22(R)-hydroxycholesterol에 의한 MKP-1 발현 기작 규명을 통한 새로운 항염 기전 연구
A novel anti-inflammatory mechanism targeting post-transcriptional control of MKP-1 expression by 22(R)-hydroxycholesterol
- 주제(키워드) MKP-1 , 22(R)-hydroxycholesterol , HuR , mGluR5 , PKCa
- 발행기관 아주대학교
- 지도교수 주일로
- 발행년도 2017
- 학위수여년월 2017. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000024144
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
MAP kinase phosphatase (MKP)-1 plays a pivotal role in controlling MAP kinase (MAPK)-dependent (patho)physiological processes. Although MKP-1 gene expression is tightly regulated at multiple levels, the underlying mechanistic details remain largely unknown. In this study, I demonstrate that MKP-1 expression is regulated at the post-transcriptional level by 22(R)-hydroxycholesterol [22(R)-HC] through a novel mechanism. 22(R)-HC induces Hu antigen R (HuR) phosphorylation, cytoplasmic translocation and binding to MKP-1 mRNA, resulting in stabilization of MKP-1 mRNA. The resulting increase in MKP-1 leads to suppression of JNK-mediated inflammatory responses in brain astrocytes. I further demonstrate that 22(R)-HC–induced phosphorylation of nuclear HuR is mediated by PKCα, which is activated in the cytosol by increases in intracellular Ca2+ levels mediated by the phospholipase C/inositol 1,4,5-triphosphate receptor (PLC/IP3R) pathway and translocates from cytoplasm to nucleus. In addition, pharmacological interventions reveal that metabotropic glutamate receptor5 (mGluR5) is responsible for the increases in intracellular Ca2+ that underlie these actions of 22(R)-HC. Collectively, my findings identify a novel anti-inflammatory mechanism of 22(R)-HC, which acts through PKCα-mediated cytoplasmic shuttling of HuR to post-transcriptionally regulate MKP-1 expression. These findings provide an experimental basis for the development of a RNA-targeted therapeutic agent to control MAPK-dependent inflammatory responses.
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TABLE OF CONTENTS
ABSTRACT ⅰ
TABLE OF CONTENTS ⅲ
LIST OF FIGURES vii
LIST OF TABLES xii
ABBREVIATIONS xiii
Ⅰ. INTRODUCTION 1
A. Inflammation 1
1. MAPK activation 1
2. MKP-1 as MAPK inhibitor 3
(1) MKP-1 gene 4
(2) MKP-1 mRNA 5
a. mRNA stabilization 5
① HuR 6
② Tristetraprolin (TTP) 6
③ Nuclear factor-90 (NF-90) 7
④ T-cell intracellular antigen-1 (TIA-1) 7
b. mRNA elongation 7
(3) MKP-1 protein 8
B. Oxysterols 8
1. Synthesis 10
(A) Enzymatic reaction 10
(B) Non-enzymatic reaction 11
2. Biological effects 11
(A) LXR-dependent mechanism 11
(B) LXR-independent mechanism 13
3. Oxysterols and neurodegenerative disorder 13
C. Aims of this study 14
Ⅱ. MATERIALS AND METHODS 15
A. Reagents 15
B. Antibodies 15
C. Cell culture 16
D. RNA interference 16
E. Plasmids 18
F. RNA isolation 19
G. Reverse transcription and quantitative real-time PCR analysis 20
H. ELISA 21
I. Subcellular fractionation 21
J. Western blotting 22
K. Immunoprecipitation (IP) 22
L. UV cross-link ribonucleoprotein (RNP)-IP 22
M. Immunostaining 23
N. In vitro PKCα kinase assay 23
O. Immune complex kinase assay 24
P. Measurement of cytosolic Ca2+ 25
Q. Cell viability assay (WST-1 assay) 25
R. Statistical analysis 25
Ⅲ. RESULTS 27
A. Some nuclear receptor ligands and oxysterols exert anti-inflammatory effects through MKP-1 expression. 27
1. Some nuclear receptor ligands induce MKP-1 expression. 27
2. 22(R)-HC-induced MKP-1 expression inhibits JNK-mediated induction of
pro-inflammatory genes. 31
B. 22(R)-HC induces HuR-dependent MKP-1 expression via mGluR5-mediated Ca2+/PKCα signaling. 36
1. 22(R)-HC induces MKP-1 expression in an LXR-independent manner. 36
2. MKP-1 induction by 22(R)-HC is not associated with post-translational regulation. 42
3. HuR mediates MKP-1 induction by 22(R)-HC. 45
4. 22(R)-HC-induced stabilization of MKP-1 mRNA depends on MKP-1 mRNA-bounded HuR shuttling from nucleus to cytoplasm. 52
5. The LXR ligands or oxsyterols show differential abilities to induce nucleocytoplasmic translocation of HuR. 59
6. PKCα mediates HuR-dependent regulation of MKP-1 expression in 22(R)-HC-treated cells. 62
7. 22(R)-HC activates PKCα indirectly. 66
8. The non-phosphorylatable mutants of HuR inhibit 22(R)-HC-induced HuR cytoplasmic translocation and MKP-1 expression. 75
9. 22(R)-HC-induced nuclear translocation of PKCα underlies cytoplasmic translocation of HuR and subsequent MKP-1 induction. 83
10. PKCα/HuR-mediated MKP-1 induction by 22(R)-HC is dependent on an increase in cytosolic Ca2+ concentration. 91
11. mGluR5 mediates 22(R)-HC-induced increases in cytosolic Ca2+ concentration. 98
12. MAPK-mediated induction of pro-inflammatory genes is inhibited by
22(R)-HC-induced MKP-1 expression, is HuR- and PKCα-dependent manner. 107
Ⅳ. DISCUSSION 113
Ⅴ. SUMMARY AND CONCLUSION 118
REFERENCES 120
국문요약 143
