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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳宏文(Hung-Wen Chen) | |
| dc.contributor.author | Ching-Wen Chang | en |
| dc.contributor.author | 張清文 | zh_TW |
| dc.date.accessioned | 2021-06-08T05:27:27Z | - |
| dc.date.copyright | 2011-08-02 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-07-28 | |
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Nucleic Acids Res, 2008. 36(11): p. 3834-46. 56. Delidaki, M., et al., Interplay of cAMP and MAPK pathways in hCG secretion and fusogenic gene expression in a trophoblast cell line. Mol Cell Endocrinol, 2011. 332(1-2): p. 213-20. 57. Oestreich, E.A., et al., Epac-mediated activation of phospholipase C(epsilon) plays a critical role in beta-adrenergic receptor-dependent enhancement of Ca2+ mobilization in cardiac myocytes. J Biol Chem, 2007. 282(8): p. 5488-95. 58. Hochbaum, D., et al., Epac, in synergy with cAMP-dependent protein kinase (PKA), is required for cAMP-mediated mitogenesis. J Biol Chem, 2008. 283(8): p. 4464-8. 59. Petersen, R.K., et al., Cyclic AMP (cAMP)-mediated stimulation of adipocyte differentiation requires the synergistic action of Epac- and cAMP-dependent protein kinase-dependent processes. Mol Cell Biol, 2008. 28(11): p. 3804-16. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24475 | - |
| dc.description.abstract | 人類胎盤特殊轉錄因子GCM1 (Glial cells missing homolog 1)屬於GCM轉錄因子基因家族，可控制融合蛋白syncytin基因表現，促使胎盤絨毛表面的滋養層細胞(cytotrophoblast)分化為融合細胞滋養層(syncytiotrophoblast)，是調控胎盤發育的重要轉錄因子之一。先前的研究指出，胎盤細胞內cAMP濃度提高時會透過訊息傳遞路徑活化GCM1的轉錄活性，促使胎盤細胞滋養層的融合，顯示cAMP可能藉由下游因子來調控GCM1活性和相關基因的表現。目前實驗室已經證實 GCM1活性可以受到不同層次的轉譯後修飾作用調控 (post-translational modification)，例如藉由cAMP/PKA/CBP路徑減少泛素化 (ubiquitination)程度以提高GCM1蛋白的穩定性；或是透過UBC9將DNA結合區域內的Lys156小類泛素化 (sumoylation)以降低對DNA的結合能力。雖然cAMP/PKA調控GCM1的機制已經非常清楚；但是對於GCM1小類泛素化的調控機制確有待研究。本研究論文證實，cAMP藉由PKA以外的傳遞路徑調控GCM1的轉錄活性。透過cAMP下游因子Epac1和Rap1活化CaMKI的磷酸酶活性進而磷酸化GCM1Ser47，當GCM1Ser47被磷酸化後，會誘使SENP1(去小類泛素化酵素)結合，移除GCM1小類泛素化修飾，增加其DNA的結合能力。此外，我們發現將GCM1表現弱化(knock-down)的胎盤細胞處理Epac1活化藥物時，會導致細胞融合能力下降。為了進一步證實GCM1Ser47的重要性，我們將GCM1表現弱化的細胞株，重新導入不受shRNA作用的GCM1表現蛋白，比較正常(wild-type)、S47A (mutant)和S47D (phosphomimetic mutant )三者之間對細胞融合能力的影響。結果顯示帶有GCM1S47D表現細胞株具有較高的融合能力，顯示GCM1Ser47磷酸化對細胞融合的重要性。綜合上述結果，我們首度證實GCM1活性受到cAMP/Epac1/Rap1訊息傳遞路徑的調控，對胎盤滋養層細胞的融合扮演不可或缺的角色；同時對於cAMP誘使胎盤滋滋養層細胞融合的作用機制，提出了新的觀點，透過GCM1的磷酸化和去小類泛素化來調控胎盤滋養層細胞的分化。 | zh_TW |
| dc.description.abstract | cAMP signaling and the placental transcription factor GCM1 regulate expression of syncytin-1 and -2 fusogenic proteins, which are critical for syncytiotrophoblast formation by trophoblast fusion. We recently revealed a cAMP/PKA/CBP signaling pathway that activates GCM1 by coordinating GCM1 phosphorylation and acetylation. In contrast, GCM1 activity is downregulated by sumoylation of Lys156. How GCM1 sumoylation is regulated was unknown. Here we identify a novel PKA-independent cAMP signaling pathway as the critical regulator of GCM1 sumoylation. We show that Epac1 and Rap1, in response to cAMP, activate CaMKI to phosphorylate Ser47 in GCM1. This phosphorylation facilitates the interaction between GCM1 and the desumoylating enzyme SENP1 and thereby leads to GCM1 desumoylation and activation. Using RNAi, we further demonstrate that 8-CPT-AM, an Epac activator, stimulates syncytin-1 and -2 gene expression and cell fusion of placental BeWo cells in a GCM1-dependent manner. Importantly, the cell fusion defect in GCM1-knockdown BeWo cells can be reversed and enhanced by the RNAi-resistant phosphomimetic GCM1S47D mutant. Our study has identified a novel
cAMP/Epac1/CaMKI/GCM1 signaling cascade that stimulates trophoblast fusion through promoting GCM1 phosphorylation and desumoylation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T05:27:27Z (GMT). No. of bitstreams: 1 ntu-100-D95b46003-1.pdf: 1121863 bytes, checksum: 7f98e93f60a6d377f6d6ea638426fa86 (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | Abstract 1
中文摘要 2 Introduction 3 Placental function 3 Early development of the human placenta 3 Placental trophoblast cell lineages 4 Human placental endogenous retrovirues: HERV-W and HERV-FRD 5 Human GCM1 regulates the expression of syncytin-1 and -2 7 GCM1 function in placentation 7 GCM1 in disease 8 GCM1 structure 9 Regulation of GCM1 activity by post-translational modification (PTM) 9 Regulation of GCM1 stability by ubiquitination 10 Negative regulation of GCM1 activity by sumoylation 10 The SUMO modification pathway 11 Desumoylation enzymes-SENPs 12 cAMP/PKA signaling pathway regulates GCM1 activity 13 cAMP/Epac1 signaling pathway mediates differentiation of human placental BeWo cells 14 Models used in the study 16 Significance and purpose 17 Materials and Methods 18 Chemicals and protein kinase inhibitors 18 Plasmid constructs 18 Cell culture, transfection, and lentivirus transduction 19 Isolation of villous cytotrophoblast from term placenta 20 GCM1 sumoylation and desumoylation 20 Regulation of interaction between GCM1 and SENP1 21 Mapping the SENP1-interacting domain in GCM1 21 CaMKI-mediated GCM1 phosphorylation and mass spectrometry 22 Chromatin immunoprecipitation (ChIP) assay 23 Rap1 activity assays 23 Immunofluorescence microscopy and cell-cell fusion analysis 24 Quantitative real-time PCR 25 Results 26 CaMKI is downstream of cAMP signaling in regulation of GCM1 activity 26 CaMKI facilitates GCM1 desumoylation by enhancing the interaction of GCM1 and SENP1 27 CaMKI mediates Ser47 phosphorylation in GCM1 29 Epac activates CaMKI to regulate the interaction of GCM1 and SENP1 31 Epac regulates placental cell fusion via GCM1 33 Discussion 35 References 53 Figures 39 Figure 1. CaMKI is involved in the regulation of GCM1 activity by cAMP. 39 Figure 2. Regulation of GCM1 desumoylation by CaMKI. 41 Figure 3. SENP1 interactions with GCM1 42 Figure 4. CaMKI mediates Ser47 phosphorylation in GCM1 to enhance the interaction between GCM1 and SENP1 43 Figure 5. Ser47 is critical for stimulation of GCM1 activity by CaMKI. 44 Figure 6. EPAC1 regulates the interaction of GCM1 and SENP1. 46 Figure 7. Regulation of Rap1 activity by Epac1. 48 Figure 8. Stimulation of Ser47 phosphorylation by Epac1 and CaMKI in placenta cells. 49 Figure 9. Regulation of placental cell fusion by Epac1. 50 Figure 10. Regulation of GCM1 activity and placental cell fusion by cAMP. 52 | |
| dc.language.iso | en | |
| dc.subject | 去小類泛素化 | zh_TW |
| dc.subject | 胎盤轉錄因子 | zh_TW |
| dc.subject | desumoylation | en |
| dc.subject | GCM1 | en |
| dc.subject | SENP1 | en |
| dc.subject | sumoylation | en |
| dc.title | 磷酸化和去小類泛素化協同調控人類胎盤轉錄因子GCM1活性 | zh_TW |
| dc.title | Coordinated Regulation of Human GCM1 Activity by Phosphorylation and Desumoylation in Placental Cell Differentiation | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 張震東(Geen-Dong Chang),張茂山(Mau-Sun Chang),李明亭(Ming-Ting Lee),張功耀(Kung-Yao Chang) | |
| dc.subject.keyword | 胎盤轉錄因子,去小類泛素化, | zh_TW |
| dc.subject.keyword | GCM1,SENP1,sumoylation,desumoylation, | en |
| dc.relation.page | 57 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2011-07-28 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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