSH and/or IFN{gamma}
To identify the cis-elements regulated by TSH and/or IFN{gamma} in the ICAM-1 promoter, we completed serial deletion and mutation analyses, as shown in Fig. 2Go. As we progressively deleted the promoter region of pCAM-1822, we localized the region between -175 and -97 as a cis-element responsible for regulation by IFN{gamma} (Fig. 2Go). As noted in Fig. 2AGo, the region between -175 and -97 bp contains two major regulatory elements known to be involved in IFN{gamma} action: GAS (-138 to -128 bp) and Sp1 (-112 to -108 bp) (22). When we mutated the GAS sequences in pCAM-1822, pCAM-431, and pCAM-175, IFN{gamma} could no longer affect the ICAM-1 promoter activity (Fig. 2BGo).

IFN{gamma}-induced reporter activities were inhibited by simultaneous treatment with TSH in the promoter constructs pCAM-1822, pCAM-431, and pCAM-175 (Fig. 2BGo). In the reporter plasmids containing mutations in the GAS element, neither induction by IFN{gamma} nor inhibition by TSH was observed (Fig. 2BGo), strongly indicating that the GAS element mediates IFN{gamma}-dependent ICAM-1 gene activation and TSH-dependent ICAM-1 gene repression. Thus, we conclude that TSH exerts its inhibitory effect on ICAM-1 gene expression through the same promoter region that is regulated by IFN{gamma}.

The GAS element contains a conserved palindromic 11-bp GAS core element, 5'-tttccgggaaa-3'. As it is known that GAS elements are binding motifs for the STAT family transcription factors STAT1, STAT3, STAT5, and STAT6 (7, 8, 9, 51), we hypothesized that DNA binding experiments using TSH with IFN{gamma}, a potent agonist for STAT-dependent transcription, will clearly show the inhibitory effect of TSH on ICAM-1 gene expression.

TSH inhibits IFN{gamma}-induced DNA-binding activities of STAT1
EMSA was used to investigate the DNA-binding activities of STAT following stimulation of FRTL-5 cells with IFN{gamma} in the presence or absence of TSH. The GAS oligonucleotides, which were derived from the rat ICAM-1 promoter (as described in Materials and Methods) (22), were used as the radiolabeled probe. The nuclear extracts obtained from quiescent cells cultured in 5H5% medium showed a STAT3 homodimer complex (Fig. 3AGo, lanes 2–5), as evidenced by the anti-STAT3 antibody-dependent supershifted band (Fig. 3AGo, lane 5). This STAT3/DNA complex was relatively labile, so it was detectable only in low poly(dI-dC) (0.5 µg/lane) reaction conditions (Fig. 3AGo, lane 2 vs. Fig. 3BGo, lane 2). Furthermore, the treatment of IFN{gamma} did not induce formation of the STAT3/DNA complex (Fig. 3BGo, lane3 and Fig. 4AGo, lane 3), which suggests that STAT3 is not involved in the IFN{gamma}-mediated induction of ICAM-1 transcription in FRTL-5 cells. However, when we treated cells with IFN{gamma}, we could detect a strong DNA-binding activity (Fig. 3BGo, lane 3) that was identified as a STAT1 homodimer by antibody supershift experiments (Fig. 3BGo, lane 4). Antibodies reacting to STAT3, interferon regulatory factor-1 (IRF-1), and IRF-2 did not affect the formation of these IFN{gamma}-induced complexes (Fig. 3BGo, lanes 5–7). In addition, this IFN{gamma}-induced GAS-binding activity of STAT1 persisted for more than 48 h (data not shown) in FRTL-5 cells. These results strongly suggest that STAT1 plays a major role in IFN{gamma}-dependent ICAM-1 gene activation, whereas STAT3 plays different roles, such as uninduced basal activities in the absence of IFN{gamma}.



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  		Figure 3. DNA-binding activities of STAT1 are strongly induced by IFN{gamma} stimulation in FRTL-5 cells. A, EMSAs for quiescent FRTL-5 cells. Nuclear extracts from TSH-starved FRTL-5 cells were prepared and incubated with the radiolabeled oligonucleotide probe containing the GAS sequence of the rat ICAM-1 promoter and 0.5 µg/ml poly(dI-dC) as described in Materials and Methods. The nuclear extracts used for lanes 3, 4, and 5 were incubated with anti-STAT1, anti-IRF-1, and anti-STAT3 antibodies, respectively, before adding the radioactive probe as described in Materials and Methods. B, EMSAs for IFN{gamma}-stimulated FRTL-5 cells. TSH-starved cells were stimulated with (+) or without (-) IFN{gamma} (100 U/ml) for 24 h, and their EMSA analyses were performed as described in A, except that 1 µg/ml poly(dI-dC) was added. The nuclear extracts used for lanes 4, 5, 6, and 7 were incubated with anti-STAT1, anti-STAT3, anti-IRF-1, and anti-IRF-2 antibodies, respectively. The arrows denote the locations of the IFN{gamma}-induced STAT-GAS complexes identified as STAT3 and STAT1 homodimer.

 


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  		Figure 4. TSH inhibits the IFN{gamma}-induced DNA-binding activities of STAT1. A, TSH-starved FRTL-5 cells were stimulated with TSH (1 x 10-10 M), IFN{gamma} (100 U/ml), or both for 24 h. EMSAs were completed as described in Fig. 3BGo. The arrow denotes the location of the IFN{gamma}-induced STAT/GAS complex identified as a STAT1 homodimer. B, Results from A were quantitated using a BAS 2000 image analyzer (Fuji Photo Film Co., Ltd.). Relative ratios of the DNA-binding activities for STAT1 were determined. Data are the mean ± SEM of values obtained from three separate experiments.

 
Next, we examined whether TSH affects the DNA-binding activities of STAT1 (Fig. 4Go). Stimulation with TSH suppressed IFN{gamma}-induced STAT1-binding activity (Fig. 4Go, A and B, lane 3 vs. lane 4). This reduced binding of STAT1 to the GAS element of ICAM-1 was tightly correlated with the decreased transcription activities of the promoter shown in Fig. 2B.

TSH inhibits the IFN{gamma}-mediated phosphorylation of Jak1, IFN{gamma} receptor {alpha}, and STAT1, but not Jak2
As the rapid activation of STAT1 by exogenous IFN{gamma}, which was demonstrated by an increase in STAT1 tyrosine 701 phosphorylation, was not blocked by treatments with protein and RNA synthesis inhibitors (Fig. 5AGo, lanes 4 and 6), we suspected that TSH down-modulated STAT1 activity at a posttranslational level. As shown in Fig. 5BGo, TSH indeed strongly inhibited IFN{gamma}-induced STAT1 tyrosine 701 phosphorylation (Fig. 5BGo, lane 4). Thus, we concluded that TSH inhibited ICAM-1 gene expression by interfering with the IFN{gamma}-induced tyrosine phosphorylation of STAT1, which is required for its DNA-binding activity.



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  		Figure 5. TSH inhibits phosphorylation of tyrosine 701 of STAT1 induced by IFN{gamma}. A, FRTL-5 thyroid cells were treated with (+) or without (-) cycloheximide (CHX) and actinomycin D (Act) for 6 h and subsequently with IFN{gamma} (100 U/ml) for another 15 min. B, TSH-starved FRTL-5 cells were treated with TSH for 4 h and subsequently with IFN{gamma} (100 U/ml) for another 15 min. C, FRTL-5 thyroid cells were treated with cycloheximide (CHX) or actinomycin D (Act) in the presence or absence of TSH for 6 h and subsequently with IFN{gamma} (100 U/ml) for another 15 min. D, TSH-starved cells were pretreated with (+) or without (-) TSH and MG132 (100 µM) or sodium orthovanadate (100 µM) for 4 h. Subsequently, they were treated with (+) or without (-) IFN{gamma} (100 U/ml) for another 15 min. Cell lysates were prepared as described in Materials and Methods. Whole cell lysates were resolved in 6% SDS-PAGE and further examined for immunoblot analyses with anti-STAT1 tyrosine 701 phosphospecific (upper panels) and anti-STAT1 (lower panels) antibodies.

 
However, this inhibition of IFN{gamma}-induced STAT1 tyrosine phosphorylation by TSH was completely blocked in the presence of cycloheximide and actinomycin D (Fig. 5CGo, lanes 3 and 4, respectively). These results strongly suggest that inhibition of STAT1 signaling by TSH requires de novo synthesis of an inhibitory protein(s). In support of this, TSH exhibited the inhibitory effects only when FRTL-5 cells were exposed to TSH for longer than 4 h (55), whereas the IFN{gamma} activation of STAT1 explained above required less than 15 min (Fig. 5BGo), reflecting the much slower kinetics of regulation via de novo synthesis compared with regulation by posttranslational modification.

As STAT1 activities can be negatively regulated by activation of tyrosine phosphatases or proteasome-mediated degradation pathways (7, 8, 9), we examined whether MG132, a proteasome inhibitor, or sodium orthovanadate, a tyrosine phosphatase inhibitor, influences the effects of TSH. Sodium orthovanadate alone slightly increased tyrosine phosphorylation of STAT1 (Fig. 5DGo, lane 3), but MG132 itself did not induce STAT1 tyrosine phosphorylation (Fig. 5DGo, lane 2). Again, TSH inhibited the IFN{gamma}-induced tyrosine phosphorylation of STAT1 in the presence of MG132 (Fig. 5DGo, lane 5) and sodium orthovanadate (Fig. 5DGo, lane 6). However, the inhibitory effect of TSH on STAT1 tyrosine phosphorylation decreased significantly in the presence of sodium orthovanadate and MG132 (compare Fig. 5BGo, lane 4, vs. Fig. 5DGo, lanes 5 and 6). These results suggest that TSH inhibits IFN{gamma} signaling not only via an upstream signaling step(s) that involves the tyrosine phosphorylation of STAT1, but also through a pathway leading to the dephosphorylation or proteasomal degradation of phosphorylated STAT1.

To understand the upstream mechanisms by which TSH inhibits the tyrosine phosphorylation of STAT1, we examined the Janus family tyrosine kinases and IFN{gamma} receptors. Phosphorylation of STAT1 is a consequence of the activation of the IFN{gamma} receptor after ligand binding, so TSH could indirectly inhibit STAT1 phosphorylation by inhibiting the activities of Jak1, Jak2, and/or IFN{gamma} receptor. First, we examined the tyrosine phosphorylation of Jak1 and Jak2. The proteins from lysates of cells treated with IFN{gamma} alone or with IFN{gamma} plus TSH were immunoprecipitated with their specific antibodies, and immunoblot analyses were performed with antiphosphopeptide-specific (Affinity BioReagents, Inc., Golden, CO) or antiphosphotyrosine antisera (Upstate Biotechnology, Inc.; Fig. 6Go). IFN{gamma}-induced Jak1 phosphorylation was inhibited when cells were stimulated with TSH (Fig. 6Go, top panel). However, the IFN{gamma}-induced tyrosine phosphorylation of Jak2 was not affected by TSH treatment (Fig. 6Go, middle panel). It was previously discovered that Jak1 is associated with the {alpha}-subunit of IFN{gamma} receptor (5). Thus, we examined IFN{gamma}-induced tyrosine phosphorylation of IFN{gamma} receptor {alpha}. As shown in the bottom panel of Fig. 6Go, TSH strongly inhibited the tyrosine phosphorylation of IFN{gamma} receptor {alpha} induced by IFN{gamma} treatment, which is probably a consequence of Jak1 inhibition.



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  		Figure 6. Selective inhibition of IFN{gamma}-dependent activation of Jak1, Jak2, and IFN{gamma} receptor {alpha} by TSH. FRTL-5 cells were treated with TSH (1 x 10-10 M) for 4 h and with IFN{gamma} (100 U/ml) for another 15 min. Cell lysates were prepared as described in Materials and Methods and immunoprecipitated with anti-Jak1, anti-Jak2, and anti-IFN{gamma} receptor {alpha} antibodies as indicated. Immunoprecipitated proteins were separated in 6% SDS-PAGE and immunoblotted with antiphosphotyrosine antibody ({alpha}pY) and their specific antibodies as indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ICAM-1 is expressed in human and rat thyroid cells and mediates lymphocyte binding to thyrocytes (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). It is excessively expressed in thyroid follicular cells from patients with Hashimoto’s thyroiditis (11, 15, 16, 19, 20) and some patients with Graves’ disease (17, 18, 19). These findings strongly indicate that ICAM-1 plays an important role in inflammation in the thyroid gland during autoimmune reactions, particularly in the lymphocyte-thyrocyte interaction. As ICAM-1 expression appears to be transcriptionally regulated (32, 33, 52, 53, 54), its induction and suppression by various physiological and pathological factors have been studied mainly through identification and subsequent analysis of regulatory signals modulating the activities of involved transcription factors, namely activating protein-1, nuclear factor-{kappa}B, STAT, and SP1 (52, 53, 54).

In this study we have shown that TSH inhibits ICAM-1 gene expression through the same GAS element that mediates induction by IFN{gamma}. We confirmed this by EMSAs, which revealed that TSH hampered the DNA-binding activities of STAT1 induced by IFN{gamma}. As STAT1 needs to be phosphorylated at tyrosine 701 to translocate to the nucleus and bind to the target promoter (7, 8, 9), we examined whether TSH interferes with the phosphorylation of STAT1 using a phosphotyrosine 701-specific antibody. Surprisingly, TSH strongly inhibited STAT1 tyrosine 701 phosphorylation induced by IFN{gamma}, implying that TSH inhibits ICAM-1 gene expression by preventing the phosphorylation of STAT1.

Next, we asked whether this inhibition of tyrosine phosphorylation of STAT1 after TSH treatment is a consequence of the decreased activities of Jak1 and/or Jak2. Antiphosphotyrosine immunoblot analyses showed that TSH inhibits the IFN{gamma}-induced phosphorylation of Jak1 and IFN{gamma} receptor {alpha}, but not Jak2. These findings suggest that the inhibitory effect generated by TSH is specifically based on the inhibition of Jak1, which is associated with the cytoplasmic domain of IFN{gamma} receptor {alpha} (5).

As shown in Fig. 5Go, the mechanisms by which TSH inhibits the tyrosine phosphorylation of Jak1 might involve the de novo induction of Jak1 inhibitors, such as suppressors of cytokine signaling (SOCS). In FRTL-5 cells, SOCS-1, SOCS-3, and CIS (cytokine-inducible SH2-containing protein) messenger RNA (mRNA) were strongly induced in a TSH-dependent manner and peaked about 4 h after TSH stimulation (55). The induction kinetics of the messages for SOCS-1, SOCS-3, and CIS are well correlated with the temporal inhibitory pattern of TSH for the IFN{gamma}-stimulated Jak1/STAT1 activities (55). Therefore, we propose that one of the mechanisms behind the TSH-mediated inhibition of IFN{gamma} signaling involves the inhibition of Jak1 by the de novo induction of SOCS or CIS protein.

Another possible mechanism of the inhibitory regulation of STAT1 by TSH may involve the deactivation of activated STAT1 by STAT1 phosphatases. Recently, a novel receptor tyrosine phosphatase (r-PTP{eta}) was identified from rat thyroid cells (56). This phosphatase has a homology with the human receptor tyrosine phosphatase DEP-1/HPTP{eta}. Treatment of rat thyroid cells with TSH resulted in a rapid increase in the mRNA level of r-PTP{eta}, which appeared within 1 h of treatment with TSH and remained elevated until 48 h (56). This phosphatase may play a role in the down-regulation of Jak/STAT by TSH. To determine whether a tyrosine phosphatase may be involved in TSH-mediated down-regulation of STAT1 tyrosine phosphorylation, immunoblot analyses with a STAT1 tyrosine 701 phosphospecific antibody were performed in the presence of the tyrosine phosphatase inhibitor, sodium orthovanadate. These results showed that orthovanadate partially blocked the TSH-mediated effects. In addition, Kim and Maniatis have shown that the STAT1 activity induced by IFN{gamma} can be negatively regulated by specific proteolytic degradation of phosphorylated STAT1 in HeLa cells (57). Thus, we examined whether pretreatment with MG132 may affect TSH-mediated inhibition of STAT1 tyrosine phosphorylation. Interestingly, our results also imply that the MG132-sensitive proteolytic pathway plays a minor, but nonetheless significant, role in the TSH-mediated inhibition of STAT1 activity. Taken together, these findings suggest that the sodium orthovanadate-sensitive tyrosine phosphatases and the MG132-sensitive proteolytic pathways are also involved in TSH signaling in FRTL-5 cells.

TSH exerts its effect by specifically binding to its G protein-coupled receptor. Activated TSH receptor increases intracellular concentrations of the second messenger cAMP (39, 40, 41, 42). cAMP has been implicated in the negative regulation of many cell-signaling pathways, including the mitogen-activated protein kinase pathway and the Jak/STAT pathway. Therefore, we completed parallel experiments to determine whether forskolin, an activator of adenyl cyclase, inhibits the tyrosine phosphorylation of Jak1, Jak2, and IFN{gamma} receptor {alpha} in FRTL-5 cells. Interestingly, forskolin (10 µM) did not affect the IFN{gamma}-induced tyrosine phosphorylation of Jaks and IFN{gamma} receptor {alpha} (data not shown), which suggests that cAMP is not involved in the TSH-mediated inhibition of the Jak/STAT pathway and ICAM-1 expression in FRTL-5 cells.

Without a doubt, TSH is one of the most important physiological factors for maintaining cell survival and differentiated functions in the thyroid gland. The constitutive and cytokine-stimulated activation of STAT1 is related to growth inhibition or apoptosis in particular cell types (12); particularly, IFN{gamma}-induced STAT1 activation is implicated in growth inhibition in FRTL-5 thyroid cells (Park, E. S., and M. Shong, unpublished data). Although there are some limitations in applying our observations to human physiology, we strongly believe that our results will provide important clues for understanding the molecular mechanisms by which TSH functions as one of the physiological modulators during an autoimmune thyroid disease.


   Footnotes
 
1 This work was supported by the Korea Research Foundation (to J.C.); Biotech 2000 Project 97-N1–02-04-A-01 (to M.S.); the Molecular Medicine Research Group Program, Ministry of Science and Technology; and a grant from the Korean Society of Internal Medicine (to M.S.). Back

Received October 25, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Davies TF 1999 The thyroid immunology of the postpartum period. Thyroid 9:675–684[Medline]
  2. Feldmann M., Dayan C, Grubeck-Loebenstein B, Rapoport B, Londei M 1992 Mechanism of Graves’ thyroiditis: implications for concepts and therapy of autoimmunity. Int Rev Immunol 9:91–106[Medline]
  3. Weetman AP 1994 The potential immunological role of the thyroid cell in autoimmune thyroid disease. Thyroid 4:493–499[Medline]
  4. Boehm U, Klamp T, Groot M, Howard JC 1997 Cellular responses to interferon-{gamma}. Annu Rev Immunol 15:749–795[CrossRef][Medline]
  5. Bach EA, Aguet M, Schreiber RD 1997 The IFN {gamma} receptor: a paradigm for cytokine receptor signaling. Annu Rev Immunol 15:563–591[CrossRef][Medline]
  6. Pestka S 1997 The interferon receptors. Semin Oncol [Suppl 9] 24:S9–18–S9–40
  7. Ihle JN 1996 STATs: signal transducers and activators of transcription. Cell 84:331–334[CrossRef][Medline]
  8. Darnell Jr JE 1997 STATs and gene regulation. Science 277:1630–1635[Abstract/Free Full Text]
  9. Ivashkiv LB 1995 Cytokines and STATs: how can signals achieve specificity? Immunity l:1–4
  10. Springer TA 1990 Adhesion receptors of the immune system. Nature 346:425–433[CrossRef][Medline]
  11. Zheng RQ, Abney ER, Grubeck-Loebenstein B, Dayan C, Maini RN, Feldmann M 1990 Expression of intercellular adhesion molecule-1 and lymphocyte function-associated antigen-3 on human thyroid epithelial cells in Graves’ and Hashimoto’s diseases. J Autoimmun 3:727–736[CrossRef][Medline]
  12. Tandon N, Dinsdale C, Tamatani T, Miyasaka M, Weetman AP 1991 Adhesion molecule expression by the FRTL-5 rat thyroid cell line. J Endocrinol 130:451–456[Abstract/Free Full Text]
  13. Weetman AP, Freeman M, Boryswicz LK, Makoba MW 1990 Functional analysis of intercellular adhesion molecule-1-expressing human thyroid cells. Eur J Immunol 20:271–275[Medline]
  14. Martin A, Huber GK, Davies TF 1990 Induction of human thyroid cell ICAM-1 (CD54) antigen expression and ICAM-1-mediated lymphocyte binding. Endocrinology 127:651–657[Abstract/Free Full Text]
  15. Bagnasco M, Caretto A, Olive D, Pedini B, Canonica GW, Betterle C 1991 Expression of intercellular adhesion molecule-1 (ICAM-1) on thyroid epithelial cells in Hashimoto’s thyroiditis but not in Graves’ disease or papillary thyroid cancer. Clin Exp Immunol 83:309–313[Medline]
  16. Tolosa E, Roura C, Catalfamo M, Marti M, Lucas-Martin A, Sanmarti A, Salinas I, Obiols G, Foz-Sala M, Pujoll-Borrell R 1992 Induction of intercellular adhesion molecule-1 but not of lymphocyte function-associated antigen-3 in thyroid follicular cells. J Autoimmun 5:107–118[CrossRef][Medline]
  17. Fukazawa H, Yoshida K, Ichinohasama R, Sawai T, Hiromatsu Y, Mori K, Kikuchi K, Aizawa Y, Abe K, Wall JR 1993 Expression of the Hermes-1 (CD44) and ICAM-1 (CD54) molecule on the surface of thyroid cells from patients with Graves’ disease. Thyroid 3:285–289[Medline]
  18. Ciampolillo A, Napolitano G, Mirakian R, Miyasaki A, Giorgino R, Bottazzo GF 1993 Intercellular adhesion molecule-1 (ICAM-1) in Graves’ disease: contrast between in vivo and in vitro results. Clin Exp Immunol 94:478–485[Medline]
  19. Nakashima M, Eguchi K, Ida H, Yamashita I, Sakai M, Origuchi T, Kawabe Y, Ishikawa N, Ito K, Nagataki S 1994 The expression of adhesion molecules in thyroid glands from patients with Graves’ disease. Thyroid 4:19–29[Medline]
  20. Arreaza G, Yoshikawa N, Mukuta T, Resetkova E, Barsuk A, Nishikawa M, Muallim C, Miller N, Jamieson C, Volpe R 1995 Expression of intercellular adhesion molecule-1 on human thyroid cells from patients with autoimmune thyroid disease: study of thyroid xenografts in nude and severe combined immunodeficient mice and treatment with FK-506. J Clin Endocrinol Metab 80:3724–3731[Abstract]
  21. Hoermann R, Spitzweg C, Poertl S, Mann K, Heufelder AE, Schumm-Draeger PM 1997 Regulation of intercellular adhesion molecule-1 expression in human thyroid cells in vitro and human thyroid tissue transplanted to the nude mouse in vivo: role of Graves’ immunoglobulins and human thyrotropin receptor. J Clin Endocrinol Metab 82:2048–2055[Abstract/Free Full Text]
  22. Park ES, You SH, Kim H, Kwon O-Y, Ro HK, Cho BY, Taniguchi S-I, Kohn LD, Shong M 1999 Hormone-dependent regulation of intercellular adhesion molecule-1 gene expression: cloning and analysis of 5'-regulatory region of rat intercellular adhesion molecule-1 gene in FRTL-5 rat thyroid cells. Thyroid 9:601–612[Medline]
  23. Alexander CL, Edward M, MacKie RM 1999 The role of human melanoma cell ICAM-1 expression on lymphokine activated killer cell-mediated lysis, and the effect of retinoic acid. Br J Cancer 80:1494–1500[CrossRef][Medline]
  24. Angelopoulou MK, Kontopidou FN, Pangalis GA 1999 Adhesion molecules in B-chronic lymphoproliferative disorders. Semin Hematol 36:178–197[Medline]
  25. Iiyama K, Hajra L, Iiyama M, Li H, DiChiara M, Medoff BD, Cybulsky MI 1999 Patterns of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression in rabbit and mouse atherosclerotic lesions and at sites predisposed to lesion formation. Circ Res 85:199–207[Abstract/Free Full Text]
  26. Beauchamp P, Richard V, Tamion F, Lallemand F, Lebreton JP, Vaudry H, Daveau M, Thuillez, C 1999 Protective effects of preconditioning in cultured rat endothelial cells: effects on neutrophil adhesion and expression of ICAM-1 after anoxia and reoxygenation. Circulation 100:541–546[Abstract/Free Full Text]
  27. Lee SJ, Benveniste EN 1999 Adhesion molecule expression and regulation on cells of the central nervous system. J Neuroimmunol 98:77–88[CrossRef][Medline]
  28. Papi A, Johnston SL 1999 Rhinovirus infection induces expression of its own receptor intercellular adhesion molecule-1 (ICAM-1) via increased NF-{kappa}B-mediated transcription. J Biol Chem 274:9707–9720[Abstract/Free Full Text]
  29. Jahnke A, Johnson JP 1994 Synergistic activation of intercellular adhesion molecule 1 (ICAM-1) by TNF-{alpha} and IFN-{gamma} is mediated by p65/p50 and p65/c-Rel and interferon-responsive factor Stat1 {alpha} (p91) that can be activated by both IFN-{gamma} and IFN-{alpha}. FEBS Lett 354:220–226[CrossRef][Medline]
  30. Jahnke A, Johnson JP 1995 Intercellular adhesion molecule 1 (ICAM-1) is synergistically activated by TNF-{alpha} and IFN-{gamma} responsive sites. Immunobiology 193:305–314[Medline]
  31. Kyrkanides S, Olschowka JA, Williams JP, Hansen JT, O’Banion MK 1999 TNF alpha and IL-1ß mediate intercellular adhesion molecule-1 induction via microglia-astrocyte interaction in CNS radiation injury. J Neuroimmunol 95:95–106[CrossRef][Medline]
  32. Naik SM, Shibagaki N, Li LJ, Quinlan KL, Paxton LL, Caughman SW 1997 Interferon {gamma}-dependent induction of human intercellular adhesion molecule-1 gene expression involves activation of a distinct STAT protein complex. J Biol Chem 10:1283–1290
  33. Caughman SW, Li LJ, Degitz K 1990 Characterization and functional analysis of interferon-{gamma}-induced intercellular adhesion molecule-1 expression in human keratinocytes and A-431 cells. J Invest Dermtol 94:22S–26S[CrossRef]
  34. Wissink S, van Heerde EC, van der Burg B, van der Saag PT 1998 A dual mechanism mediates repression of NF-{kappa}B activity by glucocorticoids. Mol Endocrinol 12:355–363[Abstract/Free Full Text]
  35. Caldenhoven E, Liden J, Wissink S, Van de Stolpe A, Raaijmakers J, Koenderman L, Okret S, Gustafsson JA, Van der Saag PT 1995 Negative cross-talk between RelA and the glucocorticoid receptor: a possible mechanism for the antiinflammatory action of glucocorticoids. Mol Endocrinol 9:401–412[Abstract/Free Full Text]
  36. Ohmori Y, Schreiber RD, Hamilton TA 1997 Synergy between interferon-{gamma} and tumor necrosis factor-{alpha} in transcriptional activation is mediated by cooperation between signal transducer and activator of transcription 1 and nuclear factor {kappa}B. J Biol Chem 272:14899–14907[Abstract/Free Full Text]
  37. Look DC, Pelletier MR, Tidwell RM, Roswit WT, Holtzman MJ 1995 Stat1 depends on transcriptional synergy with Sp1. J Biol Chem 270:30264–30267[Abstract/Free Full Text]
  38. Walter MJ, Look DC, Tidwell RM, Roswit WT, Holtzman MJ 1997 Targeted inhibition of interferon-{gamma}-dependent intercellular adhesion molecule-1 (ICAM-1) expression using dominant-negative Stat1. J Biol Chem 272:28582–28589[Abstract/Free Full Text]
  39. Kohn LD, Shimura H, Shimura Y, Hidaka A, Giuliani C, Napolitano G, Ohmori M, Laglia G, Saji M 1995 The thyrotropin receptor. Vit Horm 50:287–384[Medline]
  40. Rapoport B, Chazenbalk GD, Jaume JC, McLachlan SM 1998 The thyrotropin (TSH) receptor: interaction with TSH and autoantibodies. Endocr Rev 19:673–716[Abstract/Free Full Text]
  41. Vassart G, Dumont JE 1992 The thyrotropin receptor and the regulation of thyrocyte function and growth. Endocr Rev 13:596–611[Abstract/Free Full Text]
  42. Allgeier A, Offermanns S, Van Sande J, Spicher K, Schultz G, Dumont JE 1994 The human thyrotropin receptor activates G-proteins Gs and Gq/11. J Biol Chem 269:13733–13735[Abstract/Free Full Text]
  43. Saji M, Moriaty J, Ban T, Singer DS, Kohn LD 1992 Major histocompatibility complex class I gene expression in rat thyroid cells is regulated by hormones, methimazole, and iodide as well as interferon. J Clin Endocrinol Metab 75:871–878[Abstract]
  44. Saji M, Shong M, Napolitano G, Palmer LA, Taniguchi S-I, Ohmori M, Ohta M, Suzuki K, Kirshner SL, Giuliani C, Singer DS, Kohn LD 1997 Regulation of major histocompatibility complex class I gene expression in thyroid cells. Role of the cAMP response element-like sequence. J Biol Chem 272:20096–20107[Abstract/Free Full Text]
  45. Singer DS, Mozes E, Kirshner S, Kohn LD 1997 Role of MHC class I molecules in autoimmune disease. Crit Rev Immunol. 17:463–468
  46. Ambesi-Impiombato FS, Coon HG 1979 Thyroid cells in culture. Int Rev Cytol [Suppl] 10:163–171
  47. Kohn LD, Valente WA, Grollman EF, Aloj SM, Vitti P 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 FRTL-5. U.S. patent 4:609,622
  48. Kita Y, Takashi T, Ligo Y, Tamatani T, Miyasaki M, Horiuchi T 1992 Sequence and expression of rat ICAM-1. Biochim Biophys Acta 1131:108–110[Medline]
  49. Sanger F, Nicklen S, Coulson AR 1997 DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
  50. Giuliani C, Saji M, Napolitano G, Palmer LA, Taniguchi S-I, Shong M, Singer DS, Kohn LD 1995 Hormonal modulation of major histocompatibility complex class I gene expression involves an enhancer A-binding complex consisting of Fra-2 and the p50 subunit of NF-{kappa}B. J Biol Chem 270:11453–11462[Abstract/Free Full Text]
  51. Wen Z, Zhong Z, Darnell JE Jr 1995 Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell 82:241–250[CrossRef][Medline]
  52. de Lange P, Koper JW, Huizenga NA, Brinkmann AO, de Jong FH, Karl M, Chrousos GP, Lamberts SW 1997 Differential hormone-dependent transcriptional activation and -repression by naturally occurring human glucocorticoid receptor variants. Mol Endocrinol 11:1156–1164[Abstract/Free Full Text]
  53. Rahman A, Anwar KN, True AL, Malik AB 1999 Thrombin-induced p65 homodimer binding to downstream NF-{kappa}B site of the promoter mediates endothelial ICAM-1 expression and neutrophil adhesion. J Immunol 162:5466–5476[Abstract/Free Full Text]
  54. Degitz K, Li LJ, Caughman SW 1991 Cloning and characterization of the 5'-transcriptional regulatory region of the human intercellular adhesion molecule 1 gene. J Biol Chem 266:14024–14030[Abstract/Free Full Text]
  55. Park ES, You SH, Kim H, Park S J, Kwon O-Y, Kim YK, Ro HK, Cho BY, Chung J, Shong M 2000 Thyrotropin induces suppressors of cytokine signaling (SOCS)-1 and -3 molecules in FRTL-5 thyroid cells. Mol Endocrinol 14:440–448[Abstract/Free Full Text]
  56. Martelli ML, Trapasso F, Bruni P, Berlingieri MT, Battaglia C, Vento MT, Belletti B, Iuliano R, Santoro M, Viglietto G, Fusco A 1998 Protein tyrosine phosphatase-{eta} expression is regulated by the PKA-dependent and is downregulated by the PKC-dependent pathways in thyroid cells. Exp Cell Res 245:195–202[CrossRef][Medline]
  57. Kim TK, Maniatis T 1996 Regulation of interferon-{gamma}-activated STAT1 by the ubiquitin-proteasome pathway. Science 273:1717–1719[Abstract/Free Full Text]



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