목차

IIntroduction 1
PART Ⅰ 4
TRIM24-RIPK3 axis perturbation accelerates osteoarthritis pathogenesis 4
I. Introduction 5
II. Materials and Methods 7
A. Human OA cartilage samples and experimental OA mouse models 7
B. Reagents 7
C. Primary mouse articular chondrocytes 8
D. Cell viability assay 9
E. Histology and immunohistochemistry 9
F. Western blotting 10
G. RT-PCR and qPCR 10
H. Collagenase and aggrecanase activity assay 10
I. Microarray analyses 11
J. In silico binding assay 11
K. Gene set enrichment analysis (GSEA) 12
L. Statistical analysis 12
III. Results 13
1. RIPK3 and MLKL expression patterns in various mouse tissue. 13
2. Non-canonical role of receptor-interacting protein kinase-3 (RIPK3) is related to osteoarthritis (OA) pathogenesis. 16
3. OA-signature genes were upregulated in RIPK3-overexpressing chondrocytes. 19
4. RIPK3 modulates OA pathogenesis 22
5. RIPK3 plays a key role in OA pathogenesis. 25
6. RIPK3 upregulation may potentiate OA pathogenesis via non-canonical MLKL-independent functions. 27
7. TRIM24 negatively regulates RIPK3-mediated OA pathogenic signatures 30
8. TRIM24 may negatively regulate elevated RIPK3 expression to accelerate OA pathogenesis 33
9. Identification of drugs that correlate with RIPK3 expression using CMap 36
10. RIPK3 kinase inhibition abrogates Ripk3 activity in OA pathogenesis 40
11. RIPK3 kinase activity inhibition by AZ-628 attenuated cartilage pathophysiology 43
IV. Discussion 45
PART Ⅱ 48
Regulation of ROS-mediated RIPK3 activation under glucose deprivation 48
I. Introduction 49
II. Materials and Methods 52
1. Cell lines and culture conditions 52
2. Antibodies and chemical reagents 52
3. Plasmid construction, mutagenesis and transfection 53
4. shRNA and reverse transcription-PCR (RT-PCR) 54
5. Purification of recombinant proteins 54
6. Cytotoxicity Assays 55
7. ROS measurement 55
8. Immunoblot analysis 56
9. Immunofluorescence analysis 56
10. Statistical analysis 57
III. Results 58
1. RIPK3 is phosphorylated at ser 227, followed by MLKL activation under glucose derivation condition 58
2. RIPK3 mediates MLKL phosphorylation in RIPK1-independent manner 61
3. AMPK is dispensable for RIPK3 activation under glucose deprivation condition 64
4. Accumulated ROS induce RIPK3 phosphorylation 67
5. ROS leads to RIPK1-independent MLKL phosphorylation 70
6. RIPK3 forms oligomerization through inter-chain disulfide bond 73
7. RIPK3 forms inter-chain disulfide bond dependent oligomerization during ROS induction 76
8. RIPK3 forms oligomerization in RHIM-independent manner 79
9. Activated MLKL accelerates cell death under glucose deprivation condition. 82
IV. Discussion 85
V. Reference 88
국문 요약 107