신경교세포에서 Matrix Metalloproteinase-3의 전사 조절에 대한 연구
Transcriptional Regulation of Matrix MetalloProteinase-3 in Rat Brain Astrocytes.
- 주제(키워드) MMP-3 , LXR , AP-1 , gangliosides , astrocytes
- 발행기관 아주대학교
- 지도교수 주일로
- 발행년도 2006
- 학위수여년월 2006. 2
- 학위명 석사
- 학과 및 전공 일반대학원 의학과
- 본문언어 영어
초록/요약
Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that degrade extracellular matix (ECM) components during normal and pathogenic condition. Documented expression of MMPs contributes to tissue remodeling, atherosclerosis, and inflammatory diseases. MMPs are tight regulated by several steps including transcriptional regulation. Transcription factors, NF-？？B, Ets and activator protein (AP)-1 are reported to be involved in transcriptional regulation of MMPs. Here we demonstrated transcriptional regulations of MMP-3 in LPS-, IFN？^-, and gangliosides-activated cultured astrocytes obtained from rat brain. MMP-3 transcript and protein were increased by stimulation with LPS, IFN？^ and gangliosides. Promoter analysis revealed that sequences between -500 to start codon are important in induction of MMP-3. EMSA-based study and site-direct mutagenesis revealed that AP-1 is important in induction of MMP-3. Since three AP-1 binding sites (we described them as proximal, middle and distal, respectively according to the distances from start codon) exist in -500 promoter sites, we tested which AP-1 site is important using sequence-specific primers for EMSA. We observed that middle and distal AP-1, not proximal AP-1 are activated by all three stimulators in EMSA analysis. Further, we demonstrated that LXR activation inhibit MMP-3 transcription through interaction with AP-1.
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ABSTRACT i
TABLE OF CONTENTS iii
LIST OF FIGURES v
LIST OF TABLES vi
ABBREVIATION vii
I. INTRODUCTION 1
II. MATERIAL AND METHOD 9
A. Reagents 9
B. Cell cultures 9
C. Reverse Transcription (RT)- PCR Analysis 9
D. Western Blot Analysis 10
E. Electrophoretic Mobility Shift Assay (EMSA) 11
F. RAT MMP-3 Promoter-Reporter Constructs 13
G. Transfection 14
F. Site-Direct Mutagenesis 14
III. RESULT 16
A. Induction of MMP-3 mRNA and protein expression in rat
primary astrocyte. 16
B. Distal and middle AP-1 of MMP-3 promoter confers
transcriptional regulation of MMP-3 in rat astrocytes. 19
C. Stimuli activation of AP-1 binding function activates
MMP-3 transcript. 24
D. Both distal- and middle-AP-1 element were important on expression of MM-3 gene. 28
E. Activation up-stream of AP-1 signal pathways and MMP-3 expression only repressed by JNK inhibitor. 31
F. MMP-3 expression and MMP-3 5…-Flanking promoter function
were interfered with LXR agonists. 35
G. LXR agonists inhibited AP-1 element in MMP-3 promoter
region. 39
IV. DISCUSSION 42
V. CONCLUSION 46
REFERENCES 49
국문요약 58
