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Student Number 972204019
Author Yi-Ju Wu(吳怡儒)
Author's Email Address kathy614@livemail.tw
Statistics This thesis had been viewed 780 times. Download 577 times.
Department Life Science
Year 2010
Semester 1
Degree Master
Type of Document Master's Thesis
Language zh-TW.Big5 Chinese
Title Defining the intra-cellular localization of FoxO1 and its implication in the inhibition of myogenesis
Date of Defense 2011-01-05
Page Count 65
Keyword
  • differentiation
  • FoxO1
  • MRF family
  • myogenesis
  • Abstract FoxO1 belongs to the forkhead family that bind to their target sites by their forkhead DNA-binding domain. FoxO1 has been shown to play important roles in the regulation of cell growth, proliferation, differentiation, and longevity. Its chimeric fusion with Pax3 or Pax7, generating either Pax7-or Pax3- FoxO1, has been demonstrated as strong inhibitors of muscle cell differentiation and apoptosis. To further elucidate the roles of FoxO1 in myogenesis and its possible implication in tumorigenesis, FoxO1 was over-expressed in C2C12 myoblasts (C2C12-FoxO1) and which led to a consistently inhibitory effect on myogenic differentiation. The expression of myogenic regulatory factors, including MyoD, Myogenin and Myf5, was significantly reduced in C2C12-FoxO1 cells. In this study, we observed constitutive nuclear accumulation of FoxO1 in differentiating myoblasts. However, simultaneous nuclear exclusion of FoxO1 and MHC expression was observed in mononucleated cells ready for fusion into multinucleated myotubes. FoxO1 remains cytoplasmic in multinucleated myotubes and GPF-FoxO1 introduced into myotubes also stays in the cytoplasm, implying that nuclear exclusion of FoxO1 is prerequisite for terminal differentiation. The nuclear exclusion process of FoxO1 can be mimicked by insulin treatment, but they return to the nucleus 8 hr after the treatment. To further elucidate the roles of FoxO1 in myogenesis and its possible implication in tumorigenesis, FoxO1 was over-expressed in C2C12 myoblasts (C2C12-FoxO1) and which strongly inhibited myogenic differentiation. Both insulin treatment and over-expression of MyoD partially rescued myogenic differentiation of C2C12-FoxO1 cells, implying that direct targeting of FoxO1 on myogenic genes, including MyoD, is required for its inhibitory effect. In silico analysis and transient transfection promoter assays have identified putative FoxO1-binding sites in the 24 kb upstream regulatory region of MyoD. These putative FoxO1-binding sites will be confirmed by EMSA and chromatin immunoprecipitation assay. Our results suggest that FoxO1 inhibits myogenesis by direct targeting the regulatory elements of myogenic genes.
    Table of Content 目 錄
    中文摘要-------------------------------------------------------------------------------------------------------------- I
    ABSTRACT --------------------------------------------------------------------------------------------------------- II
    聲明(DECLARATION) ----------------------------------------------------------------------------------------- III
    誌謝------------------------------------------------------------------------------------------------------------------IV
    目錄-------------------------------------------------------------------------------------------------------------------V
    縮寫與全名對照表----------------------------------------------------------------------------------------------VIII
    藥品及材料...................................................................................................................................VIII
    第一章、緒論------------------------------------------------------------------------------------------------------- 1
    1-1 . FOXO1 (FORKHEAD BOX PROTEIN 1):........................................................................................1
    1-2. 肌肉的起源與生成(MYOGENESIS):............................................................................................5
    1-3.MYOD (MYOGENIC DIFFERENTIATION FACTOR 1):........................................................................6
    1-4.葡萄糖輸送蛋白(GLUCOSE TRANSPORTER):................................................................................6
    1-5.泛素連接酶(UBIQUITION LIGASE):...............................................................................................7
    1-6. 研究動機與目的: ........................................................................................................................8
    第二章、實驗材料與方法---------------------------------------------------------------------------------------- 9
    Ⅰ. 實驗材料......................................................................................................................................9
    1. 細胞株----------------------------------------------------------------------------------------------------- 9
    2. 菌株-------------------------------------------------------------------------------------------------------- 9
    II. 質體建構.......................................................................................................................................9
    1. 質體建構方式-------------------------------------------------------------------------------------------- 9
    2. 構築載體的修飾----------------------------------------------------------------------------------------10
    3. p-Stable-MyoD601514 ----------------------------------------------------------------------------------10
    4. p-Stable-MyoD60-EM----------------------------------------------------------------------------------- 11
    5. p-Stable-MyoD60-enhancer (human)------------------------------------------------------------------ 11
    6. p-Stable-MyoD601514-enhancer (human)------------------------------------------------------------ 11
    7. p-Stable-human Atrogin-1 promoter------------------------------------------------------------------- 11
    8. p-Stable-mouse MuRF1 promoter --------------------------------------------------------------------- 11
    9. pPyCAGIP-GFP- FOXO1-wt -------------------------------------------------------------------------- 11
    10. p-Stable-mouse Atrogin-1 promoter------------------------------------------------------------------ 11
    11. p-Stable-mouse Glut4 promoter ----------------------------------------------------------------------12
    12.聚合酶連鎖反應(Polymerase Chain Reaction, PCR) --------------------------------------------12
    III. 轉染作用 (TRANSFECTION) ........................................................................................................12
    1. 穩定細胞株的製備-------------------------------------------------------------------------------------12
    VI
    2. 過渡性轉染實驗----------------------------------------------------------------------------------------12
    Ⅳ. 螢火蟲冷光活性方法( LUCIFERASE ACTIVITY ASSAY ) ............................................................13
    Ⅴ. 葡萄糖攝取實驗(GLUCOSE UPTAKE) ........................................................................................13
    Ⅵ. 反轉錄酶反應(REVERSE TRANSCRIPTASE, RT).........................................................................14
    1. Total RNA 製備-----------------------------------------------------------------------------------------14
    2. 反轉錄酶反應(Reverse Transcription) -------------------------------------------------------------14
    3. Real-time PCR--------------------------------------------------------------------------------------------15
    Ⅶ螢光免疫染色(IMMUNOFLUORESCENCE) .....................................................................................15
    Ⅷ 細胞質與細胞核蛋白的抽取......................................................................................................16
    1. 質核蛋白的抽取----------------------------------------------------------------------------------------16
    2. 蛋白質透析----------------------------------------------------------------------------------------------16
    3. 蛋白質定量----------------------------------------------------------------------------------------------17
    Ⅸ 西方墨點轉漬法(WESTERN BLOT) .............................................................................................17
    第三章、實驗結果------------------------------------------------------------------------------------------------18
    Ⅰ FOXO1可降低細胞受到INSULIN刺激的葡萄糖攝取反應。......................................................18
    Ⅱ 比較C2C12 CONTROL及C2C12 FOXO1-AAAMONOCLONE 5 在CMB與MT時期,葡萄糖輸送蛋白表現量差異。...............................................................................................................................18
    Ⅲ FOXO1可抑制MYOD PROMOTER的轉錄活性。..........................................................................19
    Ⅳ 探討長時間細胞分化過程中,FOXO1的分布位置。.............................................................20
    Ⅴ 觀察處理 INSULIN 及LICL 細胞中,FOXO1隨著時間的變化其存在位置的改..................20
    變20
    Ⅵ FOXO1的轉錄活性對其他PROMOTER的影響。.........................................................................22
    第四章、討論------------------------------------------------------------------------------------------------------23
    Ⅰ FOXO1對肌肉細胞葡萄糖攝取的影響。..................................................................................23
    Ⅱ FOXO1抑制細胞分化與MYOGENIC FACTORS的關係。.............................................................23
    Ⅲ 持續時間觀察FOXO1在細胞分化各階段表現位置。.............................................................24
    Ⅳ 探討FOXO1的轉錄活性對其他PROMOTER的影響。................................................................25
    Ⅴ 結論.............................................................................................................................................26
    第五章、圖表 ----------------------------------------------------------------------------------------------------27
    圖一、大量表現FOXO1的穩定細胞株C2C12 FOXO1-AAA較後期代數分別處理INSULIN及LICL, 分化四天後測量葡萄糖攝取的差異。..............................................................................28
    圖二、C2C12 CONTROL及大量表現FOXO1的穩定細胞株C2C12 FOXO1-AAAMONOCLONE 5較前期代數分別處理INSULIN及LICL,分化四天後測量葡萄糖攝取的差異。...............................31
    圖三、將C2C12 CONTROL及C2C12 FOXO1-AAAMONOCLONE 5以2% HS處理刺激分化四天後,比較GLUT1與GLUT4 MRNA表現量。.............................................................................................32
    圖四、構築P-STABLE-MYOD601514-ENHANCER (HUMAN) 質體。.................................................34
    VII
    圖五、FOXO1-WT與MSX1轉錄活性對MYOD PROMOTER的影響。.............................................35
    圖六、大量表現FOXO1-WT的穩定細胞株C2C12 FOXO1-WT在2% HS中加入INSULIN及LICL刺激分化四天後,以西方墨點法與C2C12 CONTROL比較細胞核中FOXO1表現量。........................36
    圖七、C2C12 CONTROL細胞培養至CMB後,更換2% HS並處理INSULIN及LICL刺激分化,利用免疫螢光染色觀察兩天及四天細胞中FOXO1的表現位置。.......................................................38
    圖八、C2C12 FOXO1-WT細胞培養至CMB後, 更換2% HS並處理INSULIN及LICL刺激分化, 利用免疫螢光染色觀察兩天及四天細胞中FOXO1的表現位置。...................................................40
    圖九、利用免疫螢光染色觀察C2C12 FOXO1-WT在PMB及CMB時期,FOXO1在細胞中表現位置。..................................................................................................................................................41
    圖十、利用免疫螢光染色觀察C2C12 FOXO1-WT,在2% HS中隨著時間FOXO1的表現位置。43
    圖十一、利用免疫螢光染色觀察C2C12 FOXO1-WT,在2% HS加入LICL 10 MM後,FOXO1隨著時間表現位置的變化。...............................................................................................................45
    圖十二、利用免疫螢光染色觀察C2C12 FOXO1-WT,在2% HS加入INSULIN 50 MM後,FOXO1隨著時間表現位置的變化。...........................................................................................................47
    圖十三、C2C12 FOXO1-WT細胞經2% HS (加入INSULIN 50 NM) 刺激分化三天後,再加入INSULIN 50 NM。利用免疫螢光染色觀察,FOXO1隨著時間表現位置的變化。.......................49
    圖十四、將PPYCAGIP-GFP-FOXO1-WT轉染到已分化的C2C12 MYOTUBE。............................51
    圖十五、老鼠小腿肌(GASTROCNEMIUS MUSCLE) 切片FOXO1表現位置。................................52
    圖十六、質體P-STABLE-HATROGIN-1 PROMOTER與P-STABLE-MMURF1 PROMOTER的構築。.......53
    圖十七、FOXO1-WT轉錄活性對ATROGIN-1及MURF1 PROMOTER的影響。...............................54
    第六章、參考文獻 ----------------------------------------------------------------------------------------------55
    附錄一 -------------------------------------------------------------------------------------------------------------59
    附錄二 -------------------------------------------------------------------------------------------------------------60
    附錄三 -------------------------------------------------------------------------------------------------------------61
    附錄四 -------------------------------------------------------------------------------------------------------------62
    Ⅰ. 溶液及試劑配方.........................................................................................................................62
    Ⅱ. 藥品試劑....................................................................................................................................64
    Ⅲ. 酵素和限制酶.............................................................................................................................64
    Ⅳ. 抗體............................................................................................................................................64
    附錄五 -------------------------------------------------------------------------------------------------------------65
    建構質體PRIMER 對照表................................................................................................................65
    REAL-TIME PCR PRIMER 對照表......................................................................................................65
    BAC CLONE對照表 ............................................................................................................................65
    Reference Anne Bigot, et al. (2008) Replicative aging down-regulates the myogenic regulatory factors in human myoblasts. Biol. Cell 100(3):189-99
    Armoni M, et al. (2002) PAX3/forkhead homolog in rhabdomyosarcoma oncoprotein activates glucose transporter 4 gene expression in vivo and in vitro. J Clin Endocrinol Metab 87(11):5312-5324
    Armoni M, et al. (2007) Transcriptional regulation of the GLUT4 gene: from PPAR-gamma and FOXO1 to FFA and inflammation. Trends Endocrinol Metab 18(3):100-107
    Armoni M, et al. (2007) Transcriptional regulation of the GLUT4 gene: from PPAR-gamma and FOXO1 to FFA and inflammation. Trends Endocrinol Metab. 18(3):100-7
    Atsushi Asakura, et al. (1995) The regulation of MyoD gene expression: conserved element mediate expression in embryonic axial muscle. Dev Biol 171, 386-389
    Carlsson P & Mahlapuu M (2002) Forkhead transcription factors: key players in development and metabolism. Dev Biol 250(1):1-23
    Carvajal JJ & Rigby PW (2010) Regulation of gene expression in vertebrate skeletal muscle. Exp Cell Res 316(18):3014-3018
    Chang-Hook Kim, et al. (2008) β-catenin interacts with MyoD and regulates its transcription activity. Mol. Cell. Biol. 28(9):2941-2951
    Dijkers PF, et al. (2000) Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1. Curr Biol. 10(19):1201-1204
    Eric N. Olson and William H. Klein. (1994) bHLH factors in muscle development: dead lines and commitments, what to leave in and what to leave out. Genes Dev. 8(1):1-8
    Frank M. J. Jacobs, et al. (2003) FoxO6, a novel member of the FoxO class of transcription factors with distinct shuttling dynamics. J. Biol. Chem. 278:35959–35967
    Gallego Melcon S & Sanchez de Toledo Codina J (2007) Molecular biology of rhabdomyosarcoma. Clin Transl Oncol 9(7):415-419
    56
    Gaster M, et al. (2004) GLUT11, but not GLUT8 or GLUT12, is expressed in human skeletal muscle in a fibre type-specific pattern. Pflugers Arch 448(1):105-113
    Goldhamer DJ, et al. (1992) Regulatory elements that control the lineage-specific expression of myoD. Science 256(5056):538-542
    Greer EL & Brunet A (2005) FOXO transcription factors at the interface between longevity and tumor suppression. Oncogene 24(50):7410-7425
    Guttilla IK & White BA (2009) Coordinate regulation of FOXO1 by miR-27a, miR-96, and miR-182 in breast cancer cells. J Biol Chem 284(35):23204-23216
    Hansol Lee, et al. (2004) Msx1 cooperates with histone H1b for inhibition of transcription and myogenesis. Science 304(5677):1675-8
    Holman GD, et al. (1990) Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel. Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester. J Biol Chem. 265(30):18172-9.
    Hosaka T, et al. (2004) Disruption of forkhead transcription factor (FOXO) family members in mice reveals their functional diversification. PNAS. 101(9):2975-2980
    Hsiang-chung Chi (2006) The roles of FOXO transcriptional factors in skeletal muscle terminal differentiation.
    Jun Nakae, et al. (2001) The Forkhead transcription factor FoxO1 (Fkhr) confers insulin sensitivity onto glucose-6-phosphatase expression. J Clin Invest. 108(9): 1359–1367
    Kopan R, et al. (1994) The intracellular domain of mouse Notch: a constitutively activated repressor of myogenesis directed at the basic helix-loop-helix region of MyoD. Development 120(9):2385-2396
    Langland K, et al. (1997) Differential interactions of Id proteins with basic-helix-loop-helix transcription factors. J Biol Chem. 8;272(32):19785-93
    Li-Chiung Chang (2007) Effects of FoxOs over-expression on terminal myogenic differentiation.
    Maher F, et al. (1994) Glucose transporter protein in brain. FASEB J. 8(13):1003-11
    57
    Marta L. Hribal, et al. (2003) Regulation of insulin-like growth factor–dependent myoblast differentiation by Foxo forkhead transcription factors. J Cell Biol. 162(4): 535–541
    Michael M. Brent, et al. (2008) Structural Basis for DNA Recognition by FoxO1 and its Regulation by Post-Translational Modification. Structure. 16(9): 1407-1416
    Miranda S. C. et al. (2010) Transcription Factor Encyclopedia.
    Naomi Galili, et al. (1993) Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma. Nature Genetics. 5:230-235
    Ordahl CP, et al. (1992) Two myogenic lineages within the developing somite. Development. 114(2):339-53
    Philippe R. J. Bois, et al. (2003) FKHR (FOXO1a) is required for myotube fusion of primary mouse myoblasts. EMBO J. 22(5):1147-57
    Pourquie O, et al. (1996) Lateral and axial signals involved in avian somite patterning: a role for BMP4. Cell 84:461-471
    Rao AS, et al. (2005) Lithium stimulates proliferation in cultured thyrocytes by activating Wnt/beta-catenin signalling. Eur J Endocrinol. 53(6):929-38
    Sandri M, et al. (2004) Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell. 117(3):399-412
    Sato Y, et al. (2002) Morphological boundary forms by a novel inductive event mediated by Lunatic fringe and Notch during somitic segmentation. Development 129(15):3633-3644
    Shivapriya Ramaswamy, et al. (2002) A novel mechanism of gene regulation and tumor suppression by the transcription factor FKHR. Cancer Cell. 2:81-91
    Tajbakhsh S, et al. (1997) Redefining the genetic hierarchies controlling skeletal myogenesis: Pax-3 and Myf-5 act upstream of MyoD. Cell 89(1):127-138
    Tatsuo Furuyama, et al. (2000) Identification of the differential distribution patterns of mRNAs and consensus binding sequences for mouse DAF-16 homologoes. Biochem. J. 349:629-634
    58
    Uldry M & Thorens B (2004) The SLC2 family of facilitated hexose and polyol transporters. Pflugers Arch 447(5):480-489
    Waddell DS, et al. (2008) The glucocorticoid receptor and FOXO1 synergistically activate the skeletal muscle atrophy-associated MuRF1 gene. Am J Physiol Endocrinol Metab. 295(4):E785-97
    Waddell DS, et al. (2008) The glucocorticoid receptor and FOXO1 synergistically activate the skeletal muscle atrophy-associated MuRF1 gene.Am J Physiol Endocrinol Metab. 295(4):E785-97
    Weidong Zhao, et al. (2008) Expression of the muscle atrophy factor muscle atrophy F-box is suppressed by testosterone. Endocrinology. 149(11):5449-60
    Yen-Hsin Fang (2009) Defining the mechanisms of FoxO1-mediated inhibition of myogenesis.
    Ying Zhao, et al. (2010) Cytosolic FoxO1 is essential for the induction of autophagy and tumour suppressor activity. Nat Cell Biol. 12(7):665-75
    Advisor
  • Shen-Liang Chen(陳盛良)
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    Date of Submission 2011-01-18

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