- Regulatory Mechanism of p66 Shc Expression by TSH in FRTL-5 Cells.
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Young Joo Park, Eun Shin Park, Tae Yong Kim, Yun Yong Lee, Seon Hwa Lee, Do Joon Park, Chan Soo Shin, Kyoung Soo Park, Seong Yeon Kim, Hong Kyu Lee, Bo Youn Cho
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J Korean Endocr Soc. 2003;18(1):45-55. Published online February 1, 2003
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Thyroid goiters are very common, however, the mechanism of development is not fully understood. A TSH receptor has been known to activate two different signaling pathways the cAMP/protein kinase A(PKA) and phospholipase C(PLC)/protein kinase C(PKC) systems. However, both systems are limited in the degree to which they explain the discrepancy between a goiter and TSH receptor activation. It has recently been reported that the expression of p66 Shc was increased by TSH stimulation in thyrocytes, suggesting that the p66 Shc molecule may play a critical role in the transition of the TSH-induced growth signals. METHODS AND RESULTS: In this study, we examined the expression of p66 Shc by stimulation of TSH, and the regulatory mechanisms of the TSH-induced expression of the p66 Shc in FRTL-5 cells. In FRTL-5 cells, TSH could increase the expression of the p66 Shc, and the this expression was decreased to basal levels after the removal of TSH. The TSH-induced p66 Shc expression was competitively inhibited by TSH receptor blocking antibodies. The increments of the expression of the p66 Shc protein caused by TSH were both time and concentration dependent, and it was same in the mRNA levels. Cholera toxin increased the expression of the p66 Shc, while pertussis toxin did not. The activators of the cAMP/PKA pathway (8-bromo-cAMP and forskolin) also stimulated the expression of p66 Shc, and the PKA inhibitor H89 decreased the expression, while the inhibition of the PKC pathway by GF109203X, or PMA, affected the expression of p66 Shc very little. CONCLUSION: Our data suggests that p66 Shc may play an important role in regulating the growth of thyrocytes. The TSH receptor - Gs protein - adenylate cyclase - cAMP - PKA pathway mainly mediates the TSH effects on the expression of p66 Shc molecules.
- The Effects of Iodide on the Cellular Functions and Expression of Thyroid Autoantigens in Thyroid Cells.
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Young Joo Park, Eun Shin Park, Tae Yong Kim, Hye Seung Jung, Hyeong Kyu Park, Do Joon Park, Won Bae Kim, Chan Soo Shin, Kyoung Soo Park, Seong Yeon Kim, Hong Kyu Lee, Bo Youn Cho
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J Korean Endocr Soc. 2002;17(1):69-78. Published online February 1, 2002
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- BACKGROUND
Iodide has been known to control the function and the growth of the thyroid gland, and to be used as a substrate of thyroid hormone. Moreover, it has been suggested that excessive iodide stimulates the thyroid autoimmune responses. To evaluate the effects of iodide on thyrocytes, we investigated cell function and proliferation, or thyroid autoantigen expression after administering iodide to rats or FRTL-5 cells. MEHTODS AND RESULTS: Ten-weeks-old Sprague-Dawley rats were sacrificed after 7 days of NaI treatment. The expressions of thyroid autoantigens were examined by northern blot analysis. Chronic administration of iodide resulted in no effect on TSH receptor (TSHR) and thyroperoxidase (TPO) mRNA expression, while it increased thyroglobulin (TG) and diminished sodium-iodide symporter (NIS) mRNA expression. FRTL-5 cells were also treated with various concentrations of NaI. The generation of cAMP or iodide uptake was decreased, and the cellular growth was also inhibited by iodide. However, the expressions of all thyroid autoantigens (TSHR, TG, TPO, MHC class I and class II) except NIS were unchanged for 72 hours after iodide administration. The expression of NIS was mildly increased after 24 hours. CONCLUSION: Iodide resulted in decreased cell proliferation and cellular function of cAMP generation and iodide uptake. Chronic administration of iodide increased TG and diminished NIS mRNA expression in vivo but not in vitro
- The Changes of Expression of Thyroid Specific Antigens in Aging.
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Young Joo Park, Eun Shin Park, Tae Yong Kim, Sang Wan Kim, Hyeong Kyu Park, Do Joon Park, Won Bae Kim, Chan Soo Shin, Kyoung Soo Park, Seong Yeon Kim, Sang Chul Park, Hong Kyu Lee, Bo Youn Cho
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J Korean Endocr Soc. 2001;16(4-5):457-466. Published online October 1, 2001
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With the prevalence of serum antithyroglobulin(anti-TG) and antithyroperoxidase(anti-TPO) autoantibodies increasing with age, it has been suggested that changes of thyroid autoimmunity with aging are associated with endemic iodine intake. To understand the mechanism of aging-related increases of thyroid autoimmune response, we investigated the expression of thyroid specific autoantigens of aged phenotype, and compared them with those of young phenotype both in vivo and in vitro. METHODS: Sprague-Dawley rats were sacrificed at 5, 10 and 16 weeks(young), and at 23 months(aged). Their FRTL-5 thyroid cells were harvested at cell passages less than 10(fresh) or more than 30 (aged). The expression of thyroid autoantigens, sodium-iodide symporter(NIS), TSH receptor (TSHR), TG and TPO, were examined by northern blot analysis. To evaluate the effects of iodide, 1mM of NaI was added to the medium for 24 hours, and following incubation the expressions of MHC class I and class II were also examined. RESULTS: The expressions of TPO were markedly increased in the aged rats, and those of TG were moderately. However, NIS and TSHR showed no differences in their expression levels between aged rats and young rats. In vitro, there were no differences in the expressions of TG or TPO, nor of NIS or TSHR, between aged cells and fresh cells. Neither did Iodide exhibit any influence on the expression of MHC molecules in aged cells or fresh cells. CONCLUSION: The expression levels of TPO and TG were increased in aged rats, which may partially explain the mechanism of increasing thyroid autoimmunity with age.
- 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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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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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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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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