근육세포에서 산화스트레스에 의한 신호전달기작 연구
- 주제(키워드) 산화스트레스 , 근육세포 , PI3-kinase/Akt , alpha-syntrophin , Focal adhesion kinase
- 발행기관 아주대학교
- 지도교수 김혜선
- 발행년도 2011
- 학위수여년월 2011. 8
- 학위명 박사
- 학과 및 전공 일반대학원 생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000011851
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Skeletal muscle tissue continuously generates reactive oxygen species (ROS) during contraction. Although low-to-moderate levels of oxidative stress play important roles in the modulation of skeletal muscle force production, control of cell signaling pathway, and regulation of gene expression, increasing production of ROS leading to cellular oxidative stress is linked to numerous pathologies and muscle degeneration. Specially, the muscle of Duchenne muscular dystrophy (DMD), caused by deficiency of dystrophin, and mdx, mouse model of DMD, is known to be more vulnerable to oxidative stress than normal muscle. However, the signaling mechanisms to the oxidative stress in skeletal muscle have not much known yet. Myoblasts, the proliferating mononucleated cells of skeletal muscle, terminally differentiate to form multinucleated myotubes. In a medium lacking mitogenic activity, myoblasts withdraw from the cell cycle, elaborate muscle-specific gene products, and fuse to form myotubes. The expression of proteins including the signaling molecules as well as myogenic proteins dramatically changes during differentiation with the morphological changes. In addition, activities of numerous signaling pathways such as phosphoinositide 3-kinase (PI3-kinase)/Akt, p38 mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB) pathway are known to be related to myoblast differentiation. This study aims to clarify the signaling pathways under the oxidative stress in skeletal muscle cells. Firstly, the difference of susceptibility between myoblasts and myotubes was investigated under the menadione-induced oxidative stress. It was found that the undifferentiated myoblasts were more vulnerable to menadione-induced oxidative stress than the differentiated myotubes. The expression ratio of Bcl-2 to Bax, an indicator for the apoptotic cells, in myoblasts was significantly decreased by menadione. Although this decrease was not found in myotubes, when they were pre-incubated with LY294002, PI3-kinase inhibitor, myotubes also showed increased susceptibility to menadione. Moreover, the PI3-kinase activity was significantly blocked by menadione, particularly in myoblasts. Both the concentration and activity of Akt, the downstream effector protein of PI3-kinase, were also down-regulated in myoblasts. These results suggest that the PI3-kinase/Akt signaling pathway is responsible for the differential susceptibility to the menadione-induced oxidative stress between myoblasts and myotubes. Secondly, the role of α-syntrophin in myoblasts under the menadione-induced oxidative stress was investigated. Oxidative stress induces a myriad of signaling pathway in skeletal muscle cells. The muscle from DMD and mdx showed to increase the susceptibility to oxidative stress. Among the dystrophin-associated protein complex (DAPC), α-syntrophin has been known a scaffolding protein because of its multiple protein interaction domains leading to link the internal signaling pathways and DAPC. However, the precise function of α-syntrophin in muscle cells has not been clear yet. The protein level of α-syntrophin was elevated when cells were exposed to menadione. Interestingly, the cells over-expressed α-syntrophin showed to be resistant to the menadione-induced oxidative stress. On the contrary, the apoptotic signal was increased in the cells transfected α-syntrophin siRNA under the menadione-induced oxidative stress. The interaction between α-syntrophin and p85, the regulatory subunit of PI3-kinase, was increased by menadione. In addition, the phosphorylation of Akt was also increased in α-syntrophin over-expressed cells while it was decreased in α-syntrophin knock-downed cells. Furthermore, Ca2+ influx, which is known to increase when the cells were exposed to oxidative stress, decreased in the α-syntrophin over-expressed cells while that increased in the α-syntrophin knock-downed cells. These results suggest that α-syntrophin might play a pivotal role in the survival pathway under oxidative condition via regulation Ca2+ influx. Finally, based on the observation that the damaged cells changed to round shape and were detached from the culture dish under the oxidative stress, the activity of focal adhesion kinase (FAK), one of the focal adhesion molecules was studied. The 80 kDa N-terminal and the 35 kDa C-terminal fragments as well as the full-length FAK (125 kDa) were found under a normal condition. However, the cleavage products of FAK were disappeared under the oxidative stress. Calpeptin, a specific inhibitor of calpain, not only blocked the cleavage of FAK, but also reduced survival ratio of cells under the stress. When the cells were transfected with the 80 kDa N-terminal fragment, the phosphorylation of Akt and the ratio of Bcl-2/Bax increased; which means survival signaling pathway was activated by the increased expression of the fragment. These results suggest that the N-terminal fragment of FAK, a product of calpain, is involved in the survival signal under the oxidative stress in cultured myoblasts. These findings not only reveal important insight for the cell signaling pathway under oxidative stress in skeletal muscle cells but also provided new approaches of therapy for muscle degeneration caused by oxidative stress in muscular dystrophy.
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Table of Contents
Abstract 1
Table of contents 4
List of figures 7
I. Introduction 10
A. Oxidative stress in skeletal muscle 10
1. ROS production in skeletal muscle 10
2. Duchenne muscular dystrophy (DMD 13
3. Differentiation of myoblasts 15
4. Menadione 15
B. PI3-kinase/Akt signaling pathway 18
1. PI3-kinase 18
2. PI3-kinase/Akt signaling under oxidative stress 20
C. α-Syntrophin 22
D. Focal adhesion kinase (FAK) 25
1. The structure and phosphorylation of FAK 25
2. FAK signaling under oxidative stress 25
3. Calpain 26
E. The aims of this study 28
II. Materials and Methods 29
A. Materials 29
B. Cell culture 30
C. Oxidative stress 30
D. Viability assay 30
E. Immunoblotting 31
F. PI3-kinase activity assay 31
G. Immunostaining 32
H. Isolation of nuclei fraction 32
I. DNA constructs 32
J. Over-expression 33
K. Transfection of siRNA 33
L. RNA preparation and cDNA synthesis 33
M. Real-Time PCR 34
N. Immunoprecipitation 34
O. Calcium imaging 34
P. Statistical analysis 35
III. Results 36
A. The signaling pathway responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress 36
1. Undifferentiated myoblasts are more vulnerable to menadione-induced oxidative stress than differentiated myotubes 36
2. Apoptosis is induced in myoblasts under oxidative stress earlier than myotubes 38
3. PI3-kinase/Akt signaling is involved in the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress 41
4. Inhibition of PI3-kinase/Akt signaling results in decrease of Bcl-2/Bax ratio in menadione-exposed myotubes 46
5. The expression of antioxidant enzymes were not changed by menadione at low concentration 46
6. PI3-kinase/Akt pathway migrated to the nuclear fraction under the menadione-induced oxidative stress 50
B. The novel role of α-syntrophin under menadione-induced oxidative stress in myoblasts 57
1. α-Syntrophin expression level was increased by low concentration of menadione but mRNA of α-syntrophin was not changed 57
2. The cell viability and survival signaling were increased in α-syntrophin over-expressed C2 myoblasts 60
3. The survival signaling was suppressed in α-syntrophin knock-downed C2 myoblasts 64
4. α-Syntrophin regulates Ca2+ influx when the cells exposed to menadione 67
5. The interaction of α-syntrophin and p85 increased by menadione. 70
C. The survival signaling of N-terminal fragment of focal adhesion kinase under oxidative stress in cultured myoblasts 72
1. Cleavage of FAK is suppressed by oxidative stress 72
2. N-FAK is also phosphorylated on Tyr397 and the viability reduced when the FAK phosphorylation is blocked by genistein 76
3. The cleavage of FAK was blocked when calpain activity is inhibited 81
4. Calpeptin and menadione have synergistic effects on the cell death. 86
5. The cell viability and the Akt phosphorylation on Ser473 were increased when N-FAK was over-expressed 90
6. The level of N-FAK increased during myoblasts differentiation 90
7. The N-FAK is not translocalized to nuclear by oxidative stress 94
IV. Discussion 96
A. PI3-kinase/Akt signaling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress 96
B. α-Syntrophin can induce the survival signaling pathway through regulation of [Ca2+]i under menadione-induced oxidative stress in myoblasts. 99
C. N-terminal fragment of focal adhesion kinase is required for the activation of survival signaling under oxidative stress in cultured myoblasts 101
V. References 104
VI. 국문요약 113
