일시적 산화자극에 노출된 쥐신경성상세포에서의 STAT-3와 STAT-6의 조절기전에 관한 연구
Regulation of STAT-3 and STAT-6 by Transient Oxidative Stress in Rat Brain Astrocytes
- 발행기관 아주대학교
- 발행년도 2009
- 학위수여년월 2009. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의학과
- 실제URI http://www.dcollection.net/handler/ajou/000000010109
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
STAT family of transcription factors mediate in a variety of cellular processes, such as immune response, differentiation, cell survival, proliferation, or oncogenesis, following a series of extracellular signaling such as cytokine, growth factor, and hormone signaling. Accumulating evidence also supports a role for STAT proteins in mediating the cellular response to oxidative stress. Nevertheless, the regulation and the role of STAT proteins in brain oxidative stress still remain a poorly understood. Here, I determined the molecular mechanisms of indivisual STAT regulation and those governing how STAT proteins exerts its function under brain oxidative stress conditions using cultured rat brain astroctyes. The present study has shown that individual STAT proteins are differently regulated to H2O2-mediated oxidative stress. Fistly I report that H2O2 induced a decrease in the steady state level of STAT-3 phosphorylation by SHP-2 activation through lipid raft. In the presence of H2O2, SHP-2 formed a complex with STAT-3, and reduced the steady-state STAT-3 phosphorylation level. H2O2 triggered SHP-2 phosphorylation in a time- and dose-dependent manner, and led to its translocation into lipid rafts. H2O2-mediated SHP-2 phosphorylation and translocation were inhibited by filipin III, a lipid raft-disrupting agents. Interestingly, the effect of H2O2 on SHP-2 phosphorylation was cell type-specific. Remarkably, SHP-2 phosphorylation was induced strongly by H2O2 in astrocytes, but barely detectable in microglia. Secondly, I also report that H2O2 induced rapid STAT-6 activation via c-Src/JAK pathway contributed to the COX-2 dependet PGE2 production in brain astrocytes. I showed that H2O2 induced STAT6 phosphorylation was accompanied by it’s nuclear translocation and DNA binding activity. Experiments using pharmacological inhibitors and siRNA demonstrated that H2O2-induced STAT-6 phosphorylation was mediated by c-Src/JAK pathways upstreams. In addition, transfection with STAT-6 siRNA and EMSA using oligonucleotide probe containing COX-2 specific STAT-6 binding consensus sequence revealed that STAT-6 required for mediating COX-2 expression and PGE2 production in H2O2-treated astrocytes. In addition performing incubation experiment of microglia with ACM, I determined that PGE2 released from H2O2 treated-astrocytes functioned as paracrine mediator in deactivation of microglia. Finally, I showed that STAT-6 could be activated in a ROS-dependent manner and involved in COX-2 induction in the brain astrocytes under H/R condition. These results may indicate that STAT-6-COX-2 pathway is induced whenever ROS-mediated oxidative stress take place. Taken together, given the specific function of each STAT protein in a variety of cellular processes, understanding of the regulation and the role of indivisual STAT under brain oxidative stress condition may lead to the development of specific therapeutic strategies for neuronal oxidative damage.
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ABSTRACT ⅰ
TABLE OF CONTENTS ⅲ
LIST OF FIGURES ⅵ
Ⅰ. INTRODUCTION 1
A. Reactive oxygen species (ROS) 1
1. ROS in the Brain 1
B. Astrocytes 4
1. Normal structure and function of astrocytes 4
2. Astrocytes in neuronal ischemic damage 5
C. Signal Transducers and Activators of Transcription
(STAT) 6
1. Structure of STATs 7
2. Activation mechanisms of STATs 7
3. Negative regulation of STATs 8
4. STATs in oxidative stress 9
D. Aims of study 10
Ⅱ. MATERIAL AND METHOD 12
A. Material 12
B. Methods 12
1. Cell culture 12
2. Hypoxia/Reoxygenation 13
3. Western blot analysis 13
4. Immunofluorescence assay 14
5. Detergent-free discontinuous sucrose gradient
ultracentrifugation 14
6. Immunoprecipitation 15
7. Transfection of siRNA 16
8. Reverse Transcription- and Polymerase Chain
Reaction (RT-PCR) 16
9. Electrophoretic Mobility Shift Assay (EMSA) 17
10. PGE2 assay 18
11. Assessment of cell viability 18
Ⅲ. RESULTS 20
A. H2O2-induced STAT-3 dephosphorylation is mediated
by SHP-2 activation via lipid-raft 20
1. Oxidative stress by hypoxia/reoxygenation induces
SHP-2 phosphorylation in primary astrocytes 20
2. SHP-2 tyrosine phosphorylation is induced by H2O2 in
astrocytes 22
3. SHP-2 phosphorylation by H2O2 is mediated via lipid
raft 25
4. H2O2 induces translocation of SHP-2 into lipid rafts 27
5. SHP-2 reduces the steady-state STAT-3
phosphorylation level in astrocytes 31
6. SHP-2 phosphorylation is strongly induced by H2O2 in
rat primary astrocytes but barely detectable in
microglia 34
B. STAT-6 mediated astrocytic prostaglandin modulates
microglia activation under transient oxidative stress 37
1. H2O2 induces STAT-6 phosphorylation 37
2. H2O2 enhances nuclear translocation and DNA
binding activity of STAT-6 40
3. H2O2 induces STAT-6 phosphorylation via c-Src/JAK
pathways 42
4. The effect of a transient exposure of astrocytes to
H2O2 44
5. STAT-6 is required for H2O2-induced COX-2
expression and resultant prostaglandin synthesis in
brain astrocytes 44
6. Astrocyte-derived PGE2 mediated the suppression of
TNF alpha expression in microglia 48
7. ROS-dependent-STAT-6 activation induces COX-2
expression under hypoxia/reoxygenation 50
Ⅳ. DISCUSSION 53
Ⅴ. CONCLUSION 61
REFERENCES 62
국문요약 81
