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研究生： 呂婉禎
Lu, Wan-Chen
論文名稱： SH2B1β減少 PC12 細胞因氧化壓力所引起的細胞死亡
SH2B1β Reduces Oxidative Stress - Induced Cell Death in PC12 Cells
指導教授： 陳令儀
Chen, Linyi
口試委員:
學位類別： 碩士
Master
系所名稱： 生命科學暨醫學院 - 分子醫學研究所
Institute of Molecular Medicine
論文出版年： 2010
畢業學年度： 98
語文別： 英文
論文頁數： 73
中文關鍵詞： 氧化壓力
外文關鍵詞： FoxO, oxidative stress, PC12
相關次數： 點閱：2  下載：0 
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    • 氧化壓力會導致細胞死亡並與許多疾病和神經退化性疾病，諸如帕金森氏症、阿茲海默症、亨丁頓舞蹈症，息息相關。氧化壓力會產生許多活性氧分子(包括過氧化氫)而造成細胞損傷。氧化壓力會活化許多的細胞內訊息傳遞分子，例如Phosphatidylinositol 3-Kinase (PI3K)-Akt (PKB)主要與細胞存活相關，以及三條絲裂原活化蛋白激酶(MAPK)路徑，包含胞外信號調節激酶(ERK1/2)、c-jun胺基末端激酶(JNK)、p38激酶(p38MAPK)，這些激酶被證實會由氧化壓力所活化，並參與細胞存活和細胞死亡之調控。SH2B1□是一種訊息銜接蛋白，在PC12細胞中被發現能促進由神經生長因子(NGF)所調節之神經細胞分化，並且為交感神經元之存活所必需；另外SH2B1□也和調控FoxO轉錄因子有關，FoxO轉錄因子為Akt下游之基因，可調控許多與細胞凋亡或是抗氧化相關之基因，例如Fas ligand和MnSOD。我的論文主要是去測試SH2B1□在氧化壓力所誘導之反應中所扮演的角色。實驗數據顯示SH2B1□可以藉由增強MAPK和Akt的活性，進而降低FasL 基因表現量且增加MnSOD表現量來減少因氧化壓力所導致的細胞凋亡。此外我們發現在過氧化氫處理下，MAPK和Akt皆可磷酸化FoxO1和 FoxO3a，我們更進一步證實SH2B1□會降低由氧化壓力所引發之細胞死亡現象，主要是經由PI3K-AKT-FoxO路徑以及MEK-ERK1/2-FoxO路徑來保護細胞。

    致謝 Ⅰ Abstact Ⅲ 中文摘要 Ⅳ Index Ⅴ Abbreviations Ⅸ Introduction 1 Fig. Ⅰ Selected members of reactive oxygen species (ROS) 7 Fig. Ⅱ Putative pathways mediated by oxidative stress 8 Fig. Ⅲ Schematic of SH2B family members 9 Fig. Ⅳ Post-translational modification of FoxOs 10 Fig. Ⅴ Regulation of FoxO proteins in response to external and internal stimuli. 11 Objective 12 Procedures 13 Material and Methods 14 Reagents 14 Cell culture and drug treatment 15 Cell visbility assay 16 Immunoblotting 16 Western blotting using Infrared imaging system (Odyssey detection).. 18 Immunostaining 19 Inhibitor assay 19 Total RNA preparation 19 Reverse transcription and semi-quantitative real-time polymerase chain reaction 21 Measure of intracellular ROS levels 22 Statistical analysis 22 Results 23 SH2B1□ reduces oxidative-stress induced cell death 23 SH2B1□ enhances H2O2-induced activation of Akt and ERK1/2 25 SH2B1□□enahnces phosphorylation of FoxOs, reduces their nuclear localization and target gene expression 25 SH2B1□□regulates the expression of FoxO target genes 27 Discussion 29 Reference 32 Figures 40 Fig. 1 H2O2 –induced cell death in PC12-GFP and PC12-SH2B1□ cells 40 Fig. 2 H2O2 – induced cell death and neurite retraction in differentiated – PC12-GFP and PC12-SH2B1□ cells 41 Fig. 3 Viability of PC12-GFP and PC12-SH2B1□ cells treated with H2O2 42 Fig. 4 H2O2–Induced caspase 3–dependent apoptosis in PC12-GFP and PC12-SH2B1□ cells 44 Fig. 5 SH2B1□□enhanced□the phosphorylation levels of Akt in response to H2O2 Treatment 46 Fig. 6 SH2B1□□enhanced the phosphorylation levels of ERK1/2 in response to H2O2 treatment 48 Fig. 7 SH2B1□□enhanced□the phosphorylation levels of FoxO1 and FoxO3a in response to H2O2 treatment 49 Fig. 8 SH2B1□□reduced□□□□□□induced subcellular distribution of FoxO1 50 Fig. 9 SH2B1□□reduced□□□□□□induced subcellular distribution of FoxO3a 52 Fig. 10 SH2B1□□reduced□□□□□□induced nuclear localization of FoxO1 through pAkt and pERK1/2 54 Fig. 11 SH2B1□□reduced□□□□□□induced nuclear localization of FoxO3a through pAkt and pERK1/2 55 Fig. 12 SH2B1□□reduced FasL and enhanced MnSOD mRNA expression 56 Fig. 13 SH2B1□□regulated FasL mRNA expression level through pAkt, pERK 57 Fig. 14 The effect of PI3K and MEK inhibitors on MnSOD expression 58 Fig. 15 SH2B1□□enhanced Bcl2 mRNA expression level 59 Fig. 16 Overexpressing SH2B1□□enhances catalase mRNA expression level and reduces H2O2-induced ROS level 60 Tables 62 Table 1. Reverse transcription protocol used in this thesis 62 Table 2. Reaction temperature and time of reverse transcription 63 Table 3. Sequences of the Q-PCR primers used in this thesis 64 Table 4. Reaction temperature and time of Q-PCR 65 Apendix 66 Fig. A1 The H2O2–induced phosphorylation of JNK in PC12-GFP and PC12-SH2B1□ cells 66 Fig. A2 Effect of PI3K and MEK inhibitors on the subcellular distribution of FoxO3a 68 Fig. A3 Effect of inhibiting pJNK on FasL and MnSOD mRNA expression 69 Fig. A4 SH2B1□□reduced cell death in response to H2O2 treatment 70 Fig. A5 Effect of PI3K, MEK, JNK inhibitors on catalase mRNA expression 71 Fig. A6 Effect of PI3K, MEK, JNK inhibitors on Bcl2 mRNA expression 72 Fig. A7 Effect of PI3K, MEK, JNK inhibitors on Bim mRNA expression 73

    1. Degterev, A. and J. Yuan, Expansion and evolution of cell death programmes. Nat Rev Mol Cell Biol, 2008. 9(5): p. 378-90.
    2. Kitanaka, C. and Y. Kuchino, Caspase-independent programmed cell death with necrotic morphology. Cell Death Differ, 1999. 6(6): p. 508-15.
    3. Benn, S.C. and C.J. Woolf, Adult neuron survival strategies--slamming on the brakes. Nat Rev Neurosci, 2004. 5(9): p. 686-700.
    4. Krantic, S., et al., Apoptosis-inducing factor: a matter of neuron life and death. Prog Neurobiol, 2007. 81(3): p. 179-96.
    5. Boujrad, H., et al., AIF-mediated programmed necrosis: a highly regulated way to die. Cell Cycle, 2007. 6(21): p. 2612-9.
    6. Vandenabeele, P., W. Declercq, and T.V. Berghe, Necrotic cell death and 'necrostatins': now we can control cellular explosion. Trends Biochem Sci, 2008. 33(8): p. 352-5.
    7. Barnham, K.J., C.L. Masters, and A.I. Bush, Neurodegenerative diseases and oxidative stress. Nat Rev Drug Discov, 2004. 3(3): p. 205-14.
    8. Giorgio, M., et al., Hydrogen peroxide: a metabolic by-product or a common mediator of ageing signals? Nat Rev Mol Cell Biol, 2007. 8(9): p. 722-8.
    9. Burdon, R.H., D. Alliangana, and V. Gill, Hydrogen peroxide and the proliferation of BHK-21 cells. Free Radic Res, 1995. 23(5): p. 471-86.
    10. Burdon, R.H., V. Gill, and D. Alliangana, Hydrogen peroxide in relation to proliferation and apoptosis in BHK-21 hamster fibroblasts. Free Radic Res, 1996. 24(2): p. 81-93.
    11. Bladier, C., et al., Response of a primary human fibroblast cell line to H2O2: senescence-like growth arrest or apoptosis? Cell Growth Differ, 1997. 8(5): p. 589-98.
    12. Davies, K.J., The broad spectrum of responses to oxidants in proliferating cells: a new paradigm for oxidative stress. IUBMB Life, 1999. 48(1): p. 41-7.
    13. Kim, B.Y., M.J. Han, and A.S. Chung, Effects of reactive oxygen species on proliferation of Chinese hamster lung fibroblast (V79) cells. Free Radic Biol Med, 2001. 30(6): p. 686-98.
    14. Day, R.M. and Y.J. Suzuki, Cell proliferation, reactive oxygen and cellular glutathione. Dose Response, 2005. 3(3): p. 425-42.
    15. Martindale, J.L. and N.J. Holbrook, Cellular response to oxidative stress: signaling for suicide and survival. J Cell Physiol, 2002. 192(1): p. 1-15.
    16. Nindl. G, Hydrogen Peroxide - From Oxidative Stressor to Redox Regulator . Cellscience Reviews, 2004. 1(2)
    17. Andersen, J.K., Oxidative stress in neurodegeneration: cause or consequence? Nat Med, 2004. 10 Suppl: p. S18-25.
    18. Fatokun, A.A., T.W. Stone, and R.A. Smith, Oxidative stress in neurodegeneration and available means of protection. Front Biosci, 2008. 13: p. 3288-311.
    19. Wang, X., et al., The cellular response to oxidative stress: influences of mitogen-activated protein kinase signalling pathways on cell survival. Biochem J, 1998. 333 ( Pt 2): p. 291-300.
    20. Yoshizumi, M., et al., Ebselen attenuates oxidative stress-induced apoptosis via the inhibition of the c-Jun N-terminal kinase and activator protein-1 signalling pathway in PC12 cells. Br J Pharmacol, 2002. 136(7): p. 1023-32.
    21. Nair, V.D., et al., Early single cell bifurcation of pro- and antiapoptotic states during oxidative stress. J Biol Chem, 2004. 279(26): p. 27494-501.
    22. Fujita, Y., et al., Pramipexole protects against H2O2-induced PC12 cell death. Naunyn Schmiedebergs Arch Pharmacol, 2006. 372(4): p. 257-66.
    23. Wu, X.J., et al., Propofol attenuates oxidative stress-induced PC12 cell injury via p38 MAP kinase dependent pathway. Acta Pharmacol Sin, 2007. 28(8): p. 1123-8.
    24. Zhuang, S., J.T. Demirs, and I.E. Kochevar, p38 mitogen-activated protein kinase mediates bid cleavage, mitochondrial dysfunction, and caspase-3 activation during apoptosis induced by singlet oxygen but not by hydrogen peroxide. J Biol Chem, 2000. 275(34): p. 25939-48.
    25. Greene, L.A., Nerve growth factor prevents the death and stimulates the neuronal differentiation of clonal PC12 pheochromocytoma cells in serum-free medium. J Cell Biol, 1978. 78(3): p. 747-55.
    26. Sofroniew, M.V., C.L. Howe, and W.C. Mobley, Nerve growth factor signaling, neuroprotection, and neural repair. Annu Rev Neurosci, 2001. 24: p. 1217-81.
    27. Rui, L., et al., Identification of SH2-Bbeta as a substrate of the tyrosine kinase JAK2 involved in growth hormone signaling. Mol Cell Biol, 1997. 17(11): p. 6633-44.
    28. Qian, X., et al., Identification and characterization of novel substrates of Trk receptors in developing neurons. Neuron, 1998. 21(5): p. 1017-29.
    29. Rui, L., J. Herrington, and C. Carter-Su, SH2-B is required for nerve growth factor-induced neuronal differentiation. J Biol Chem, 1999. 274(15): p. 10590-4.
    30. Herrington, J., et al., SH2-B is required for growth hormone-induced actin reorganization. J Biol Chem, 2000. 275(17): p. 13126-33.
    31. Zhang, Y., et al., Interaction of SH2-Bbeta with RET is involved in signaling of GDNF-induced neurite outgrowth. J Cell Sci, 2006. 119(Pt 8): p. 1666-76.
    32. Wang, X., et al., SH2-B is a positive regulator of nerve growth factor-mediated activation of the Akt/Forkhead pathway in PC12 cells. J Biol Chem, 2004. 279(1): p. 133-41.
    33. Chen, L. and C. Carter-Su, Adapter protein SH2-B beta undergoes nucleocytoplasmic shuttling: implications for nerve growth factor induction of neuronal differentiation. Mol Cell Biol, 2004. 24(9): p. 3633-47.
    34. Maures, T.J., J.H. Kurzer, and C. Carter-Su, SH2B1 (SH2-B) and JAK2: a multifunctional adaptor protein and kinase made for each other. Trends Endocrinol Metab, 2007. 18(1): p. 38-45.
    35. Chen, L., et al., SH2B1beta (SH2-Bbeta) enhances expression of a subset of nerve growth factor-regulated genes important for neuronal differentiation including genes encoding urokinase plasminogen activator receptor and matrix metalloproteinase 3/10. Mol Endocrinol, 2008. 22(2): p. 454-76.
    36. Huang, H. and D.J. Tindall, Dynamic FoxO transcription factors. J Cell Sci, 2007. 120(Pt 15): p. 2479-87.
    37. Van der Horst, A. and B.M. Burgering, Stressing the role of FoxO proteins in lifespan and disease. Nat Rev Mol Cell Biol, 2007. 8(6): p. 440-50.
    38. Birkenkamp, K.U. and P.J. Coffer, FoxO transcription factors as regulators of immune homeostasis: molecules to die for? J Immunol, 2003. 171(4): p. 1623-9.
    39. Medema, R.H., et al., AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature, 2000. 404(6779): p. 782-7.
    40. Kajihara, T., et al., Differential expression of FoxO1 and FoxO3a confers resistance to oxidative cell death upon endometrial decidualization. Mol Endocrinol, 2006. 20(10): p. 2444-55.
    41. Maiese, K., Z.Z. Chong, and Y.C. Shang, OutFoxOing disease and disability: the therapeutic potential of targeting FoxO proteins. Trends Mol Med, 2008. 14(5): p. 219-27.
    42. Tang, E.D., et al., Negative regulation of the forkhead transcription factor FKHR by Akt. J Biol Chem, 1999. 274(24): p. 16741-6.
    43. Burgering, B.M. and G.J. Kops, Cell cycle and death control: long live Forkheads. Trends Biochem Sci, 2002. 27(7): p. 352-60.
    44. Burgering, B.M. and R.H. Medema, Decisions on life and death: FoxO Forkhead transcription factors are in command when PKB/Akt is off duty. J Leukoc Biol, 2003. 73(6): p. 689-701.
    45. Van Der Heide, L.P., M.F. Hoekman, and M.P. Smidt, The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation. Biochem J, 2004. 380(Pt 2): p. 297-309.
    46. Barthel, A., D. Schmoll, and T.G. Unterman, FoxO proteins in insulin action and metabolism. Trends Endocrinol Metab, 2005. 16(4): p. 183-9.
    47. Carter, M.E. and A. Brunet, FoxO transcription factors. Curr Biol, 2007. 17(4): p. R113-4.
    48. Burgering, B.M., A brief introduction to FoxOlogy. Oncogene, 2008. 27(16): p. 2258-62.
    49. Brenkman, A.B., et al., Mdm2 induces mono-ubiquitination of FoxO4. PLoS ONE, 2008. 3(7): p. e2819.
    50. Essers, M.A., et al., FoxO transcription factor activation by oxidative stress mediated by the small GTPase Ral and JNK. Embo J, 2004. 23(24): p. 4802-12.
    51. Hu, Y., et al., ERK phosphorylates p66shcA on Ser36 and subsequently regulates p27kip1 expression via the Akt-FoxO3a pathway: implication of p27kip1 in cell response to oxidative stress. Mol Biol Cell, 2005. 16(8): p. 3705-18.
    52. Asada, S., et al., Mitogen-activated protein kinases, Erk and p38, phosphorylate and regulate Foxo1. Cell Signal, 2007. 19(3): p. 519-27.
    53. Davila, D. and I. Torres-Aleman, Neuronal death by oxidative stress involves activation of FoxO3 through a two-arm pathway that activates stress kinases and attenuates insulin-like growth factor I signaling. Mol Biol Cell, 2008. 19(5): p. 2014-25.
    54. Calnan, D.R. and A. Brunet, The FoxO code. Oncogene, 2008. 27(16): p. 2276-88.
    55. Yang, J.Y., et al., ERK promotes tumorigenesis by inhibiting FoxO3a via MDM2-mediated degradation. Nat Cell Biol, 2008. 10(2): p. 138-48.
    56. van der Horst, A., et al., FoxO4 transcriptional activity is regulated by monoubiquitination and USP7/HAUSP. Nat Cell Biol, 2006. 8(10): p. 1064-7
    57. Taylor, J.M. and P.J. Crack, Impact of oxidative stress on neuronal survival. Clin Exp Pharmacol Physiol, 2004. 31(7): p. 397-406.
    58. Calnan DR. and A. Brunet, The FoxO code. Oncogene, 2008. 27: p. 2276–2288.
    59. Kaestner, K.H., W. Knochel, and D.E. Martinez, Unified nomenclature for the winged helix/forkhead transcription factors. Genes Dev, 2000. 14(2): p. 142-6.
    60. Nakamura, T. and K. Sakamoto, Forkhead transcription factor FoxO subfamily is essential for reactive oxygen species-induced apoptosis. Mol Cell Endocrinol, 2008. 281(1-2): p. 47-55.
    61. Keita T., et al., Stat3 confers resistance against hypoxia/reoxygenation-induced oxidative injury in hepatocytes through upregulation of Mn-SOD. Journal of Hepatology, 2004. 41: p. 957-965.
    62. Lin, W. F., et al., SH2B1□ enhances fibroblast growth factor 1 (FGF1)-induced neurite outgrowth through MEK-ERK1/2-STAT3-Egr1 pathway. Cell Signal, 2009. 21: p. 1060-1072.
    63. Catherine B., Christopher E H. and P Brigitte, FoxO3a induces motoneuron death through the Fas pathway in cooperation with JNK. BMC Neuroscience, 2004. 5: p. 48.
    64. Imada K. and W.J. Leonard, The Jak-STAT pathway. Mol Immunol, 2000. 37: p. 1-11.
    65. James E. and Jr. Darnell, STATs and Gene Regulation. Science, 1997. 277: p. 1630–1635.
    66. Stephanou A., et al., Opposing actions of STAT-1 and STAT-3 on the Bcl-2 and Bcl-x promoters. Cell Death Differ, 2000. 7: p. 329–330.
    67. Yu H. M., et al., Role of the JAK-STAT pathway in protection of hydrogen peroxide preconditioning against apoptosis induced by oxidative stress in PC12 cells. Apoptosis, 2006. 11: p. 931-941.
    68. Marcin K., et al., Akt ModulatesSTAT3-mediated Gene Expression a through FKHR(FoxO1a) -dependent Mechanism. JBC, 2003. 278(7): p. 5242-5249.
    69. Long C., et al., Hydrogen peroxide-induced neuronal apoptosis is associated with inhibition of protein phosphatase 2A and 5, leading to activation MAPK pathway. The International Journal of Biochemistry & Cell Biology, 2009(41): p. 1284-1295.
    70. Regina M. D., et al., Cell proliferation, reactive oxygen and cellular glutathione. International hormesis Society, 1995. 3: p. 425-442.
    71. Yao R. and Cooper GM. Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science, 1995. 267: p. 2003–6.
    72. Schneider A., et al., Nf-kappab is activated and promotes cell death in focal cerebral ischemia. Nat. Med, 1999. 5: p. 554–9.
    73. Flohe L., et al., Redox regulation of NF-kappa B activation. Free Radic. Biol. Med, 1997. 22: p. 1115–26.
    74. Clemens JA., et al., Drug-induced neuroprotection from global ischemia is associated with prevention of persistent but not transient activation of nuclear factor-kappaB in rats. Stroke, 1998. 29: p. 677–82.
    75. Datta SR, Brunet A, and ME. Greenberg, Cellular survival: A play in three akts. Genes Dev, 1999. 13: p. 2905–27.
    76. James W., et al., Nrf2:INrf2 (Keap1) signaling in oxidative stress. Free Radical Biology & Medicine, 2009. 47: p. 1304–1309.

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