근원세포의 증식과 분화에서의 phosphatidylinositol 3-kinase의 기능 연구
Functional Analysis of Phosphatidylinositol 3-kinase during the Proliferation and the Differentiation of Myoblasts
- 주제(키워드) PI3-kinase , myoblasts , proliferation , differentiation
- 주제(KDC) 470
- 주제(DDC) 570
- 발행기관 아주대학교
- 지도교수 김혜선
- 발행년도 2007
- 학위수여년월 2007. 8
- 학위명 박사
- 학과 및 전공 일반대학원 생명과학과
- 본문언어 영어
초록/요약
Myoblast undergoes a series of events of proliferation, cell cycle exit, alignment and elongation, fusion and formation of straight muscle fiber during differentiation. Phosphatidylinositol 3-kinase (PI3-kinase) is activated by a variety of extracellular stimuli, which impacts a number of cellular process including cell growth, proliferation, differentiation, migration, and survival. This paper discusses the function of PI3-kinase in proliferation and differentiation of myoblast. LY294002, an inhibitor of PI3-kinase, decreased the proliferation of L6 myoblasts and delayed the cell cycle progression from G1 to S phase. The expression of cyclin D1 and cdk4 were decreased by LY294002. These results suggest that PI3-kinase regulate the proliferation of L6 myoblasts by promoting the expression of cyclin D1 and cdk4. When cultured in low growth factor-containing medium, L6 myoblasts exit cell cycle and undergo a well-defined program of differentiation. Phosphorylation of eukaryotic elongation factor 2 (eEF2) was related to the differentiation of chick embryonic muscle cells in culture. The extent of eEF2 phosphorylation declined shortly after differentiation induction of L6 myoblasts, when cells prepare for terminal differentiation by withdrawing from the cell cycle. This decrease in phosphorylation, however, was blocked by LY294002 and wortmannin, inhibitors of PI3-kinase. These inhibitors have previously been shown to strongly block the differentiation of myoblasts. Therefore, I hypothesized that PI3-kinase plays an important role in the withdrawal from the cell cycle by regulating eEF2 phosphorylation in the early stages of differentiation. To test this hypothesis, L6 myoblasts were synchronized at the G2/M phase of the cell cycle using nocodazole and then cultured in either fresh differentiation medium (DM) or growth medium (GM). Released cells accumulated in the G0/G1 phase in DM and progressed to the S phase in GM. Cyclin D1 was more rapidly degraded in cells cultured in DM than in GM. The extent of eEF2 phosphorylation was seen to decrease more prominently in DM than in GM. Inhibitors of PI3-kinase or Akt, or mTOR increased eEF2 phosphorylation, but PI3-kinase became more activated when eEF2 phosphorylation declined. These results suggest that the regulation of L6 myoblast differentiation by PI3-kinase is related to eEF2 phosphorylation. PI3-kinase induces transcription of myogenin mRNA in myoblast differentiation. Here, I examined another mechanism regulating expression of myogenin by PI3-kinase. When LY294002 was added even after expression of myogenin, the fusion of myoblasts did not increase more than the level at the time treated with LY294002 and myogenin protein was decreased without change of its mRNA level. However, the mRNA and the protein of both GAPDH and b-actin were not changed by LY294002. LY294002 inhibited the rate of global protein synthesis through down-regulation of the activity of S6-kinase, S6, eIF4E, and eEF2. Cycloheximide, inhibitor of translation, rapidly decreased protein level of myogenin without the change of mRNA level but did not affected the level of protein and mRNA of both GAPDH and b-actin. These results shows that a half-life of myogenin protein is greatly shorter than those of GAPDH and b-actin proteins and the inhibition of protein synthesis at the translational level by LY294002 causes rapid depletion of myogenin protein which has a short half-life. This finding represents the first identification that PI3-kinase regulates expression of myogenin post-transcriptionally. Genistein (4',5,7-trihydroxyisoflavone) is a tyrosine kinase inhibitor. Although the agent has shown to inhibit myoblast differentiation, neither intracellular target(s) as a tyrosine kinase inhibitor nor action mechanism of the agent is well known. Here I studied the effect of genistein on the differentiation of myoblasts. Genistein strongly but reversibly blocked both myoblast fusion and synthesis of the muscle-specific proteins. The agent also reversibly reduced the phosphorylation level of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase, and its interaction with p85, the regulatory subunit of PI3-kinase. In addition, genistein indirectly inhibited PI3-kinase activity and blocked calcium influx which is required for myoblast fusion. However, both genistein-induced inhibition of cell fusion and calcium influx were abrogated by the lipid products of PI3-kinase. These results demonstrate that genistein can exert their effect on the signaling pathway from FAK to calcium influx via PI3-kinase in the differentiation of myoblasts. These data, together with the observation that PI3-kinase signaling is involved in the cell cycle progression of myoblast from G1 to S, induction of myogenin, sustaining the expression of myogenin, and cytosolic calcium elevation, suggest that PI3-kinase signaling plays important key roles in the proliferation and the differentiation of myoblasts.
more목차
Abstract 1
Table of Contents 4
List of Figures 7
I. Introduction 9
A. Skeletal muscle differentiation 9
B. PI3-kinase 18
C. Translational regulation of protein synthesis 24
D. The regulation of eEF2 phosphorylation 28
E. Extracellular matrix, integrin, and FAK 30
F. The purpose of this study 33
Ⅱ. Materials and Methods 34
A. Materials 34
B. Cell culture, cell counting and fusion index 35
C. Western blotting (Immunoblotting) 35
D. Immunoprecipitation 36
E. eEF2 Phosphorylation 37
F. Cell cycle analysis 37
G. PI3-kinase assay 38
H. Pulse metabolic labeling 38
I. Semi-quantitative RT-PCR (qRT-PCR) 39
J. Lipid preparation 39
K. Ca2+ imaging 40
Ⅲ. Results 41
A. LY294002 blocks the differentiation of L6 myoblasts 41
B. LY294002, an inhibitor of PI3-kinase, suppresses the proliferation of L6 myoblasts 45
1. LY294002 delayed the proliferation of L6 myoblasts 45
2. LY294002 inhibits the translation from G1 to S phase 49
3. LY294002 decreases the expression of cyclin D1 and cdk4 49
C. Phosphorylation of eukaryotic elongation factor 2 can be regulated by PI 3-kinase in the early stages of myoblast differentiation 53
1. PI3-kinase regulates eEF2 phosphorylation in the early stages of L6 myoblast differentiation 53
2. PI3-kinase is involved in regulation of the cell cycle by eEF2 phosphorylation 56
D. PI3-kinase can regulate expression of myogenin in post-transcriptional level 60
1. LY294002 blocks the differentiation of L6 myoblasts 60
2. LY294002 inhibits the expression of myogenin at the post-transcriptional level 63
3. LY294002 alters the phosphorylation of translational factors 65
4. LY294002 impairs the rate of global protein synthesis 67
5. Myogenin is rapidly depleted under the inhibition of mRNA translation by cycloheximide 69
E. Lipid products of PI3-kinase abrogate genistein-induced fusion inhibitioin in myoblasts 71
1. Genistein inhibits differentiation of L6 myoblasts 71
2. Genistein inhibits phosphorylation of FAK 72
3. Genistein indirectly inhibits PI3-kinase activity 77
4. Genistein as well as LY294002 impairs Ca2+ influx in L6 myoblasts 80
5. Lipid products of PI3-kinase eliminate genistein-induced inhibition of Ca2+ influx and cell fusion 82
6. Genistein does not inhibit Akt-depedent PI3-kinase pathway 88
Ⅳ. Discussion 90
A. PI3-kinase is required to transition from G1 to S during proiferation of L6 myoblasts 90
B. PI3-kinase is required to exit from cell cycle for the differentiation of L6 myoblasts 91
C. PI3-kinase activity is required to maintain a constant level of myogenin during myoblast differentiation 92
D. Products of PI3-kinase are involved in the intracellular Ca2+ influx pre-requisite for myoblast fusion 94
E. Roles of PI3-kinase during skeletal muscle differentiation 98
Ⅴ. References 100
Ⅵ. 국문 요약 118
Fig. 1. Schematic representation of skeletal muscle formation in the limb, with the different stages and genes potentially involved at each stage 10
Fig. 2. Model of satellite cell dynamics 11
Fig. 3. Scheme of skeletal muscle differentiation 16
Fig. 4. The phosphatidylinositol 3-kinase (PI3-kinase) family 19
Fig. 5. Structure and signaling of the class IA and IB PI3-kinases 20
Fig. 6. Upstream and downstream of Akt 23
Fig. 7. Regulation of translational initiation through mTOR signaling pathway 26
Fig. 8. Activation of eEF2 by insulin, GPCR agonists and other stimuli 29
Fig. 9. Structure of FAK and FAK mediated signaling 32
Fig. 10. LY294002 blocks the fusion of L6 myoblasts 43
Fig. 11. LY294002 inhibits the expression of muscle specific genes 44
Fig. 12. The effect of LY294002 on proliferation of L6 myoblasts 47
Fig. 13. LY294002 increases population doubling time 48
Fig. 14. LY294002 inhibits the transition from G1 to S phase 51
Fig. 15. LY294002 decreases the expression of cyclinD1, Cdk4 and pRb 52
Fig. 16. Phosphorylation of eEF2 during myoblast differentiation 55
Fig. 17. Phosphorylation of eEF2 and activity of PI3-kinase in synchronized cells 58
Fig. 18. LY294002 blocks the differentiation of L6 myoblasts 61
Fig. 19. Effect of LY294002 on the differentiation of L6 myoblasts when it was treated after expression of myogenin 62
Fig. 20. LY294002 decreases the protein level of myogenin but not the mRNA level 64
Fig. 21. LY294002 alters the phosphorylation of translation factors 66
Fig. 22. LY294002 impairs the rate of protein synthesis 68
Fig. 23. Cycloheximide decreases myogenin protein prior to GAPDH and b-actin 70
Fig. 24. Effect of genistein on differentiation of L6 myoblasts 73
Fig. 25. Phosphorylation of FAK 75
Fig. 26. Association of FAK with p85 78
Fig. 27. Effect of genistein on PI3-kinase activity 79
Fig. 28. Ca2+ imaging of L6 myoblasts 81
Fig. 29. Restoration of Ca2+ influx by PI-3,4,5-P3 83
Fig. 30. Restoration of cell fusion by addition of lipid products of PI 3-kinase 86
Fig. 31. Effect of genistein on the Akt phosphorylation 89
Fig. 32. Model of functions of PI3-kinase in the proliferation and the differentiation of myoblasts 99
