Title Page
ABSTRACT
Contents
PART I : XIAP is essential for shear stress-enhanced Tyr-576 phosphorylation of FAK 14
1. ABSTRACT 15
2. INTRODUCTION 16
3. MATERIALS AND METHODS 18
1) Cell culture 18
2) Transfection of BAECs with xiap siRNA 18
3) Drug treatment 18
4) Shear stress studies 18
5) Preparation of cell lysates 19
6) Western blotting 19
7) Cytotoxicity assay 19
8) Immunofluorescence 19
4. RESULTS 21
1) Shear stress stimulates Tyr-576 phosphorylation of FAK 21
2) XIAP knockdown inhibits shear stress-enhanced Tyr-576 23
3) Phosphorylation of Tyr-576 mediates shear stress-dependent ERK activation 25
4) XIAP is essential to maintain cytosolic FAK and allow interaction with Src kinase 27
5. DISCUSSION 29
6. REFERENCES 32
PART II : XIAP reverses various functional activities of FRNK in endothelial cells 35
1. ABSTRACT 36
2. INTRODUCTION 37
3. MATERIALS AND METHODS 39
1) Cell culture 39
2) Cloning of FAK and construction of deletion mutant genes 39
3) Transfection of BAECs with fak mutant genes 39
4) Preparation of cell lysates 39
5) Western blotting 40
6) Cell adhesion assay 40
7) Immunofluorescence 40
8) Immunoprecipitation 41
9) Co-transfection of BAECs with fak mutant genes and xiap siRNA 41
10) Shear stress studies 41
11) Cell migration 42
4. RESULTS 43
1) FRNK recruits into FAs and enhances cell adhesion 43
2) FRNK binds with XIAP 45
3) XIAP knockdown inhibits FRNK recruitment into FAs and FRNK-enhanced cell adhesion 47
4) XIAP depletion reverses an inhibitory effect of FRNK on shear stress-induced ERK activation 49
5) XIAP depletion reverses FRNK-inhibited endothelial cell migration 49
5. DISCUSSION 51
6. REFERENCES 55
PART III : Cytokeratin 1 controls vascular functions by anti-apoptosis, stabilization of cell adhesion and promotion of shear-sensing 58
1. ABSTRACT 59
2. INTRODUCTION 60
3. MATERIALS AND METHODS 63
1) Cell culture 63
2) KRS expression and purification 63
3) In vitro KRS binding assay 63
4) Immunofluorescence 64
5) Transfection of plasmid 64
6) Transfection of siRNA 65
7) Proliferation 65
8) Measurement of apoptosis 65
9) Preparation of cell lysates 66
10) Western blotting 66
11) Endothelial cell Adhesion 66
12) Immunoprecipitation 67
13) CKI expression and purification 67
14) In vitro CK1 binding assay 68
15) Shear stress studies 68
15) Adhesion of THP-1 68
4. RESULTS 69
1) CK1 is an essential surface molecule interacting with a pro-atherogenic factor, KRS. 69
2) Overexpression of CK1 has an anti-apoptotic effect in endothelial cells via PI₃K/Akt pathway 71
3) CK1 knockdown plays crucial roles in endothelial cell apoptosis 75
4) CK1 is an important factor for maintenance of normal cell shape 78
5) CK1 is involved in the interaction between actin and integrins 81
6) CK1 enhances endothelial cell adhesion to laminin via its head/central domain 83
7) CK1 knock-down cells maintain adhesion to laminin by forming actin fiber 84
8) CK1 interacts with laminin through integrin α6 87
9) CK1 plays a critical role in shear stress response of endothelial cells via its head/central domain of CK1 89
10) CK1 prevents monocytes binding to endothelial cells via NO production. 93
5. DISCUSSION 96
6. REFERENCES 102
국문초록 106
PART I : XIAP is essential for shear stress-enhanced Tyr-576 phosphorylation of FAK 12
Figure 1. Shear stress enhances phosphorylation of Tyr-576 of FAK. 22
Figure 2. XIAP knockdown blocks shear-enhanced phosphorylation of Tyr-576 of FAK. 24
Figure 3. DCA inhibits shear-enhanced phosphorylation of Tyr-576 of FAK. 26
Figure 4. XIAP is essential for the interaction between FAK and Src kinase. 28
Figure 5. Schematic diagram showing a role of XIAP in Tyr-576 phosphorylation of FAK. 31
PART II : XIAP reverses various functional activities of FRNK in endothelial cells 12
Figure 1. FRNK in FA increases endothelial cell adhesion. 44
Figure 2. XIAP interacts with FAK via FRNK domain. 46
Figure 3. XIAP knockdown blocks the FRNK-enhanced adhesion. 48
Figure 4. XIAP depletion reverses inhibitory effects of FRNK. 50
Figure 5. Schematic diagram for roles of FRNK in shear stress-stimulated ERK activation and endothelial cell migration. 54
PART III : Cytokeratin 1 controls vascular functions by anti-apoptosis, stabilization of cell adhesion and promotion of shear-sensing 12
Figure 1. KRS interacts with CK1 at endothelial cell surface. 70
Figure 2. CK1 overexpression inhibits TNFα- or resveratrol-induced apoptosis through Akt/PI₃K pathway. 74
Figure 3. CK1 knockdown decreases cell proliferation and increases TNFα- or resveratrol-induced apoptosis. 77
Figure 4. CK1 knockdown stimulates the formation of actin fiber, making endothelial cells sensitive to apoptosis. 80
Figure 5. Actin fibers formed by CK1 knockdown are colocalized with integrin α6. 82
Figure 6. CK1 plays a key role in cell adhesion to laminin-coated plate. 86
Figure 7. CK1 interacts with laminin through integrin α6. 88
Figure 8. CK1 is a possible shear-sensing receptor. 92
Figure 9. CK1 is expressed at low level in atherogenic patients, suggesting its preventive benefits against the development of atherosclerosis. 95
Figure 10. A schematic diagram indicating roles of CK1 in cell survival, cell adhesion, and shear stress response. 101