( 您好!臺灣時間:2024/04/19 14:55
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::


研究生(外文):Peng-Hsuan Lai
論文名稱(外文):SIRT1 regulates differentiation of myoblasts through modulating MURF3 expression
指導教授(外文):Suh-Hang JuoChia-Yang Li
口試委員(外文):Tsan-Ju ChenBin Huang
外文關鍵詞:SIRT1MURF3skeletal muscledifferentiation
  • 被引用被引用:0
  • 點閱點閱:278
  • 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
肌肉老化的特徵是肌肉生成能力降低,進而導致肌肉質量下降。Sirtuin 1 (SIRT1) 屬於第三類組蛋白去乙醯酶,具有抑制細胞凋亡以及減緩細胞老化的作用。先前的研究指出白藜蘆醇活化SIRT1 有助於肌原母細胞的分化並減緩老化引起的肌肉疾病「肌少症」。因此,本篇研究的目的為探討SIRT1在骨骼肌的分化中所扮演的角色。我們利用小鼠肌原母細胞株C2C12進行實驗,發現以過氧化氫誘導細胞老化,細胞內的SIRT1表現量下降,並且伴隨著肌原母細胞的分化能力下降以及肌管 (Myotube) 形成減少。為了進一步釐清SIRT1與肌肉分化的相關訊息調控機轉,我們透過生物訊息分析軟體 (Ingenuity Pathway Analysis) 推測SIRT1可能透過MURF3影響肌肉的分化。然而,在MURF3的胺基酸序列中,我們未找到可能的去乙醯化位置,故推測SIRT1是間接透過轉錄因子RAR調控MURF3的基因表現。由染色質免疫沉澱實驗顯示,SIRT1藉由轉錄因子RARA結合於MURF3的啟動子而調控其基因的轉錄。綜合以上結果,本研究證實,在肌肉老化的過程中,SIRT1透過RARA/MURF3的訊息傳遞路徑影響肌肉細胞的分化能力。
Muscle senescence is characterized by decreasing of myogenic ability which may result in muscle mass loss. Sirtuin 1 (SIRT1), known as a histone deacetylase, has been reported to implicate in anti-apoptotic and anti-aging effects. In addition, SIRT1 activator (resveratrol) had been reported to promote myoblast differentiation and attenuate age-induced sacropenia. Accordingly, this study aimed to examine the role of SIRT1 in skeletal myoblasts differentiation. Our results demonstrated that SIRT1 levels were decreased in H2O2-induced senescent myoblast. Moreover, down-regulation of SIRT1 reduced myogenic capability and impaired myotube formation. To clarify this mechanism of SIRT1 and muscular differentiation, Ingenuity Pathway Analysis (IPA) was applied to predict muscle-specific RING finger protein 3 (MURF3) as a downstream molecule of SIRT1, however, none of potential acetylation site was identified in MURF3 peptide. Therefore, we hypothesized that SIRT1 might indirectly regulate MURF3 by interacting with a nuclear receptor RAR. MURF3 is an E3 ubiquitin ligase involved in the function of ubiquitin-proteasome system and is essential for microtubule maintenance and myoblast differentiation. Knockdown of endogenous SIRT1 reduced the expression and function of MURF3. By using chromatin immunoprecipitation assay, we further demonstrated that RARA was associated with MURF3 and regulated by SIRT1 activator. Taken together, our results suggest that SIRT1/RARA/MURF3 pathway plays an important role in myoblast differentiation during muscle senescence.
致謝 2
Abstract 3
摘要 5
Abbreviation list 6
Contents 7
Introduction 9
1.1 Differentiation program of myogenic precursor cells 9
1.2 Muscle senescence and the age-related muscle disease (sarcopenia) 10
1.3 Literature review of SIRT1 (Sirtuin 1) and MURF (muscle-specific RING finger protein) genes 11
1.3.1 Role of SIRT1 in the physiology and age-related muscle disease 11
1.3.2 Role of MURF family in physiology and age-related muscle disease 14
The aim of study 16
Materials and methods 17
3.1 Cell culture 17
3.2 Methylene blue assay 17
3.3 Senescence-associated β-galactosidase assay 18
3.4 RNA isolation and quantitative real-time PCR (qPCR) 19
3.5 RNA interference 21
3.6 Western blot 22
3.7 Immunofluorescence 23
3.8 ChIP assay 24
3.9 Animal models 26
3.10 Statistical analysis 26
Results 27
4.1 SIRT1 is down-regulated during muscle senescence 27
4.2 Silence of SIRT1 impairs the differentiation potential of C2C12 cells 32
4.3 SIRT1 increases the MURF3 expression level 36
4.4 SIRT1 regulates MURF3 expression level by modulating RARA transcription factor 38
4.5 Resveratrol attenuates the defects of muscle differentiation induced by H2O2 44
Discussion 48
References 52
1Grefte, S., Kuijpers-Jagtman, A. M., Torensma, R. & Von den Hoff, J. W. Skeletal muscle development and regeneration. Stem Cells Dev 16, 857-868, doi:10.1089/scd.2007.0058 (2007).
2Bentzinger, C. F., Wang, Y. X. & Rudnicki, M. A. Building muscle: molecular regulation of myogenesis. Cold Spring Harb Perspect Biol 4, doi:10.1101/cshperspect.a008342 (2012).
3McCullagh, K. J. & Perlingeiro, R. C. Coaxing stem cells for skeletal muscle repair. Adv Drug Deliv Rev 84, 198-207, doi:10.1016/j.addr.2014.07.007 (2015).
4Yablonka-Reuveni, Z. The skeletal muscle satellite cell: still young and fascinating at 50. J Histochem Cytochem 59, 1041-1059, doi:10.1369/0022155411426780 (2011).
5Karagounis, L. G. & Hawley, J. A. Skeletal muscle: increasing the size of the locomotor cell. Int J Biochem Cell Biol 42, 1376-1379, doi:10.1016/j.biocel.2010.05.013 (2010).
6Ali, S. & Garcia, J. M. Sarcopenia, cachexia and aging: diagnosis, mechanisms and therapeutic options - a mini-review. Gerontology 60, 294-305, doi:10.1159/000356760 (2014).
7Melton, L. J., 3rd et al. Epidemiology of sarcopenia. J Am Geriatr Soc 48, 625-630 (2000).
8von Haehling, S., Morley, J. E. & Anker, S. D. An overview of sarcopenia: facts and numbers on prevalence and clinical impact. J Cachexia Sarcopenia Muscle 1, 129-133, doi:10.1007/s13539-010-0014-2 (2010).
9Morley, J. E., Anker, S. D. & von Haehling, S. Prevalence, incidence, and clinical impact of sarcopenia: facts, numbers, and epidemiology-update 2014. J Cachexia Sarcopenia Muscle 5, 253-259, doi:10.1007/s13539-014-0161-y (2014).
10Walston, J. D. Sarcopenia in older adults. Curr Opin Rheumatol 24, 623-627, doi:10.1097/BOR.0b013e328358d59b (2012).
11Sousa-Victor, P., Garcia-Prat, L., Serrano, A. L., Perdiguero, E. & Munoz-Canoves, P. Muscle stem cell aging: regulation and rejuvenation. Trends Endocrinol Metab 26, 287-296, doi:10.1016/j.tem.2015.03.006 (2015).
12Nehlin, J. O., Just, M., Rustan, A. C. & Gaster, M. Human myotubes from myoblast cultures undergoing senescence exhibit defects in glucose and lipid metabolism. Biogerontology 12, 349-365, doi:10.1007/s10522-011-9336-5 (2011).
13Saunders, L. R. & Verdin, E. Sirtuins: critical regulators at the crossroads between cancer and aging. Oncogene 26, 5489-5504, doi:10.1038/sj.onc.1210616 (2007).
14Han, S. H. Potential Role of Sirtuin as a Therapeutic Target for Neurodegenerative Diseases. Journal of Clinical Neurology 5, 120-125, doi:10.3988/jcn.2009.5.3.120 (2009).
15Lin, S. J., Defossez, P. A. & Guarente, L. Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae. Science 289, 2126-2128 (2000).
16Ramis, M. R., Esteban, S., Miralles, A., Tan, D. X. & Reiter, R. J. Caloric restriction, resveratrol and melatonin: Role of SIRT1 and implications for aging and related-diseases. Mech Ageing Dev 146, 28-41, doi:10.1016/j.mad.2015.03.008 (2015).
17Boily, G. et al. SirT1 Regulates Energy Metabolism and Response to Caloric Restriction in Mice. Plos One 3, doi:ARTN e1759
10.1371/journal.pone.0001759 (2008).
18Alcendor, R. R. et al. Sirt1 regulates aging and resistance to oxidative stress in the heart. Circ Res 100, 1512-1521, doi:10.1161/01.RES.0000267723.65696.4a (2007).
19Merksamer, P. I. et al. The sirtuins, oxidative stress and aging: an emerging link. Aging (Albany NY) 5, 144-150, doi:10.18632/aging.100544 (2013).
20Joseph, A. M. et al. Short-term caloric restriction, resveratrol, or combined treatment regimens initiated in late-life alter mitochondrial protein expression profiles in a fiber-type specific manner in aged animals. Exp Gerontol 48, 858-868, doi:10.1016/j.exger.2013.05.061 (2013).
21Jackson, J. R., Ryan, M. J., Hao, Y. & Alway, S. E. Mediation of endogenous antioxidant enzymes and apoptotic signaling by resveratrol following muscle disuse in the gastrocnemius muscles of young and old rats. Am J Physiol Regul Integr Comp Physiol 299, R1572-1581, doi:10.1152/ajpregu.00489.2010 (2010).
22Montesano, A., Luzi, L., Senesi, P., Mazzocchi, N. & Terruzzi, I. Resveratrol promotes myogenesis and hypertrophy in murine myoblasts. J Transl Med 11, 310, doi:10.1186/1479-5876-11-310 (2013).
23Zhou, Y. et al. SIRT1 inhibits adipogenesis and promotes myogenic differentiation in C3H10T1/2 pluripotent cells by regulating Wnt signaling. Cell Biosci 5, 61, doi:10.1186/s13578-015-0055-5 (2015).
24Witt, S. H., Granzier, H., Witt, C. C. & Labeit, S. MURF-1 and MURF-2 target a specific subset of myofibrillar proteins redundantly: towards understanding MURF-dependent muscle ubiquitination. J Mol Biol 350, 713-722, doi:10.1016/j.jmb.2005.05.021 (2005).
25Spencer, J. A., Eliazer, S., Ilaria, R. L., Jr., Richardson, J. A. & Olson, E. N. Regulation of microtubule dynamics and myogenic differentiation by MURF, a striated muscle RING-finger protein. J Cell Biol 150, 771-784 (2000).
26Centner, T. et al. Identification of muscle specific ring finger proteins as potential regulators of the titin kinase domain. J Mol Biol 306, 717-726, doi:10.1006/jmbi.2001.4448 (2001).
27Lilienbaum, A. Relationship between the proteasomal system and autophagy. Int J Biochem Mol Biol 4, 1-26 (2013).
28Lecker, S. H., Goldberg, A. L. & Mitch, W. E. Protein degradation by the ubiquitin-proteasome pathway in normal and disease states. J Am Soc Nephrol 17, 1807-1819, doi:10.1681/ASN.2006010083 (2006).
29Ciechanover, A. The ubiquitin-proteasome pathway: on protein death and cell life. EMBO J 17, 7151-7160, doi:10.1093/emboj/17.24.7151 (1998).
30Kitajima, Y. et al. Proteasome dysfunction induces muscle growth defects and protein aggregation. J Cell Sci 127, 5204-5217, doi:10.1242/jcs.150961 (2014).
31Bell, R. A., Al-Khalaf, M. & Megeney, L. A. The beneficial role of proteolysis in skeletal muscle growth and stress adaptation. Skelet Muscle 6, 16, doi:10.1186/s13395-016-0086-6 (2016).
32Baraibar, M. A. & Friguet, B. Changes of the proteasomal system during the aging process. Prog Mol Biol Transl Sci 109, 249-275, doi:10.1016/B978-0-12-397863-9.00007-9 (2012).
33Carrard, G., Bulteau, A. L., Petropoulos, I. & Friguet, B. Impairment of proteasome structure and function in aging. Int J Biochem Cell Biol 34, 1461-1474 (2002).
34Ferrington, D. A., Husom, A. D. & Thompson, L. V. Altered proteasome structure, function, and oxidation in aged muscle. FASEB J 19, 644-646, doi:10.1096/fj.04-2578fje (2005).
35Clavel, S. et al. Atrophy-related ubiquitin ligases, atrogin-1 and MuRF1 are up-regulated in aged rat Tibialis Anterior muscle. Mech Ageing Dev 127, 794-801, doi:10.1016/j.mad.2006.07.005 (2006).
36Fielitz, J. et al. Myosin accumulation and striated muscle myopathy result from the loss of muscle RING finger 1 and 3. J Clin Invest 117, 2486-2495, doi:10.1172/JCI32827 (2007).
37Fielitz, J. et al. Loss of muscle-specific RING-finger 3 predisposes the heart to cardiac rupture after myocardial infarction. Proc Natl Acad Sci U S A 104, 4377-4382, doi:10.1073/pnas.0611726104 (2007).
38Guo, L., Xie, B. & Mao, Z. Autophagy in premature senescent cells is activated via AMPK pathway. Int J Mol Sci 13, 3563-3582, doi:10.3390/ijms13033563 (2012).
39Ota, H. et al. Sirt1 inhibitor, Sirtinol, induces senescence-like growth arrest with attenuated Ras-MAPK signaling in human cancer cells. Oncogene 25, 176-185, doi:10.1038/sj.onc.1209049 (2006).
40Lee, K. P. et al. miR-431 promotes differentiation and regeneration of old skeletal muscle by targeting Smad4. Genes Dev 29, 1605-1617, doi:10.1101/gad.263574.115 (2015).
41McLean, R. R. & Kiel, D. P. Developing consensus criteria for sarcopenia: an update. J Bone Miner Res 30, 588-592, doi:10.1002/jbmr.2492 (2015).
42Fulle, S. et al. The contribution of reactive oxygen species to sarcopenia and muscle ageing. Exp Gerontol 39, 17-24 (2004).
43Mohamed, J. S., Wilson, J. C., Myers, M. J., Sisson, K. J. & Alway, S. E. Dysregulation of SIRT-1 in aging mice increases skeletal muscle fatigue by a PARP-1-dependent mechanism. Aging (Albany NY) 6, 820-834, doi:10.18632/aging.100696 (2014).
44Lam, Y. Y., Peterson, C. M. & Ravussin, E. Resveratrol vs. calorie restriction: data from rodents to humans. Exp Gerontol 48, 1018-1024, doi:10.1016/j.exger.2013.04.005 (2013).
45Koltai, E. et al. Exercise alters SIRT1, SIRT6, NAD and NAMPT levels in skeletal muscle of aged rats. Mech Ageing Dev 131, 21-28, doi:10.1016/j.mad.2009.11.002 (2010).
46Sin, T. K., Yung, B. Y. & Siu, P. M. Modulation of SIRT1-Foxo1 signaling axis by resveratrol: implications in skeletal muscle aging and insulin resistance. Cell Physiol Biochem 35, 541-552, doi:10.1159/000369718 (2015).
47Olive, M. et al. New cardiac and skeletal protein aggregate myopathy associated with combined MuRF1 and MuRF3 mutations. Hum Mol Genet 24, 6264, doi:10.1093/hmg/ddv311 (2015).
48Martin, B. et al. The LIM-only protein FHL2 interacts with beta-catenin and promotes differentiation of mouse myoblasts. J Cell Biol 159, 113-122, doi:10.1083/jcb.200202075 (2002).
49Scholl, F. A., McLoughlin, P., Ehler, E., de Giovanni, C. & Schafer, B. W. DRAL is a p53-responsive gene whose four and a half LIM domain protein product induces apoptosis. J Cell Biol 151, 495-506 (2000).
50Furst, D. O. et al. Filamin C-related myopathies: pathology and mechanisms. Acta Neuropathol 125, 33-46, doi:10.1007/s00401-012-1054-9 (2013).
51Zhu, G. H. et al. Activation of RXR and RAR signaling promotes myogenic differentiation of myoblastic C2C12 cells. Differentiation 78, 195-204, doi:10.1016/j.diff.2009.06.001 (2009).
52Chen, J. & Li, Q. Implication of retinoic acid receptor selective signaling in myogenic differentiation. Sci Rep 6, 18856, doi:10.1038/srep18856 (2016).
第一頁 上一頁 下一頁 最後一頁 top