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배양근원세포에서의 산화스트레스에 의한 근육소실 기작에 관한 연구
- Alternative Title
- The mechanism of muscle deterioration under oxidative stress in cultured muscle cells
- Author(s)
- 최수진
- Alternative Author(s)
- Su Jin Choi
- Advisor
- 김혜선
- Department
- 일반대학원 생명과학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2018-02
- Language
- eng
- Keyword
- dystrophin; oxidative stress; transduction; Nrf2; sulforaphane
- Alternative Abstract
- 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.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Bioscience > 4. Theses(Ph.D)
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