跳到主要內容

臺灣博碩士論文加值系統

(3.95.131.146) 您好!臺灣時間:2021/07/26 04:15
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:陳明宏
研究生(外文):Ming-Hung Chen
論文名稱:轉錄因子GCM1及MTF1調控PGF基因機制之探討
論文名稱(外文):Regulation of PGF gene expression by GCM1 and MTF1
指導教授:陳宏文陳宏文引用關係
口試委員:張震東黃娟娟張功耀
口試日期:2015-07-16
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:66
中文關鍵詞:PGFGCM1MTF1胎盤滋養層細胞
外文關鍵詞:PGFGCM1MTF1placentatrophoblast cell
相關次數:
  • 被引用被引用:0
  • 點閱點閱:68
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
PGF (Placental growth factor)為血管內皮生長因子家族其下的一員。在正常懷孕的期間PGF會高度表現在胎盤裡。過去的研究顯示PGF在血管新生(vasculogenesis)與血管的衍生(angiogenesis)上,藉由旁分泌作用在內皮細胞扮演著關鍵的角色。此外,PGF也在早期胚胎發育表現並控制早期胚胎發育。胎盤專一表現的轉錄因子GCM1 (Glial cells missing 1)對於胎盤細胞的分化過程很重要。最近,我們利用ChIP-chip (chromatin immunoprecipitation-on-chip)的實驗確認PGF為GCM1的目標基因。雖然在過去文獻中已知在胎盤細胞裡GCM1在轉錄上會調控PGF的表現,但PGF如何被調控的機制尚不清楚。
在其他的研究顯示, MTF1 (metal-responsive transcription factor 1)參與在BeWo細胞(由滋養層絨毛膜所衍生的細胞株)裡,誘導PGF的表現。MTF1為反應於各種環境因子的轉錄因子,包括金屬負載、缺氧以及氧化壓力。因此,在我們的研究中也探討MTF1是否涉及PGF表在胎盤細胞裡。由我們實驗結果表明,PGF mRNA和蛋白表現在GCM1剔除以及缺氧處理下的BeWo細胞中都很顯著地減少;相反地,於JAR細胞過度表現GCM1時,其結果有增多的現象。此外,我們利用冷光報導基因試驗在HEK 293T (人類胚胎腎細胞株)細胞進行實驗,發現GCM1會促進位在-1000到-345以及+5到+100區域的PGF啟動子活性。進一步地藉由點突變的方式,我們進一步發現了對於GCM1上調PGF啟動子活性的GCM1結合位點。另一方面,在MTF1剔除和缺氧條件下的細胞,PGF mRNA表現有稍有下降。但是,在我們的結果裡,我們卻無法觀察到MTF1調節PGF啟動子活性。綜合以上的實驗結果,於本篇論文中得到以下推論。在胎盤細胞裡,可能主要由GCM1轉錄因子結合到PGF基因啟動子5端非編碼區內的位置後,去調控開啟PGF基因;而在非胎盤細胞裡,可能才是藉由MTF1轉錄因子去啟動PGF基因。


PGF is a member of VEGF family. PGF is highly expressed in placenta during normal pregnancy. Previous study showed that it plays a pivotal role for vasculogenesis and angiogenesis in endothelial cells via paracrine action. In addition, PGF is also expressed during early embryonic development and control early embryogenesis. The placenta-specific transcription factor GCM1 is critical for placental cell differentiation. We have recently identified PGF as a GCM1 target gene by ChIP-chip analysis. Although GCM1 has been showed to regulate PGF expression transcriptionally, the mechanism of PGF expression is still not clear.
Other study demonstrate that MTF1 takes part in the induction of PGF expression in Bewo cell, the trophoblast-derived choriocarcinoma cell line. MTF1 is a transcription factor that responds to a variety of stresses including metal load, hypoxia and oxidative stress. Here, we also investigated whether MTF1 is involved in PGF expression. We demonstrated that the PGF mRNA and protein levels are significantly decreased in GCM1-knockdown and hypoxia Bewo cells. Conversely, the results were increased in GCM1-overexpressed JAR cells. Furthermore, GCM1 stimulated the PGF promoter activity by luciferase reporter construct harboring the PGF promoter region from -1000 to -345 and +5 to +100 relative to the transcriptional start site. By site-directed mutagenesis, we further identified a GCM1-binding site that is essential for GCM1 to upregulate PGF promoter activity. On the other hand, the PGF mRNA level was slightly decreased on MTF1 -knockdown and hypoxia condition. But we failed to observe that MTF1 regulate PGF promoter activity. Based on the above results, we get the following inference in this paper. In placental cells, it may mainly bind to PGF promoter 5’ UTR region and turn on PGF gene by GCM1 transcription factor. In the non-placental cells, MTF1 may transcriptionally regulate PGF gene.


目錄 …………………………………………………………………………….. I
圖表目錄 ………………………………………………………………………III
中文摘要 ………………………………………………………………………IV
英文摘要 ……………………………………………………………………….V

第一章 緒論
1.1 胎盤 …………………………………………………..……………………..1
1.2 GCM 轉錄因子 ……………………………………………..………….......3
1.3 胎盤生長因子 ………………………………………………..……………..8
1.4 MTF 轉錄因子 ………………………………………………….…….......11
1.5 研究動機 …………………………………………………………….…….14

第二章 材料與方法
2.1 構築重組質體 ……………………………………………….……….……15
2.2 細胞株培養與轉染 ………………………………………….……….……19
2.3 SDS聚丙醯胺凝膠電泳 …………………………………………..………20
2.4 西方墨點法 ………………………………………………………..………21
2.5 Luciferase 冷光報導基因活性檢測 ………………………………..…….22

第三章 實驗結果
3.1 GCM1與MTF1轉錄因子調控胎盤細胞中PGF基因的表現 ………….23
3.2 GCM1以及MTF1轉錄因子對於PGF啟動子之轉錄活性 …………….24
3.3 GCM1轉錄因子結合PGF基因啟動子特定區域之分析 ……………… 25
3.4 突變後的片段對於GCM1轉錄因子結合PGF基因之影響 ……………26
3.5 染色質免疫沉澱分析 GCM1以及MTF1對於PGF基因啟動子之影響.27

第四章 討論與總結 ……………………………………………………………..…28

第五章 圖表 ………………………………………………………………………...34

第六章 參考文獻 …………………………………………………………………...56


1. Gude, N., et al., Growth and function of the normal human placenta. Thromb Res, 2004.114(5-6): p. 397-407.
2. Cross, J. C., Placental function in development and disease. Reprod Fertil Dev,2006.18(1-2): p. 71-6.
3.Huooertz, B., and Borges, M., Placenta trophoblast fusion. Metods Mol Biol, 2008.475: p. 135-47.
4.Lubghi, L., et al., Control of human trophoblast function. Reprod Biol Endocrinol,2007. 5: p. 6.
5.Hosoya, T., et al., glial cells missing: a binary switch between neuronal and glial determination in Drosophila. Cell, 1995. 82(6): p. 1025-36.
6.Jones, B.W., et al., glial cells missing a genetic switch that controls glial versus
neuronal fate. Cell, 1955. 82(6): p. 1013-23.
7.Wegner, M., and Riethmacher, D., Chronicles of a switch hunt: gcm genes in development. Trends Genet, 2001. 17(5): p. 286-90.
8.Kim, J. et al., Isolation and characterization of mammalian homologs of the Drosophila genes glial cells missing. Proc Natl Acad Sci USA, 1998. 95(21): p. 12364-9.
9.Kanemura, Y., et al., Isolation and expression analysis of a novel human homologue of the Drosophila glial cells missing (gcm) gene. FEBS Lett, 1999. 442(2-3): p. 151-6.
10.Cohen, S.X., et al., Structure of the GCM domain-DNA complex: a DNA-binding domain with a novel fold and mode of target site recognition. EMBO J,
2003.22(8): p. 1835-45.
11.Schreiber, J., Enderich, J., and Wegner, M., Structural requirements for DNA
binding of GCM proteins. Nucleic Acids Res, 1998. 26(10): p. 2337-43.
12.Akiyama, Y., et al., The gcm-motif: a novel DNA-binding motif conserved in
Drosophila and mammals. Proc Natl Acad Sci USA, 1996. 93(25): p. 14912-6.
13.Schreiber, J., et al., Placental failure in mice lacking the mammalian homolog of
glial cells missing, GCMa. Mol Cell Biol, 2000. 20(7): p. 2466-74.
14.Hashemolhosseini, S., and Wgner, M., Impacts of a new transcription factor family: mammalian GCM proteins in health and disease. J Cell Biol, 2004. 166(6): p.
765-8.
15.Anson-Cartwright, L., et al., The glial cells missing-1 protein is essential for branching morphogenesis in the chorioallantoic placenta. Nat Genet, 2000. 25(3):
p. 311-4.
16.Günther. T, et al., Genetic ablation of parathyroid glands reveals another source of parathyroid hormone. Nature. 2000.406(6792): p. 199-203.
17.Baczyk, D., et al., Glial cell missing-1 transcription factor is required for the differentiation of the human trophoblast. Cell Death Differ, 2009.16(5): p. 719-27.
18.Yamada, K., et al., A GCM motif protein is involved in placenta-specific expression
of human aromatase gene. J Biol Chem, 1999. 274(45): p. 32279-86.
19.Yu, C., et al., GCMa regulates the syncytin-mediated trophoblastic fusion. J Biol
Chem, 2002. 277(51): p. 50062-8.
20.Chen, C.P., et al., Functional characterization of the human placental fusogenic
membrane protein syncytin 2. Biol Reprod, 2008. 79(5): p. 815-23.
21.Esnault, C., et al., A placenta-specific receptor for the fusogenic, endogenous retrovirus derived, human syncytin-2. Proc Natl Acad Sci USA, 2008.105(45): p.
17532-7.
22.Liang, C.Y., et al., GCM1 regulation of the expression of syncytin 2 and its cognate receptor MFSD2A in human placenta. Biol Reprod, 2010. 83(3): p. 387-95.
23.Malassine, A., et al., Expression of the fusogenic HERV-FRD Env glycoprotein (syncytin 2) in human placenta is restricted to villous cytotrophoblastic cells.
Placenta, 2007. 28(2-3): p. 185-91.
24.Vargas, A., et al., Syncytin-2 plays an important role in the fusion of human
trophoblast cells. J Mol Biol, 2009. 392(2): p. 301-18.
25.Athanassiades, A., and Lala, P.K., Role of placenta growth factor (PlGF) in human extravillous trophoblast proliferation, migration and invasiveness. Placenta,
1998.19(7): p. 465-73.
26.De Falco, S., The discovery of placenta growth factor and its biological activity.
Exp Mol Med, 2012. 44(1): p. 1-9.
27.Dwsai, J., et al, Signal transduction and biological function of placenta growth factor in primary human trophoblast. Biol Reprod, 1999. 60(4): p. 887-92.
28.Chang, M., et al., Glial cell missing 1 regulates placental growth factor (PGF) gene transcription in human trophoblast. Biol Reprod. 2008. 78(5): p. 841-51.
29.Wang, L.J., et al., High-temperature requirement protein A4 (HtrA4) suppresses the fusogenic activity of syncytin-1 and promotes trophoblast invasion. Mol Cell
Biol, 2012. 32: p. 3707-17.
30.Chang, C.W., Chang G.D. and Chen, H., A novel cyclic AMP/Epac1/CaMKI signaling cascade promotes GCM1 desumoylation and placental cell fusion. Mol
Cell Biol, 2011. 31(18): p. 3820-31.
31.Chang, C.W., et al., Stimulation of GCMa transcriptional activity by cyclic AMP/protein kinase A signaling is attributed to CBP-mediated acetylation of
GCMa. Mol Cell Biol, 2005. 25(19): p. 8401-14.
32.Chuang, H.C., et al., Histone deacetylase 3 binds to and regulates the GCMa
transcription factor. Nucleic Acid Res, 2006. 34(5): p. 1459-69.
33.Chiang, M.H., et al., Mechanism of hypoxia-induced GCM1 degradation: implications for the pathogenesis of preeclampsia. J Biol Chem,
2009.284(26): p. 17411-9.
34.Chou, C.C., et al., Small ubiquitin-like modifier modification regulates the DNA binding activity of glial cell missing Drosophila homolog a. J Biol Chem,
2007. 282(37): p. 27239-49.
35.Maglione, D., et al., Isolation of a human placenta cDNA coding for a protein related to the vascular permeability factor. Proc Natl Acad Sci USA, 1991.
88(20): p. 9267-71.
36.DiSalvo, J., et al., Purification and characterization of a naturally occurring vascular endothelial growth factor, placenta growth factor heterodimer. J Biol
Chem, 1995. 270(13): p. 7717-23.
37.Iyer, S., et al., The Crystal Structure of Human Placenta Growth Factor-1 (PlGF-1), an Angiogenic Protein, at 2.0 Å Resolution. J Biol Chem, 2001. 276(15):
p. 12153-61.
38.Cao, Y., et al., Placenta growth factor: identification and characterization of a novel isoform generated by RNA alternative splicing. Biochem Biophys Res.
Commun.1997, 235(3): p.493-8.
39.Yang, W., et al., Evidence of a novel isoform of placenta growth factor (PlGF-4) expressed in human trophoblast and endothelial cells. J Reprod Immunol, 2003.
60(1): p.53-60.
40.Park, J.E., et al., Placenta growth factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but
not to Flk-1/KDR. J Biol Chem, 1994. 269(41): p. 25646-54.
41.Migdal, M., et al., Neuropilin-1 is a placenta growth factor-2 receptor. J Biol
Chem, 1998. 273(35): p. 22272-8.
42.DiPalma, T., et al., The placenta growth factor gene of the mouse. Mamm Genome,
1996. 7(1): p. 6-12.
43.Tchaikovski, V., Fellbrich, G., Waltenberger, J., The molecular basis of VEGFR-1 signal transduction pathway in primary human monocytes. Arterioscler Thromb
Vasc Biol, 2008. 28(2): p. 322-8.
44.Yajima, I., et al., Arsenite-mediated promotion of anchorage-independent growth of HaCat cells through placental growth factor. J Invest Dermatol, 2015. 135(4): p.
1147-56.
45.Carmeliet, P., et al., Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in
pathological conditions. Nat Med, 2001. 7(5): p. 575-83.
46.Gigante, B., et al., Placenta growth factor is not required for exercise-induced
angiogenesis. Angiogenesis, 2004. 7(3): p. 277-84.
47.Munaut, C., et al., Hypoxia is responsible for soluble vascular endothelial growth factor receptor-1 (VEGFR-1) but not for soluble endoglin induction in villous
trophoblast. Hum Reprod, 2008. 23(6): p. 1407-15.
48.Depoix, C., Tee, M.K. and Taylor, R.N., Molecular regulation of human placental growth factor (PlGF) gene expression in placental villi and trophoblast cells is mediated via the protein kinase A pathway. Reprod Sci, 2011. 18(3): p. 219-28.
49.Tayade, C., et al., Genetic deletion of placenta growth factor in mice alters uterine
NK cells. J Immunol, 2007. 178(7): p. 4267-75.
50.Ziche. M., et al., Placenta growth factor-1 is chemotactic, mitogenic, and
angiogenic. Lab Invest, 1997.76(4): p. 517-31.
51.Yonekura, H., et al., Placenta growth factor and vascular endothelial growth factor B and C expression in microvascular endothelial cells and pericytes. Implication in autocrine and paracrine regulation of angiogenesis. J Biol Chem, 1999.274(49):
p. 35172-8.
52.Bellik, L., et al., Intracellular pathways triggered by the selective FLT-1-agonist placental growth factor in vascular smooth muscle cells exposed to hypoxia. Br J
Pharmacol, 2005. 146(4): p. 568-75.
53.Chaballe, L., et al., Involvement of placental growth factor in Wallerian
degeneration. Glia, 2011a. 59(3): p. 379-96.
54.Selvaraj, S.K., et al., Mechanism of monocyte activation and expression of proinflammatory cytochemokines by placenta growth factor. Blood, 2003.102(4):
p. 1515-24.
55.Lin, Y.L., Liang, Y.C. and Chiang, B.L., Placental growth factor down-regulates type 1 T helper immune response by modulating the function of dendritic cells. J
Leukoc Biol, 2007. 82(6): p. 1473-80.
56.Failla, C.M., et al., Placenta growth factor is induced in human keratinocytes
during wound healing. J Invest Dermatol, 2000.115(3): p. 388-95.
57.Adini, A., et al., Placental growth factor is a survival factor for tumor endothelial
cells and macrophages. Cancer Res, 2002.62(10): p. 2749-52.
58.Van de Veire, S., et al., Further pharmacological and genetic evidence for the efficacy of PlGF inhibition in cancer and eye disease. Cell, 2010.141(1): p. 178-90.
59.Rolny, C., et al. HRG inhibits tumor growth and metastasis by inducing macrophage polarization and vessel normalization through downregulation of
PlGF. Cancer Cell, 2011. 19(1): p. 31-44.
60.Kerber, M., et al., Flt-1 signaling in macrophages promotes glioma growth in vivo.
Cancer Res, 2008. 68(18): p. 7342-51.
61.Ding, Y., et al., NFAT1 mediates placental growth factor induced myelomonocytic cell recruitment via the induction of TNF-a. J Immunol, 2010. 184(5): p. 2593-601.
62.Roncal, C., et al., Short-term delivery of anti-PlGF antibody delays progression of atherosclerotic plaques to vulnerable lesions. Cardiovasc Res, 2010. 86(1): p.
29-36.
63.Yoo, S.A., et al., Role of placenta growth factor and its receptor flt-1 in rheumatoid inflammation: A link between angiogenesis and inflammation. Arthritis Rheum
60(2): p. 345-54.
64.Hou, H.H., et al., Elastase induced lung epithelial cell apoptosis and emphysema
through placenta growth factor. Cell Death Dis, 2013. 4(9): p. e793.
65.Patel, N., et al., Placenta growth factor (PlGF), a novel inducer of plasminogen activator inhibitor-1 (PAI-1) in sickle cell disease (SCD). J Biol
Chem, 2010. 285(22): p. 16713-22.
66.Bischof, P. and Irminger-Finger, I., The human cytotrophoblastic cell, a mononuclear chameleon. Int J Biochem Cell Biol, 2005. 37(1): p. 1-16.
67.Serfling, E., et al., Metal-dependent SV40 viruses containing inducible enhancer from the upstream region of metallothionein genes. EMBO J, 1985.4(13b): p. 3851-9.
68. Westin, G. and Schaffner, W., A zinc-responsive factor interacts with a metal-regulated enhancer element (MRE) of the mouse metallothionein-I gene.
EMBO J, 1988. 7(12): p. 3763-70.
69. Heuchel, R., et al., The transcription actor MTF-1 is essential for basal and heavy metal-induced metallothionein gene expression, EMBO J.1994. 13(12): p. 2870-5.
70. Green, C.J., et al., Placenta growth factor gene expression is induced by hypoxia in fibroblasts: a central role for metal transcription factor-1. Cancer Res, 2001. 61(6): p. 2696-703.
71. Murphy, B.J., et al., Activation f metallothionein gene expression by hypoxia involves metal response elements and metal transcription factor-1. Cancer Res, 1990. 59(6): p. 1315-22.
72. Murphy, B.J., et al., The metal-responsive transcription actor-1 contributes to HIF-1 activation during hypoxic stress. Biochem Biophys Res. Commun, 2005. 337(3): p. 860-7.
73. Dalton, T.P., et al., Oxidative stress activates metal-responsive transcription factor-1 binding activity. Occupancy in vivo of metal response elements in the metallothionein-I gene promoter. J Biol Chem, 1996. 271(42): p. 26233-41.
74. Andrew, G.K., Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem Pharmacol, 2000. 59(1): p. 95-104.
75.Bahadorani, S., et al., Overexpression of metal-responsive transcription factor (MTF-1) in Drosophila melanogaster ameliorates life-span reductions associated with oxidgative stress and metal toxicity, Neurobiol Aging, 2010. 31(7): p. 1215-26.
76. Günes, C., et al., Embryonic lethality and liver degeneration in mice lacking the metal-responsive transcriptional activator MTF-1, EMBO J, 1998. 17(10): p. 2846-54.
77. Wang, Y., et al., Metal-responsive transcription factor-1 (MTF-1) is essential for embryonic liver development and heavy metal detoxification in the adult liver. FASEB J, 2004. 18(10): p. 1071-9.
78. Egli, D., et al., Knockout of “metal-responsive transcription factor” MTF-1 in Drosophila by homologous recombination reveals its central role in heavy metal homeostasis. EMBO J, 2003.22(1): p. 100-8.
79. Guo, L., et al., STAT5-glucocorticoid receptor interaction and MTF-1 regulate the expression of ZnT2 (Slc30a2) in pancreatic acinar cells. Proc Natl Acad Sci U S A, 2010. 107(7): p. 2818-23.
80. Langmade, S.J., et al., The transcription factor MTF-1 mediates metal regulation of the mouse ZnT1 gene. J Biol Chem, 2000.275(44): p. 34803-9.
81. Wimmer, U., et al, Two major branches of anti-cadmium defense in the mouse: MTF-1/metallothioneins and glutathione. Nucleic Acids Res, 2005.33(18): p. 5715-27.
82. Kaler, P., et al., Molecular cloning and functional characterization of novel zinc transporter rZip10 (Slc39a10) involved in zinc uptake across rat renal brush-border membrane. Am J Physiol Renal Physiol, 2007.292(1): p. F217-29.
83. Troadec, M.B., et al., Induction of FPN1 transcription by MTF-1 reveals a role for ferroportin in transition metal efflux. Blood, 2010. 116(22): p. 4657-64.
84. Balesaria, S., et al., Divalent metal-dependent regulation of hepcidin expression by MTF-1. FEBS Lett, 20105.84(4): p. 719-25.
85. Wadsworth, J.D., et al., Molecular biology of prion propagation. Curr OpinGenet Dev, 1999.9(3): p. 338-45.
86. Bellingham, S.A., et al., Regulation of prion gene expression by transcription factors SP1 and metal transcription factor-1. J Biol Chem, 2009.284(4): p. 1291-301.
87. McHugh, P.C., Wright, J.A. and Brown, D.R., Transcriptional regulation of the beta-synuclein 5′-promoter metal response element by metal transcription factor-1. PLoS One, 2001. 6(2): p. e17354.
88. Cramer, M., et al., NF-κB contributes to transcription of placenta growth factor and interacts with metal responsive transcription factor-1 in hypoxic human cells. Biol Chem, 2005. 386(9): p. 865-72.
89. Nishimoto, F., et al., Metal transcription factor-1 is involved in hypoxia-dependent regulation of placenta growth factor in trophoblast-derived cells. Endocrinology,2009. 150(4): p. 1801-8.
90. Radtke, F., et al., Cloned transcription factor MTF-1 activates the mouse metallothionein I promoter. EMBO J, 1993. 12(4): p. 1355-62.
91. Chen, W.Y., et al., Molecular cloning and developmental expression of zinc finger transcription factor MTF-1 gene in zebrafish, Danio rerio. Biochem Biophys Res Commun, 2002. 291(4): p. 798-805.
92. Stuart, G.W., et al., Identification of multiple metal regulatory elements in mouse metallothionein-I promoter by assaying synthetic sequences. Nature,1985. 317(6040): p. 828-31.
93. Chen, J., et al., Placenta growth factor, PLGF, influences the motility of lung cancer cells, the role of Rho associated kinase, Rock1. J Cell Biochem, 2008.105(1): p. 313-20.
94. Shi, Y., et al., The metal-responsive transcription factor-1 protein is elevated in human tumors. Cancer Biol Ther, 2010 .9(6): p. 469-76.


QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關點閱論文