探討FoxO1在肌肉生成細胞中的表現位置變化及抑制肌肉細胞分化的機制; Defining the intra-cellular localization of FoxO1 and its implication in the inhibition of myogenesis

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Title:	探討FoxO1在肌肉生成細胞中的表現位置變化及抑制肌肉細胞分化的機制;Defining the intra-cellular localization of FoxO1 and its implication in the inhibition of myogenesis
Authors:	吳怡儒;Yi-Ju Wu
Contributors:	生命科學研究所
Keywords:	肌肉分化;myogenesis;differentiation;FoxO1;MRF family
Date:	2011-01-18
Issue Date:	2011-06-04 15:52:12 (UTC+8)
Publisher:	國立中央大學
Abstract:	Forkhead box protein 1 (簡稱FoxO1) 又稱為FoxO1，屬於Forkhead family蛋白質成員中的〝O〞subclass。FoxO1為轉錄因子(transcription factor) 在結構上具有一段高保留性的forkhead DNA binding domain (DBD)，此DNA binding domain包含三個α-helix及三個β-strand及兩個wings；其中α-helix負責辨識並接觸到DNA的major groove。FoxO1可調控細胞的生長、繁殖、肌肉細胞的分化及壽命。FoxO1會與Pax3及Pax7發生染色體異位，而產生Pax3-FoxO1和Pax7- FoxO1融合蛋白；此功能類似致癌蛋白的融合蛋白會誘使正常的細胞轉型成癌細胞、抑制肌肉細胞分化及抑制細胞凋亡。為了要探討FoxO1與肌肉細胞分化的關係，我們觀察大量表現FoxO1的穩定細胞株C2C12- FoxO1，發現FoxO1有抑制細胞分化的能力；並且在肌肉分化過程中會表現的調控因子MyoD、Myogenin及Myf5表現也被抑制。在本篇研究利用GFP-FoxO1觀察到在尚未分化的肌肉細胞中，FoxO1表現累積在細胞核；但在開始表現MHC的單顆細胞中發現FoxO1移動到細胞質，並且在已分化的肌管中FoxO1也表現在細胞質。表示當細胞分化前FoxO1在細胞核內進行轉錄功能；直到細胞開始分化時，FoxO1移動到細胞質，因此推測已失去轉錄能力。接著透過免疫螢光染色持續觀察肌肉細胞分化過程中FoxO1的移動方式，發現在更換分化培養液或加入insulin及LiCl刺激細胞分化的藥劑時，FoxO1表現量會先減少並且轉移到細胞質中；但在8小時後FoxO1便漸漸的聚集回細胞核，直到細胞分化成肌管才又送出至細胞質中。由實驗室之前的研究指出，當FoxO1大量表現時會抑制細胞中MyoD 的表現，因此我們更近一步利用過渡性轉染實驗得知，FoxO1 可能作用於MyoD promoter而抑制其表現。比較持續表現FoxO1的C2C12 FoxO1-AAA和C2C12的葡萄糖攝取，得知FoxO1會減少細胞因insulin的刺激而影響葡萄糖攝取，且葡萄糖輸送蛋白GLUT4的表現量也會受到抑制。未來將著重於FoxO1對於MyoD下游基因的影響分析。 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.
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