정이숙 김미영 2008-08 9340 https://aurora.ajou.ac.kr/handle/2018.oak/7716 학위논문(박사)--아주대학교 일반대학원 :분자과학기술학과,2008. 8 본 연구에서는 허혈성 심근세포 손상과정에서 변화되는 유전자들을 탐색하고 기능상관관계를 관찰하여 허혈성 세포사멸의 원인이 되는 유전자를 발굴하고, 나아 가 그 유전자에 의한 심근세포사멸 신호전달체계를 규명함으로써 허혈성 심근손상 에 대한 새로운 치료방안을 모색하고자 하였다. 허혈/재관류성 심근경색 동물모델의 심장과 허혈/저산소 자극에 노출된 심근세 포에서 변화되는 유전자들을 microarray와 real-time PCR 기법을 이용하여 검색 하였다. 다음으로, 발굴된 손상관련 유전자 후보군을 대상으로, siRNA, 유전자 과발 현 (overexpression) 기법 등을 이용하여 유전자-기능상관관계를 조사함으로써 허 혈/저산소성 심근세포사멸 관련 마커유전자를 도출하였다. 나아가, 분자생물학적 기 법을 이용하여 유전자에 의한 세포사멸 조절기전을 밝히고자 하였다. 심근경색 동물모델의 심장과 허혈/저산소 자극에 노출된 심근세포에서 Gadd45β 유전자가 저산소성 심근세포사멸의 매개인자로 작용한다는 것과 Gadd45β의 상위 신호전달물질로서 p38이 관여한다는 것을 알 수 있었다. Gadd45β 유전자의 발현을 위한 전사인자(transcription factor)로서는 NF-kB가 아 닌 p53이 관여할 가능성이 EMSA, p53 siRNA 및 Gadd45β reporter assay 등의 결과로부터 제시되었다. p53 단백질의 발현과 인산화된 p53의 발현이 저산소에 의 해 증가하였으며 이에 반하여, p53의 mRNA 경우는 발현 변화가 관찰되지 않았다. 이는 p53 단백질 안정화가 인산화를 통하여 일어나며 이러한 안정화가 Gadd45β 유전자의 발현을 증가시키는데 중요할 것임을 시사하고 있다. p53 mutant plasmid 를 이용하여 p53 인산화의 부위를 조사해본 결과 p53의 Ser15와 Ser20위치의 인 산화가 Gadd45β의 mRNA 발현 유도에 필수적임을 알 수 있었다. Gadd45β mRNA 조절인자로서의 p38과 p53 전사인자간의 상관관계를 알아본 결과 p38에 의해 p53 이 인산화되는 것을 확인하였다. 이상의 결과를 요약하면, 심근세포에서 허혈/저산소 자극에 의해 p38이 활성 화되고 이로 인해 p53 단백질이 인산화됨으로써 p53 단백질의 안정화가 일어나고, 안정화된 인산화 p53 단백질이 Gadd45β 유전자의 전사인자로 작용하여 Gadd45β 의 mRNA 및 단백질 발현을 증가시키며 결과적으로 허혈/저산소성 심근세포 고사 가 일어난다는 것을 알 수 있었다. 결론적으로, 본 연구를 통하여 허혈/저산소성 심 근세포 사멸과정의 새로운 매개유전자로 발굴된 Gadd45β는 심근세포 사멸 치료법 의 새로운 타겟으로 활용될 수 있다고 사료된다. ABSTRACT = i TABLE OF CONTENTS = iii LIST OF FIGURES = vii LIST OF TABLE = ix LIST OF ABBREVIATION = x I. INTRODUCTION = 1 A. Myocardial ischemic injury = 1 B. The role of p38 in ischemic injury = 2 C. p53 = 3 1. p53 as a transcription factor = 3 2. Regulation of p53 = 4 3. The role of p53 = 5 D. The role of Gadd45 family = 6 II. Aims of the study = 9 III. METHODS = 10 A. Ischemia/reperfusion injury in vivo = 10 B. Cell culture and hypoxia model = 11 C. Preperation of fluorescent DNA probe and hybridization = 12 D. Scanning and data analysis = 13 E. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) = 13 F. Real-time quantitative PCR analysis = 14 G. Western blot analysis for Atf3, Gadd45β and Ddit4 = 15 H. In situ terminal deoxynucleotidyl transferase UTP nick and labeling (TUNEL) = 15 I. Cell viability measurement = 16 J. siRNA preperation and transfection = 16 K. Cell transfection = 17 L. Plasmids = 17 M. Caspase-3 activity = 18 N. Electorophoretic mobility shift assay (EMSA) = 19 O. Luciferase assay = 20 P. Assay for detecting ubiquitination of p53 = 20 Q. Statistical analysis = 20 IV. RESULTS = 21 A. cDNA chip analysis = 21 B. Induction of Atf3, Gadd45β and Ddit4 during hypoxia = 25 C. Effects of siRNAs for Atf3, Gadd45β and Ddit4 on hypoxia-inducedcell death = 28 D. Effects of siRNAs for Atf3, Gadd45β and Ddit4 on hypoxia-induced apoptotic cell death = 28 E. Effect of ectopic Gadd45β overexpression on hypoxia-induced celldeath = 32 F. Effect of ectopic Gadd45β overexpression on hypoxia-induced apoptoticcell death = 32 G. Phosphorylation of p38 during hypoxia = 35 H. Roles of p38 siRNA in hypoxia-induced cell death = 35 I. Effect of ectopic p38 overexpression on hypoxia-induced cell death = 35 J. Link between Gadd45β and p38 activation = 39 K. NF-kB DNA-binding to Gadd45β promoter = 42 L. p53 DNA-binding to Gadd45β promoter = 42 M. Regulation of the Gadd45β promoter activity by p53 = 45 N. Effect of p53 on Gadd45β transcriptional regulation = 45 O. Effects of siRNAs for p53 on hypoxia-induced cell death = 48 P. Effect of ectopic p53 overexpression on hypoxia-induced cell death = 48 Q. Induction of p53 during hypoxia = 51 R. Phophorylation of p53 at Ser15 or Ser20 during hypoxia = 51 S. Stabilization of p53 by inhibition of ubiquitination = 51 T. Regulation of the Gadd45β promoter activity by phophorylation of p53at Ser15 or Ser20 = 55 U. Effect of phosphorylation of p53 at Ser15 or Ser20 on Gadd 45βtranscriptional regulation = 55 V. Link between p38 and p53 phosphorylation = 58 V. DISCUSSION = 60 VI. CONCLUSION = 67 REFERENCES = 68 국문초록 = 80 LIST OF FIGURES = 23 Fig. 1. Changes in transcript levels in vivo ischemia = 23 Fig. 2. Changes in transcript levels in vitro hypoxia = 24 Fig. 3. Time-dependent changes of Atf3, Gadd45? and Ddit4 mRNA expression during hypoxia in primary cardiomyocytes and in H9c2 cells. = 26 Fig. 4. Time-dependent changes of Atf3, Gadd45? and Ddit4 protein expression during hypoxia in primary cardiomyocytes and in H9c2 cells. = 27 Fig. 5. Effect of Atf3, Gadd45β and Ddit4 siRNA transfection on mRNA and protein expression. = 29 Fig. 6. Effect of Atf3, Gadd45β and Ddit4 siRNA on hypoxia-induced cardiomyocyte death. = 30 Fig. 7. Effect of Atf3, Gadd45β and Ddit4 siRNA on hypoxia-induced apoptotic cell death. = 31 Fig. 8. Effect of ectopic Gadd45β overexpression on hypoxia-induced cardiomyocyte death. = 33 Fig. 9. Effect of ectopic Gadd45β overexpression on hypoxia-induced apoptotic cell death. = 34 Fig. 10. Western blot analysis for phosphorylation of p38 in H9c2 cells during hypoxia = 36 Fig. 11. Effect of p38 siRNA on hypoxia-induced cardiomyocyte death. = 37 Fig. 12. Effect of ectopic p38 overexpression on hypoxia-induced cardiomyocyte death. = 38 Fig. 13. Effect of Gadd45β siRNA on hypoxia-induced phosphorylation of p38 = 40 Fig. 14. Effect of p38 siRNA on Gadd45β expression during hypoxia in H9c2 cells = 41 Fig. 15. NF-kB DNA binding to the Gadd45β promoter = 43 Fig. 16. p53 DNA binding to the Gadd45β promoter = 44 Fig. 17. Regulation of the Gadd45β promoter activity by p53 in H9c2 cells = 46 Fig. 18. Effect of p53 on Gadd45β transcriptional regulation = 47 Fig. 19. Effect of p53 siRNA on hypoxia-induced cardiomyocyte death. = 49 Fig. 20. Effect of ectopic p53 overexpression on hypoxia-induced cardiomyocyte death = 50 Fig. 21. Expression of p53 protein during hypoxia in H9c2 cells. = 52 Fig. 22. Western blot analysis for phosphorylation of p53 at Ser15 or Ser20 in H9c2 cells during hypoxia = 53 Fig. 23. Stabilization of p53 by inhibition of ubiquitination = 54 Fig. 24. Regulation of the Gadd45β promoter activity by phosphorylation of p53 at Ser15 or Ser20 in H9c2 cells. = 56 Fig. 25. Effect of phosphorylation of p53 at Ser15 or Ser20 on Gadd45β transcriptional regulation = 57 Fig. 26. Effect of p38 siRNA on hypoxia-induced phosphorylation of p53 at Ser15 or Ser20 = 59 Fig. 27. Schematic diagram for the signal pathway of Gadd45β-induced apoptosis during hypoxia = 68 LIST OF TABLE = 22 Tab. 1. Genes with elevated expression in myocardial ischemia/hypoxia = 22 eng The Graduate School, Ajou University 아주대학교 논문은 저작권에 의해 보호받습니다. 허혈성 심근세포 손상과정에서 Gadd45β의 역할과 조절기전 Kim. Mi-Young Thesis 아주대학교 일반대학원 Kim. Mi-Young 일반대학원 분자과학기술학과 2008. 8 Master http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000009340 Gadd45β mediator apoptotic cell death myocardial ischemia/hypoxia Apoptotic death plays a critical role in cardiomyocytes loss during ischemic heart diseases, including myocardial infarction (MI). Recent studies have underlined the negative role of cardiomyocyte apoptosis in cardiac function and demonstrated a causal relationship between cardiomyocyte apoptosis and MI. Therefore, clarifying the molecular mechanisms involved in cardiomyocyte apoptotic cascades, and identifying optimal strategy for its intervention have significant clinical implications. In the present study, to examine gene profiling during myocardial ischemic/hypoxic injury, DNA microarray and RT-PCR were performed in heart from rat MI model and cardiomyocytes exposed to hypoxia. Using molecular biology techniques of siRNA and plasmid transfection, the function of hypoxia-responsive genes were evaluated during hypoxia-induced apoptotic cell death. Furthermore, signaling pathways for the proapoptotic gene-induced cell death during ischemia/hypoxia were investigated. The results from this study suggest that Gadd45β may mediate apoptotic cell death induced by ischemia/hypoxia in cardiomyocytes, and that p38 is an upstream signaling molecule for Gadd45β. The results from EMSA, p53 siRNA and Gadd45β reporter assay, suggest that p53, rather than NF-kB, acts as a transcription factor for Gadd45β mRNA expression. This study further showed that phosphorylation of p53 at Ser15 or Ser20 may be essential for p53 stabilization and activation, leading to increased transcriptional activity of Gadd45β during ischemia/hypoxia, and that the phosphorylation of p53 at Ser15 and Ser20 is induced by p38 which is activated by hypoxia In summary, these results suggest that the signaling pathways for Gadd45β-mediated apoptosis during ischemia/hypoxia in cardiomyocytes involve p38 activation → phosphorylation of p53 at Ser15 or Ser20 → Gadd45β mRNA and protein expression. In conclusion, Gadd45β can be applied for a novel therapeutic target for the ischemic heart disease.