TSH 자극에 의한 FRTL-5 세포에서 p66 Shc의 발현 조절 기전

박영주,박은신,김태용,이윤용,이선화,박도준,신찬수,박경수,김성연,이홍규,조보연 대한내분비학회 2003 Endocrinology and metabolism Vol.18 No.1

연구배경: 갑상선종은 임상적으로 매우 흔하게 나타나지만, 아직까지 그 발생기전에 대하여 알려진 바가 적다. 갑상선 세포의 TSH 수용체 신호 전달 체계가 갑상선종의 발생에 중요한 역할을 할 것으로 생각되고 있으나, 기존에 잘 알려진 신호전달 기전인 AMP/A 단백질 활성화효소 (PKA) 체계나 PKC 체계만으로는 설명하기 어려운 점이 많다. 최근 TSH가 갑상선 세포에서 p66 Shc 단백질의 발현 증가를 유도하는 것이 보고되어, p66 Shc이 TSH에 의한 세포 증식 신호전달에 있어서 중요한 역할을 담당할 가능성이 시사되었다. 목적: 본 연구에서는 TSH 자극에 따른 갑상선 세포에서의 p66 Shc의 발현 변화를 관찰하여, p66 Shc 단백질과 갑상선 세포의 증식 신호와의 연관성을 알아보고자 하였다. 또한 TSH가 p66 Shc 단백질의 발현을 조절하는 것과 연관된 신호전달 과정을 살펴보아, TSH의 p66 Shc 발현 조절 기전을 밝히고자 하였다. 방법 및 결과: FRTL-5 세포에서 TSH에 의해서 p66 Shc의 발현의 증가하고, TSH를 제고하였을 때에는 다시 기저 수준으로 감소하였다. TSH에 의한 p66 Shc의 발현 증가는 TSH 수용체 차단항체에 의하여 경쟁적으로 억제되었다. TSH에 의한 p66 Shc의 발현은 TSH를 처리한 시간과 농도에 의존적으로 증가하였고, 이러한 현상은 p66 Shc mRNA의 발현에서도 동일하게 관찰되었다. 콜레라 독소는 p66 Shc의 발현을 증가시켰으나 백일해 독소는 TSH에 의한 p66 Shc의 발현에 아무런 영향을 미치지 못하였다. cAMP/PKA 자극제(8-bromo-cAMP, forskolin)를 처리하였을 때 p66 Shc의 발현이 증가하고 cAMP/PKA 억제제(H89)에 의하여 TSH에 의한 효과가 억제되었으나, C단백질 활성화 효소 억제제(PMA, GF109203X)는 아무런 영향을 미치지 못하였다. 결론: FRTL-5 세포에서는 TSH 자극에 의존적으로 p66 Shc의 발현이 조절되었으며, 이러한 TSH의 p66 Shc 발현 조절은 주로 TSH 수용체 - Gs 단백질 - adenylate cyclase - cAMP - PKA의 신호 전달 경로를 통하여 이루어지는 것으로 생각되어진다. Background: 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 She 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 & 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 increase 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 regulation 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 (J Kor Soc Endocrinol 18:45∼55, 2003).

가령 (Aging)에 따른 갑상선 특이항원 발현의 변화

박영주,박은신,김태용,김상완,박형규,박도준,김원배,신찬수,박경수,김성연,박상철,이홍규,조보연 대한내분비학회 2001 Endocrinology and metabolism Vol.16 No.4

Background: 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, 1 mM 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

ARP Spoofing 공격과 대응방법에 관한 연구

황현욱(Hwang Hyun - Uk),박은신(Park Eun - Shin),박종백(Park Jong - Baek) 한국통신학회 2001 한국통신학회 학술대회논문집 Vol.2001 No.11

시호 약배양에 의한 캘러스 형성 및 식물체 재분화

박충헌,성낙술,이승택,정해곤,박은신 한국육종학회 1995 한국육종학회지 Vol.27 No.4

Thyrotropin은 STAT1(Signal Transducers and Activation of Transcription 1) 의 활성을 저해하여 Interferon-r에 의한 유전자 발현을 억제한다

이강욱,김영건,노흥규,송민호,김호,박은신,유순희,한희정,주원찬,원진호,임규,권오유 대한내분비학회 1998 Endocrinology and metabolism Vol.13 No.4

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 (J Kor Soc Endocrinol 13:536-553, 1998).

요오드가 갑상선 세포의 기능 및 자가항원 발현에 미치는 영향

김원배,박도준,박경수,김성연,이홍규,김태용,박영주,박은신,정혜승,박형규,신찬수,조보연 대한내분비학회 2002 Endocrinology and metabolism Vol.17 No.1

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. Methods 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

각종 간질환에서 RT - PCR을 이용한 G형 간염바이러스 RNA의 검출률

한광협,최원,박영년,황영웅,류왕식,박은신,이관식,전재윤,문영명,박찬일 대한간학회 1997 Clinical and Molecular Hepatology(대한간학회지) Vol.3 No.2

Background / Aim : Recently, nucleotide sequences from a novel virus, termed hepatitis G virus(HGV), were identified in serum from a patient with cryptogenic hepatitis and suggested as agent of non A-E hapatitis HGV has been isolated from patients with various liver disease but clinical implications of this new agent remain largely unresolved . In Korea, the etiology of substantial fraction of hepatitis has remined undefined rate of HGV. Methods : To determine the implication of HGV, medical records of 115 patients with various liver diseases were reviewed. Of 115 patients, 63 were male and 52 were female. Their mean age was 44 years(19-74) and their mean AST and ALT were 121.3±278.7 IU/L and 172.2±253.3 IU/L, infected with hepatitis B and/or C virus and 20(17.4%) had non-viral identifiable liver diseases. Results : 1.HGV RNA was detected in 15 (13.0%) patients of 115 patients. 2. Among the 15 HGV RNA positive cases, 7 were male and 8 were female. Their mean age was 48 years(19-72) and their mean AST and ALT were 71.9±45.2 IU/L and 97.4±66.8 IU/L, respectively. 3 . HGV RNA was detected in 8 (13.85) of 58 patients without obvious causes if their liver diseases and in 7(18.9%) of 37 patients infected with HBV and/or HCV. However, HGV RNA was not detected from 20 patients with non-viral liver diseases such as alcoholic liver diseases, autoimmune hepatitis, PBC, or fatty liver. 4. HGV RNA was detected in 5 (19.2%) of 26 patients with acute hepatitis in 6 (9.4%) of 64 patients with chronic hepatitis, in 1 (14.3%) of 7 patients with liver cirrhosis, and in 3 (27.3%) of 11 patients with hepatocellular carcinoma. 5. There was no statistically significant difference in sex, age, history of transfusion, serum ALT level, etiologies and status of HGV infection is quite high among the patients who have no specific cause of acute or chronic liver diseases and HGV can be confected with HBV and/or HCV infection in Korea.

갑상선 세포에서 Peroxiredoxin에 의한 과산화수소 및 아포프토시스 조절

김현진,이태훈,김도희,권오유,김영건,송민호,노흥규,박수정,김호,박은신,정효균,서재미,채수홍 대한내분비학회 2000 Endocrinology and metabolism Vol.15 No.1

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 20?g 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 500?M 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(J Kor Soc Endocrinol15:55-69, 2000).

갑상선 세포에서 전사보조활성인자인 CBP와 CIITA는 인터페론-감마 활성 부위에 대하여 서로 다른 조절 작용을 나타낸다

유순희,김현진,김도희,김영건,송민호,노흥규,박수정,김호,박은신,채수흥,권오유,한희정 대한내분비학회 1999 Endocrinology and metabolism Vol.14 No.3

Background: In the previous studies, we identified that the interferon-γ 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-γ), 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 (J Kor Soc Endocrinol 14:493-504, 1999).