阻断PI3K/AKT通路通过激活FoxO1抑制 猪骨骼肌卫星细胞分化

doi:10.3864/j.issn.0578-1752.2014.01.016

摘要/Abstract

摘要： 【目的】在骨骼肌生长或损伤刺激下，骨骼肌卫星细胞被激活、增殖分化形成肌管，促进骨骼肌的生长发育或修复组织创伤。FoxO1负调控骨骼肌的生成，但在骨骼肌卫星细胞分化过程中的作用未见报道。因此，笔者探索FoxO1对猪骨骼肌卫星细胞分化的影响，希望为深入研究FoxO1调控骨骼肌生长发育的作用机理奠定基础。【方法】以1—3日龄健康大白猪为材料，采用单根肌纤维法分离培养猪骨骼肌卫星细胞，接种第2天、第4天和第6天在倒置显微镜下观察细胞形态并拍照。在细胞分化第8天，用免疫荧光染色方法染肌管，DAPI染核，并在荧光倒置显微镜下观察拍照。待细胞汇合至70%—80%时，将培养基换成含50 nmol•L-1 渥曼青霉素（wortmannin，WM）的分化培养基，分别于细胞分化第0天、第4天和第8 天收集细胞，提取总RNA和总蛋白，采用real-time qPCR和Western blotting方法检测WM对FoxO1以及骨骼肌卫星细胞分化标志基因表达的影响。【结果】猪骨骼肌卫星细胞在接种第2天开始贴壁，呈梭形。第4天细胞数量增加，部分发生融合。第6天时细胞呈方向性生长。第8天细胞进一步融合形成肌管。WM处理组的FoxO1 mRNA表达水平未发生显著变化（P＞0.05），非磷酸化的FoxO1蛋白表达显著高于对照组（P＜0.05），而p-FoxO1蛋白表达较对照组显著下降（P＜0.05）。WM处理组的细胞在分化第8天，虽然也出现了蜂窝状生长，但是与对照组相比细胞未呈方向性生长并形成肌管。Western blotting结果显示，WM明显抑制猪骨骼肌卫星细胞分化早期标志基因MyoD、中后期标志基因MyoG和末期标志基因MyHC蛋白的表达。【结论】以WM阻断PI3K信号通路能使FoxO1去磷酸化，抑制猪骨骼肌卫星细胞的分化，延迟肌管的形成，并降低成肌分化标志基因MyoD、MyoG和MyHC的表达。总之，阻断PI3K信号通路通过激活FoxO1抑制猪骨骼肌卫星细胞分化。

关键词: PI3K/AKT通路 , FoxO1 , 骨骼肌卫星细胞 , 成肌分化 , 猪

Abstract: 【Objective】Skeletal satellite cells are activated by some specific stresses such as development and trauma, and differentiate and form myotubes to participate in the development or repair of skeletal muscle. FoxO1 negatively controls the genesis of skeletal muscle, but the molecular mechanisms by which FoxO1 funcions in the differentiation of satellite cells have not been reported so far. This experiment was conducted to explore the effects of FoxO1 on porcine skeletal muscle satellite cells differentiation, aiming to provide new theoretical reference for further research. 【Method】Extensor digitorum longus of 1 to 3-day-old piglets were used to isolate the skeletal muscle satellite cells and the cells were observed and pictures were taken by inverted microscope on day 2, day 4 and day 6, respectively. The cells were stained by immunofluorescence staining and DAPI nuclear staining on day 8 of differentiation, and observed under a fluorescence microscope. Meanwhile, the medium was replaced with differentiation medium containing 50 nmol•L-1 wortmannin (wortmannin, WM) differentiation medium when the cells density reached 70% -80% confluence, the cells were collected on day 0, day 4, and day 8, respectively. Total RNA and total protein were extracted, and Real-time qPCR and Western blotting were performed to measure the alterations in the expression of FOXO1 and myogenic differentiation marker genes caused by WM supplement. 【Result】Porcine skeletal muscle satellite cells became adherent to the dish bottem, spindle-shaped on day 2. Cell number increased on day 4 and some cells started to fuse. On day 6, cell started to grow with directivity. On day 8, cell further fused to form myotubes, further fused to form myotubes. There was no significant difference in mRNA expression level of FoxO1 between WM treatment group and the control (P＞0.05), unphosphorylated FoxO1 increased significantly (P＜0.05) with WM treatment, whereas phosphorylation level of FoxO1 dropped drastically (P＜0.05). Although on day 8 the cells displayed an alveolate morphology after treated with WM, they failed to show directional growth and formation of myotubes. Moreover, Western blotting results demonstrated that WM decreased the protein level of MyoD (early myogenic marker), MyoG (middle-stage marker), and MyHC (late marker) significantly.【Conclusion】Results of the study suggest that inhibition of PI3K signaling pathway by WM blocks results in FoxO1 phosphorylation, suppression of porcine skeletal muscle satellite cell differentiation, delay of the formation of myotubes, and down-regulation of myogenic differentiation marker genes, such as MyoD, MyoG, and MyHC. Take together, blockade of PI3K signaling pathway suppresses porcine skeletal muscle satellite cell differentiation through the activation of FoxO1.

Key words: PI3K/AKT signaling , FoxO1 , skeletal muscle satellite , myogenic differentiation , porcine

史新娥, 吴国芳, 宋子仪, 路宏朝, 贾龙, 朱嘉宇, 杨公社. 阻断PI3K/AKT通路通过激活FoxO1抑制猪骨骼肌卫星细胞分化[J]. 中国农业科学, 2014, 47(1): 154-160.

SHI Xin-E, WU Guo-Fang, SONG Zi-Yi, LU Hong-Chao, JIA Long, ZHU Jia-Yu, YANG Gong-She. Inhibition of PI3K/AKT Pathway Suppressing Porcine Skeletal Muscle Sattelite Differentiation Through Activation of FoxO1 Transcription Factor[J]. Scientia Agricultura Sinica, 2014, 47(1): 154-160.

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参考文献

西北农林科技大学动物科技学院动物脂肪沉积与[1]Nakae J, Kitamura T, Kitamura Y, Biggs W H 3rd, Arden K C, Accili D. The forkhead transcription factor Foxo1 regulates adipocyte differentiation. Developmental Cell , 2003, 4(1): 119-129.

[2]Brunet A, Sweeney L B, Sturgill J F, Chua K F, Greer P L, Lin Y, Tran H, Ross S E, Mostoslavsky R, Cohen H Y, Hu L S, Cheng H L, Jedrychowski M P, Gygi S P, Sinclair D A, Alt F W, Greenberg M E. Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science, 2004, 303(5666): 2011-2015.

[3]Kamei Y, Miura S, Suzuki M, Kai Y, Mizukami J, Taniguchi T, Mochida K, Hata T, Matsuda J, Aburatani H, Nishino I, Ezaki O. Skeletal muscle FOXO1 (FKHR) transgenic mice have less skeletal muscle mass, down-regulated Type I (slow twitch/red muscle) fiber genes, and impaired glycemic control. Journal of Diological Chemistry, 2004,279(39):41114-41123.

[4]Ni Y G, Berenji K, Wang N, Oh M, Sachan N, Dey A, Cheng J, Lu G, Morris D J, Castrillon D H, Gerard R D, Rothermel B A, Hill J A. FoxO transcription factors blunt cardiac hypertrophy by inhibiting calcineurin signaling. Circulation, 2006, 114, 1159-1168.

[5]Ni Y G, Wang N, Cao D J, Sachan N, Morris D J, Gerard R D, Kuro-O M, Rothermel B A, Hill J A. FoxO transcription factors activate Akt and attenuate insulin signaling in heart by inhibiting protein phosphatases. Proceedings of the National Academy of Sciences of the United States of America. 2007, 104(51):20517-20522.

[6]Naïmi M, Gautier N, Chaussade C, Valverde A M, Accili D, Van Obberghen E. Nuclear Foxol controls and integrates key signaling pathways in hepatocytes. Endocrinology, 2007, 148(5): 2424-2434.

[7]Roy S K, Srivastava R K, Shankar S. Inhibition of PI3K/AKT and MAPK/ERK pathways causes activation of FOXO transcription factor, leading to cell cycle arrest and apoptosis in pancreatic cance. Journal of Molecular Signaling, 2010, 5:10.

[8]Shankar S, Chen Q, Srivastava R K. Inhibition of PI3K/Akt and MEK/ERK pathways act synergistically to enhance antiangiogenic effects of EGCG through activation of FOXO transcription factor. Journal of Molecular Signaling, 2008, 3:7.

[9]Sykes S M, Lane S W, Bullinger L, Kalaitzidis D, Yusuf R, Saez B, Ferraro F, Mercier F, Singh H, Brumme K M, Acharya S S, Scholl C, Tothova Z, Attar E C, Fröhling S, DePinho R A, Armstrong S A, Gilliland D G, Scadden D T. AKT/FOXO Signaling enforces reversible differentiation blockade in myeloid leukemias. Cell, 2011, 146(5): 697-708.

[10]Bondeva T, Pirola L, Bulgarelli-Leva G, Rubio I, Wetzker R, Wymann M P. Bifurcation of lipid and protein kinase signals of PI3Kgamma to the protein kinases PKB and MAPK. Science,1998, 282(5387): 293-296.

[11]刘月光, 史新娥, 沈清武, 袁媛, 杨秋梅, 高晓娟, 陈宗正, 杨公社. 利用单根肌纤维法分离和培养猪骨骼肌卫星细胞及其成肌诱导分化. 农业生物技术学报, 2011, 19(5) : 856-863.

Liu Y G, Shi X E, Shen Q W, Yuan Y, Yang Q M, Gao X J, Chen Z Z,Yang G S. Isolation and culture of porcine skeletal muscle satellite cells by single muscle fiber and their muscle differentiation.Journal of Agricultural Biotechnology, 2011, 19(5) : 856-863.

[12]Molkentin J D, Olson E N. Defining the regulatory networks for muscle development. Current Opinion in Genetics & Development , 1996, 6(4): 445-453.

[13]Nakae J, Cao Y, Hakuno F, Takemori H, Kawano Y, Sekioka R, Abe T, Kiyonari H, Tanaka T, Sakai J, Takahashi S, Itoh H. Novel repressor regulates insulin sensitivity through interaction with Foxo1. EMBO Journal, 2012, 31(10): 2275-2295.

[14]Kimura K D, Tissenbaum H A, Liu Y, and Ruvkun G. daf-2, an insulin receptor-like gene that regulates longevity and dia-pause in Caenorhabditis elegans. Science, 1997, 217: 942-946.

[15]Lin K, Dorman J B, Rodan A, Kenyon C. daf-16: An HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science, 1997, 27: 1319-1322.

[16]Ogg S, Paradis S, Gottlieb S, Patterson G I, Lee L, Tissenbaum H A, Ruvkun G. The fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C elegans. Nature, 1997, 389: 994-999.

[17]Hedrick S M. The cunning little vixen: Foxo and the cycle of life and death. Nature Immunology, 2009, 10:1057-1063.

[18]Peter S Z, Terence A P, Zipora Y R. The skeletal muscle satellite cell: the stem cell that came in from the cold. Journal of Histochemistry and Cytochemistry, 2006, 54(11): 1177-1191.

[19]van der H A, Burgering B M. Stressing the role of FoxO proteins in lifespan and disease. Nature Reviews Molecular Cell Biology, 2007,8:440-450.

[20]Bois P R J, Grosveld G C. FKHR (FOXO1a) is required for myotube fusion of primary mouse myoblasts. Embo Journal, 2003, 22(5): 1147-1157.

[21]Hribal M L, Nakae J, Kitamura T, Shutter J R, Accili D. Regulation of insulin-like growth factor-dependent myoblast differentiation by Foxo forkhead transcription factors. Journal of Cell Biology , 2003, 162(4): 535-541.

[22]袁媛, 史新娥, 刘月光, 杨公社. 高表达FoxO1 抑制猪骨骼肌成肌细胞的分化.生物工程学报, 2010, 26(12): 1668-1673.

Yuan Y, Shi X E, Liu Y G, Yang G S. Over-expression of FoxO1 inhibits the differentiation of porcine skeletal muscle myoblast. Chinese Journal of Biotechnology, 2010, 26(12): 1668-1673. (in Chinese)

[23]Pownall M E, Gustafsson M K, Emerson C P. Myogenic regulatory factors and the specification of muscle progenitors in vertebrate embryos. Annual Review of Cell and Developmental Biology, 2002, 18: 747-783.

[24]Liu C M, Yang Z, Liu C W.Effect of RNA oligonucleotide targeting Foxo1 on muscle growth in normal and cancer cachexia mice. Cancer Gene Therapy , 2007, 14(12) : 945-952.

[25]Kitamura T, Kitamura Y I, Funahashi Y, Shawber C J, Castrillon D H, Kollipara R, DePinho RA, Kitajewski J, Accili D. A Foxo/Notch pathway controls myogenic differentiation and fiber type specification. Journal of Clinical Investigation , 2007, 117(9): 2477-2485.

[26]Yuan Y, Shi X E, Liu Y G, Yang G S. FoxO1 regulates muscle fiber- type specification and inhibits calcineurin signaling during C2C12 myoblast differentiation. Molecular and Cellular Biochemistry, 2011, 348(1/2):77-87.