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研究生:沈裕傑
研究生(外文):Yu-Jie Shen
論文名稱:探討SOCS3在WNT以及JAK-STAT訊息傳導途徑交互作用中所扮演的角色
論文名稱(外文):Role of SOCS3 in the crosstalk between Wnt and JAK-STAT pathways
指導教授:呂健惠
指導教授(外文):Chien-Hui Lieu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系暨研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:47
中文關鍵詞:細胞訊息抑制激酶抑制區
外文關鍵詞:suppressors of cytokine signaling 3kinase inhibitory region
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Wnt/β-catenin 以及 JAK/STAT 這兩條訊息傳導路徑與細胞存活、增生、分化以及細胞凋亡有著密切的相關性。當這兩條訊息傳導路徑失去調控後,則可能會進一步的誘導血癌或淋巴癌的發生。在人類紅血球血癌所建立的HEL 細胞株已發現其帶有同源JAK2 V617F 突變,並會促使JAK2/STAT5訊息傳導途徑持續活化並誘導下游SOCS3的大量表現。先前實驗室已發現β-catenin在T淋巴癌細胞以及人類紅血球血癌中不正常的堆積。因此,在這我們想進一步的探討在HEL以及Jurkat細胞株中,SOCS3在WNT與JAK/STAT交互作用中扮演着什麼挑控角色。先前的研究已發現,在血癌細胞株中JAK-2 knockdown以及Janus kinase 2 (JAK2) inhibitor AG490處理後,JAK2 活性會被抑制且進一步的減少Wnt訊息傳導的表現,亦証實Wnt 以及 JAK/STAT 訊息傳導的確有交互作用。 而在本篇論文研究中,我們利用免疫沉澱法的實驗發現JAK-2下游表現的SOCS3分子與Wnt 訊息傳導的GSK-3分子有結合交互作用,因此假設當SOCS3與GSK-3結合後,會誘導GSK-3的ubiquitination以及degradation,並且間接的增加β-catenin的表現量。在我們的研究中,證實了當SOCS3 overexpression 或使用GSK-3 kinase 抑制劑BIO刺激之下,皆可以明顯的逆轉AG490所造成之β-catenin表現量下降的效果。然而,SOCS- knockdown 或 overexpression 皆不會減少GSK-3的表現量。相對的,SCOS3會減少GSK-3的激脢活性並進一步的影響β-catenin的表現。因此證實了在AG490刺激之下,會經由活化GSK-3激脢的活性,而促使β-catenin的降解。綜合以上的論點,我們證實了SOCS3的確能與GSK-3結合交互作用,而減少GSK-3激脢的活性並阻止β-catenin的降解增加其表現量。SOCS3這個蛋白質分子在T淋巴癌細胞或人類紅血球血癌中,或許是ㄧ個正向調控WNT訊息傳導的重要分子。
Wnt/β-catenin and JAK/STAT signaling pathways are involved in cellular survival, proliferation, differentiation and apoptosis. Dysregulation of these signaling pathways was implicated in leukemogenesis. Our lab have found β-catenin abnormally accumulated in both acute T cell leukemia Jurkat cell line and homozygous JAK2 V617F mutated HEL cell line. In this study, we further explored that the role of SOCS3 in the crosstalk between WNT and JAK/STAT in HEL and Jurkat cell lines. In previous study, using Janus kinase 2 (JAK2) inhibitor AG490 and JAK-2 knockdown clone, we have demonstrated that inhibition of JAK2 activity significantly reduced Wnt signaling in leukemia cell lines. In the present study, we found that SOCS-3, the downstream gene of JAK-2, was co-precipitated with GSK-3 protein. Therefore, we proposed that SOCS3 can directly bind to GSK-3 that facilitated GSK-3 ubiquitination and degradation, and then indirectly increased the level of β-catenin protein. Our study confirmed that both overexpression of SOCS-3 and addition of BIO, GSK-3 kinase inhibitor can reverse AG490 effects on β-catenin degradation. However, neither knockdown nor overexpression of SOCS-3 did not reduce GSK-3 level, Insteadly, overexprewssion of SOCS-3 decreased the kinase activity of GSK-3, which related to β-catenin expression. The current work demonstrates that GSK-3 kinase activity down-regulates β-catenin under treatment of AG490. Taken together, our work suggests that SOCS-3 can directly interact with GSK-3, which resulted in reduction or/and increase the stability of β-catenin. SOCS3 is an important positive regulator of WNT signaling in erythroleukemia and T cell leukemia.
Abbreviations 2
中文摘要: 4
Abstract 5
Introduction 6
1. Canonical Wnt signaling pathway 6
2. Glycogen synthase kinase 3 (GSK-3) 7
3. JAK-STAT signaling pathway 7
4. JAK family and V 617F mutation 8
5. STAT and SOCS families 8
6. JAK/STAT signaling in leukemia 9
7. Hypothesis 10
Materials and Methods 12
1. Cells and cell culture 12
2. Antibodies and reagents 12
3. Western blotting 13
4. Immunoprecipitation 13
5. Isolation of mRNA and RT-PCR 14
6. VSV-G pseudotyped lentivirus-shRNA system 14
7. Plasmids and transfections 15
8. GSK-3 in vitro kinase assay 16
Results 17
1. SOCS-3 and β-catenin was down-regulated and GSK-3α/β was up-regulated in JAK-2 knockdown cell line. 17
2. SCOS-3 can interact with GSK-3 in Jurkat and HEL cell lines. 17
3. GSK-3 degradation can be inhibited by MG132 18
4. Knockdown of SOCS-3 decreased the β-catenin concentration. 18
5. Socs-3 overexpression can induce β-catenin accumulation in Jurkat cell line 18
6. Socs-3 overexpression can reverse AG490 effects on β-catenin 19
7. GSK-3 inhibitor can reverse AG490 effects on β-catenin. 19
8. SCOS-3 down-regulated GSK-3 kinase activity. 20
Discussion 21
Reference 26
Figures 29
Figure 1. Effects of JAK-2 knockdown on the expression of β-catenin and SOCS-3 related genes. 29
Figure 2. SOCS-3 interacts with GSK-3 in leukemia cell line. 30
Figure 3. MG132 inhibited GSK-3 degradation in HEL and K562 cell lines. 31
Figure 4. Effects of SOCS-3 knockdown on the expression of β-catenin and SOCS-3 related genes. 32
Figure 5. Expression of GSK-3 α/β and SOCS-3 in different leukemia cell lines. 33
Figure 6. SOCS-3 over-expression increased β-catenin concentration in Jurkat cell line. 34
Figure 7. Socs-3 overexpression can reverse AG490 effects on β-catenin in Jurkat cell line. 35
Figure 8. Effect of GSK-3 inhibitor on β-catenin degradation. 36
Figure 9. GSK-3 plays a key role in regulating β-catenin degradation under treatment of AG490. 37
Figure 10. SCOS-3 down-regulated GSK-3 kinase activity. 38
Appendix 39
Appendix 1. β-catenin acumulation in Jurkat and HEL leukemia cell lines 39
Appendix 2. JAK-2 inhibitor AG490 reduces the β-catenin concentration in Jurkat and HEL cell. 40
Appendix 3. Effect of JAK-2 knockdown on the expression of β-catenin and β-catenin related genes 41
Appendix 4.β-catenin distrubtion in Jurkat and HEL cells treated with 50μM AG490 by immunoflorescence. 42
Appendix 5. Three myeloid leukemic cell lines have different status of JAK-STAT signaling pathway. 43
Appendix 6. Microporation-Transfection efficiency of Jurkat cell line 43
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