- Peroxiredoxin I and II are Involved in Hydrogen Peroxide Regulation in FRTL-5 Thyroid Cells.
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Ho Kim, Tae Hoon Lee, Eun Shin Park, Jae Mi Suh, Soo Jung Park, Hyo Kyun Chung, Hyun Jin Kim, Soo Hong Chae, Do Hee Kim, O Yu Kwon, Young Kun Kim, Min Ho Shong, Heung Kyu Ro
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J Korean Endocr Soc. 2000;15(1):55-69. Published online January 1, 2001
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 Abstract
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- BACKGROUND
Peroxiredoxins (Prx) play an important role in regulating cellular differentiation and proliferation in several types of mammalian cells. One mechanism for this action involves modulation of hydrogen peroxide (H2O2)-mediated cellular responses. This report examines the expression of Prx I and Prx II in thyroid cells and their roles in eliminating H2O2 produced in response to TSH. METHODS: The expression of Prx-I and Prx-II were quantiated in FRTL-5 after stimulation with Thyroid stimulating hormone (TSH), Forskolin (FSK), Methimazole (MMI) and hydrogen peroxide (H2O2). Transient transfections were carried out with FRTL-5 cells at 80% confluency and 20microgram of pCRprx I and pCRprx II or equivalent molar amounts of the pCR3.1TM basic vector. Transient transfection used an electroporation technique. Intracellular H2O2 was assayed in FRTL-5 cells with a fluorescent dye, 2', 7'-dichlorofluoresceindiacetate (DCFH-DA). Apoptosis of cells were evaluated by using an detection kit (Promega, Inc., Madison, WI). RESULTS: Prx I and Prx II are constitutively expressed in FRTL-5 thyroid cells. Prx I expression, but not Prx II expression, is stimulated by exposure to TSH and H2O2. In addition, methimazole (MMI) induces a high level of Prx I mRNA and protein in these cells. Overexpression of Prx I and Prx II enhance the elimination of H2O2 produced by TSH in FRTL-5 cells. Treatment with 500microM H2O2 causes apoptosis in FRTL-5 cells as evidenced by standard assays of apoptosis (i.e., terminal deoxynucleotidyl transferase deoxyuridine triphosphate-biotin nick end-labeling (TUNEL), BAX expression and PARP cleavage. Overexpression of Prx I and Prx II reduces the amount of H2O2-induced apoptosis measured by these assays. CONCLUSION: These results suggest that Prx I and Prx II are involved in the removal of H2O2 in thyroid cells, and can protect these cells from undergoing apoptosis. These proteins are likely to be involved in the normal physiological response to TSH-induced production of H2O2 in thyroid cells.
- Differential Roles of Transcriptional Coactivators: CBP and CIITA on GAS (Interferon-r Activated Site) - Mediated Transcription in Thyroid Cells.
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Eun Shin Park, Ho Kim, Soon Hee You, Soo Jung Park, Hyun Jin Kim, Soo Heung Chae, Do Hee Kim, Hee Jeong Han, O Yu Kwon, Young Kun Kim, Minbo Shong, Heung Kyu Ro
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J Korean Endocr Soc. 1999;14(3):493-504. Published online January 1, 2001
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- BACKGROUND
In the previous studies, we identified that the interferon-gamma activated sequence (GAS) in the 5-flanking region of rat ICAM-1 gene is major element for interferon-y-inducible expression of the gene in rat thyroid cells, FRTL-5. We here, investigated the role of transcriptional coactivators, CBP (CREB binding protein) and CIITA (class II transactivator) in the modulation of the activity of GAS which could interacts with signal transducers and activators of transcription-1 and 3 (STAT1 and STAT3). METHODS: The expression of CBP RNA and protein were quantitated in FRTL-5 after stimulation with interferon-y (IFN-gamma), thyroid stimulating hormone (TSH), forskolin and methimazole. Direct association of CBP with STAT were analyzed by irnmunoprecipitation. The transcriptional roles of CBP and CIITA in the regulation of GAS were assessed by the cotransfection with their expression vectors with reporters; 5-deletion constructs of rat ICAM-1 promoter or 8xGAS-luc constructs, into FRTL-5 thyroid cells. RESULTS: The level of CBP RNA and protein were not changed by the treatment with TSH, IFN-y, forskolin and methimazole in FRTL-5, FRT and BRL liver cells. The CBP could be directly associated with STAT1. Furthernmore, the overexpression of CBP significantly increases the both promoter activities; rat ICAM-1 gene promoter which has GAS element and 8xGAS-luc cassette constructs. However the cotransfection of CI1TA decreased the constitutive and CBP-mediated transactivation of rat ICAM-1 promoter and SxGAS-luc cassette constructs. CONCLUSION: We identified that the two transcriptional coactivators; CBP and CIITA has differential roles in the regulation of transcriptional activity of GAS drived promoter. CBP increases the GAS activity through the direct binding with STATl, but CIITA inhibited the CBP-mediated transactivation of GAS activity.
- Thyrotropin Suppresses INF-r Mediated Gene Expression by Inhibiting Signal Transducer and Activation of Transcription 1(STAT1) Activity in FRTL-5 Cells.
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Min Ho Song, Young Kun Kim, Heung Kyu Ro, Eun Shin Park, Soon Hee Yoo, Ho Kim, Kang Wook Lee, Hee Jung Han, Won Chan Joo, Jin Ho Won, Kyu Lim, Oh Yoo Kwon
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J Korean Endocr Soc. 1998;13(4):536-553. Published online January 1, 2001
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Abstract
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- BACKGROUND
The proinflammatory cytokine, IFN-y has been shown to exert pleiotropic effects in a variety of pathophysiologic conditions in autoimmune thyroid disease. The thyrocyte response to IFN-y is mediated two distinct classes of proteins, Janus kinases(Jakl and Jak2) and Signal Transducers and Activation of Transcription(STATl). The activation of STAT 1 is involved in the regulation of many interferon stimulated genes, such as MHC class II, intercellular adhesion molecules-1(ICAM-1) and MHC class II transactivator(CIITA) after the binding to the GASgFN- pactivated site) of the gene promoters. Recently we found TSH/forskolin inhibits IFN-y stimulated maximal expression of ICAM-1 in FRTL-5 cell. IFN-y action is localized between -175 bp and -97 bp from the start of translation of ICAM-1 gene which contains regulatory elements known to be involved in IFN-y action in other eukaryotic cells, palindromic IFN-y activated site(GAS)(5-TTTCCGGGAAA-3) which could bind STAT1, STAT3, STAT5, STAT6. Furthermore, the addition of TSH and forskolin causes a decrease in ICAM-1 promoter activity and its action was localized in GAS. These findings suggested TSH/cAMP signaling pathways downregulate IFN-y activated Janus kinase-STAT signaling path. We wanted to explore the possible involvement of elevated cAMP in the negative regulation of IFN-y induced STAT1 activation in thyroid cells. METHOD: We made several 5-deletion constructs of rat ICAM-1 promoter and analyzed the promoter activities by measuring the luciferase activity after tranfection into FRTL-5 cells. The protein/DNA complex was measured by electrophoretic mobility shift analysis using labeled oligonucleotide. We checked the level of total and phosphorylated STATl protein by immunoblot analysis using specific antibodies. RESULTS: Stimulation of IFN-y in FRTL-5 cells resulted in rapid activation of STATl/DNA binding activity, which was apparent after several minute of stimulation, maintains its activity until 48 h. Incubation of cells with TSH result in suppression of IFN-p mediated STAT1/DNA binding activity throughout the time course of activation by IFN-y. Addition of TSH into 5H maintained FRTL-5 cells did not change the total amount of latent STAT1 amount and also not affect IFN-y mediated production of total STAT1 until 4 h. IFN-y(100 U/mL) rapidly induced phosphorylation of STAT1 within 30 min. and maintained its level without significant change until 48 hours. Cells treated with TSH dramatically lowered the level of IFN-y induced production and phosphorylation of STAT1 after 12 h, 24 h, 36 h, and 48 h but TSH had no effect on the level of phosphorylated STATl within 4 h after IFN-y stimulation. The proteasome inhibitor, MG132 and phosphatase inhibitor, sodium orthovanadate did not block the TSH or forskolin mediated downregulation of phosphorylated STAT1. CONCLUSION: These results indicate a regulatory mechanism which TSH signaling can modulate the prolonged activation of Jak/Stat by IFN-y. We identified one of mechanisms related to TSH mediated negative suppression of the ICAM-1 gene; TSH/cAMP signaling pathways downregulate the cytokine activated Janus kinase-STAT signaling path.
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