배양근원세포에서의 산화스트레스에 의한 근육소실 기작에 관한 연구
The mechanism of muscle deterioration under oxidative stress in cultured muscle cells
- 주제(키워드) dystrophin , oxidative stress , transduction , Nrf2 , sulforaphane
- 발행기관 아주대학교
- 지도교수 김혜선
- 발행년도 2018
- 학위수여년월 2018. 2
- 학위명 박사
- 학과 및 전공 일반대학원 생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000026896
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Duchenne muscular dystrophy (DMD) is an X chromosome-linked disorder caused by a mutation in the dystrophin gene. Dystrophin, a large protein (427 kDa) localized in the sarcolemma of skeletal muscle, connects the intracellular sarcolemmal cytoskeleton such as -actin with the extracellular matrix via the dystrophin protein complex. The complex proteins are known to provide scaffolding for various signaling proteins. Many previous studies reported that the skeletal muscles of DMD patients are more susceptible to oxidative stress than those of healthy people. However, not much has been known about the responsible mechanism of the differential susceptibility to the oxidative stress. This study aims to clarify the signaling pathway(s) in myoblasts and myotubes under menadione-induced oxidative stress. In order to study the response signaling pathway, dystrophin knock-down (DysKD) cell line was established by transfection of dystrophin shRNA lentiviral particles into C2 cells. Because the expression of dystrophin was found to be successfully decreased and the DysKD cells presented some characters similar with the mdx mice, an animal model largely studied in DMD. The DysKD myotubes are shown to be more vulnerable to menadione-induced oxidative stress than control myotubes. To study the mechanism we focused on the nuclear erythroid 2-related factor 2 (Nrf2) which is a transcription factor that regulates the expression of phase II antioxidant enzymes by binding to the antioxidant response element (ARE). Nrf2 normally exists in the cytoplasm interacting with Kelch-like ECH-associated protein 1 (KEAP1), which suppresses Nrf2 activity. However, when cells are exposed to stimuli like oxidative stress, Nrf2 is released from the KEAP1 complex and translocates into the nucleus, where it regulates the activation of ARE-mediated gene expression. Therefore, the signaling pathway related to the Nrf2 activity is considered pivotal in studying the intracellular signal transduction under oxidative stress. Although there are several reports about signaling pathway of Nrf2 activation to oxidative stress in various fields, studies regarding the role of Nrf2 as a key regulator in the dystrophic muscles of DMD have not been carried out yet. Nrf2 regulates the expression of antioxidant enzymes such as NAD(P)H:quinone dehydrogenase 1 (NQO1), glutathione S-transferase (GST), superoxide dismutase (SOD), and heme oxygenase-1 (HO-1). Under menadione-induced oxidative stress, the translocation of Nrf2 to the nucleus was significantly decreased in the DysKD myotubes. In addition, the expressions of NQO1 and HO-1 were diminished in the DysKD myotubes compared with the control myotubes. These results suggest that the signaling pathway related with the Nrf2-induced expression of antioxidant enzymes is involved to regulate cell survival under menadione-induced oxidative stress in the DysKD myotubes. The phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway is well-known to be closely involved in cell survival signaling, cell differentiation, and cell transformation. Recently, PI3-kinase/Akt pathway has been reported to mediate Nrf2 activation. The phosphorylation of Akt was significantly decreased in the DysKD myotubes compared to the control myotubes and this signaling pathway affected to verify responses in the DysKD myotubes under oxidative stress. Pre-incubation with LY294002, a PI3-kinase inhibitor, or API-2, an Akt inhibitor, blocked the activation of Nrf2. In addition, the binding of Nrf2 to ARE site of Bcl-2 gene was decreased particularly in the DysKD myotubes, resulting in inhibition of Bcl-2 expression. These results suggest that the reduced activation of Nrf2 in the DysKD myotubes may be mediated from the malfunction of PI3-kinase/Akt signaling pathway. Based on the observation that the activation of Nrf2 is a regulatory cellular response to menadione-induced oxidative stress, the effect of sulforaphane (SFN), a Nrf2 activator, was analyzed. As expected, the cell survival was increased by SFN-pretreatment. In addition, the expression of antioxidant enzymes such as HO-1, SOD, and NQO1 was significantly up-regulated in both the control myotubes and the DysKD myotubes. SFN increased not only Akt activation but also Nrf2 translocation to the nucleus in the DysKD myotubes; which results in the increase of Bcl-2 protein. These results suggest that the malfunction of Nrf2 pathway might be the responsible pathway to the oxidative stress-induced muscle damage in DMD and this biological response to menadione can be prevented with the pre-treatment of SFN. In this study, the PI3-kinase/Akt/Nrf2/ARE signaling pathway to elicit the differential response to oxidative stress between control and DysKD myotubes was determined and the mechanism responsible for muscle deterioration under oxidative stress was investigated. These results can provide a new information for understanding mechanisms of muscle deterioration in dystrophic muscle and be investigating novel therapeutic molecules for defending oxidative stress.
more목차
I. Introduction 1
A. Duchenne muscular dystrophy (DMD) 1
B. Dystrophin associated protein complexes and their structure 3
1. The structure of dystrophin associated protein complexes (DAPCs) 3
2. The structure of dystrophin 5
3. The structure of syntrophin 7
C. Oxidative stress 9
1. Menadione 9
2. Nuclear erythroid factor 2-related factor 2 (Nrf2) 11
3. Sulforaphane (SFN) 13
D. PI3-kinase/Akt signaling pathway 14
1. PI3-kinase 14
2. PI3-kinase/Akt signaling pathway under oxidative stress 15
E. The aims of this study 16
II. Materials and Methods 17
A. Materials 17
B. Cell culture and fusion index 17
C. Calculation of fusion index 18
D. Oxidative stress 18
E. Transfection of dystrophin shRNA lentivirus particles 18
F. WST assay 19
G. Lactate dehydrogenase (LDH) cytotoxicity assay 19
H. Isolation of nuclei fraction 19
I. Immunoblot assay 20
J. Chromatin immunoprecipitation (ChIP assay) 20
K. Immunofluorescence assay 21
L. Reverse transcription-Polymerase chain reaction (RT-PCR) 21
M. Real time-Polymerase chain reaction (qRT-PCR) 24
N. Statistical analysis 26
III. Results 27
A. The establishment of dystrophin knock-down mouse skeletal muscle (DysKD) cells 27
1. The deficiency of dystrophin protein was shown the decreased differentiation 30
2. The characterization of DysKD mouse skeletal muscle cells 34
B. Apoptosis was induced in myoblasts and myotubes under menadione-induced oxidative stress. 39
1. Menadione-induced oxidative stress elicited different expressions of antioxidant enzymes to result in cell death. 39
2. The dystrophin knock-down (DysKD) cells were more vulnerable to menadione-induced oxidative stress than control cells. 43
C. The signaling pathway responsible for the differential susceptibility of control and DysKD myotubes to menadione-induced oxidative stress. 51
1. Translocation of nuclear erythroid 2-related factor 2 (Nrf2) to the nucleus was mediated by menadione-induced oxidative stress. 51
2. The translocation of Nrf2 from the cytosol to the nucleus was down-regulated in DysKD myotubes under oxidative stress condition. 54
3. PI3-kinase/Akt signaling was involved in the differential susceptibility of control and DysKD myotubes 58
4. Inhibition of PI3-kinase/Akt signaling resulted in a decrease the translocation to the nucleus and the activity of Nrf2 in menadione-exposed myotubes 60
5. Menadione-induced oxidative stress elicited the translocation of phosphorylated Akt and Akt from the cytosol to the nucleus 63
6. Nuclear Nrf2 binds to the promoter of Bcl-2 gene at antioxidant response elements (AREs) in skeletal muscle cells. 66
D. Sulforaphane (SFN), the isothiocyanate materials from vegetables, could induce cell survival by regulating Nrf2 activity via PI3-kinase/Akt signaling pathway. 68
1. SFN in low concentration could prevent cell death under oxidative stress in skeletal muscle cells. 68
2. SFN could elicit up-regulation of antioxidant enzymes and positively regulate activation of Akt and Nrf2. 72
IV. Discussion 77
V. References 80
VI. 국문요약 86
