Title Page
Contents
ABSTRACT 8
1. Introduction 10
2. Materials and methods 14
2.1. Reagents 14
2.2. Cells and cell culture 14
2.3. Tumor necrosis factor receptor blockage 14
2.4. Endothelial tube formation assay 14
2.5. Reverse transcription (RT)-PCR 15
2.6. Target gene qPCR screening and STRING analysis 15
2.7. Immunoblot analysis 15
2.8. Expression of siRNA 16
2.9. Generation of human TIE2 promoter-reporter constructs 16
2.10. TIE2 promoter-reporter assay 17
2.11. Electrophoretic mobility shift assay (EMSA) 17
2.12. DNA affinity precipitation assay (DAPA) 18
2.13. Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) 18
2.14. Statistical analysis 19
3. Results 20
3.1. TNFα induces differentiation of bone-marrow-derived mesenchymal stem cells (BM-MSCs) into endothelial cells 20
3.2. TIE2 is one of the central endothelial related genes induced by TNFα stimulation in BM-MSCs 23
3.3. TNFα induces TIE2 expression in BM-MSCs 25
3.4. Knockdown of TIE2 expression using siRNA abrogates TNFα-induced endothelial differentiation of BM-MSCs 27
3.5. TNFα increases TIE2 promoter activity through activating STAT binding sequence on the 5′-regulatory region of the TIE2 gene 31
3.6. STAT3 binds directly to the STAT binding sequence of the TIE2 promoter and induces transcription 37
3.7. STAT3 inhibition or knockdown abrogates TNFα-induced TIE2 expression 39
3.8. Knockdown of STAT3 expression abrogates TNFα-induced endothelial tube formation 42
3.9. TNFα induces TIE2 expression through p38 MAPK-activated JAK/STAT signaling pathway 45
3.10. Inhibition of p38 MAPK abrogates TNFα-induced endothelial differentiation of BM-MSCs 51
Discussion 53
Conclusion 55
References 57
Abstract (in Korean) 61
Fig. 1. TNFα induces capillary-like tube formation of BM-MSCs. 21
Fig. 2. TNFα upregulates mRNA expression of endothelial marker genes. 22
Fig. 3. qPCR screening and protein-protein interaction network analysis reveal TIE2 as one of the hub proteins related to angiogenesis regulated by TNFα... 24
Fig. 4. TNFα induces TIE2 expression in BM-MSCs. 26
Fig. 5. Knockdown of TIE2 expression using siRNA abrogates TNFα-induced TIE2 expression. 28
Fig. 6. Knockdown of TIE2 expression using siRNA abrogates TNFα-induced capillary-like tube formation of BM-MSCs. 29
Fig. 7. TNFα increases TIE2 promoter activity. 33
Fig. 8. STAT binds to the STAT binding sequence of the TIE2 promoter. 34
Fig. 9. STAT3 regulates the TNFα-induced increase of TIE2 promoter-reporter activity. 36
Fig. 10. STAT3 binds to SBS of the TIE2 promoter at the cellular level. 38
Fig. 11. STAT3 inhibitor abrogates TNFα-induced TIE2 expression. 40
Fig. 12. STAT3 knockdown abrogates TNFα-induced TIE2 expression. 41
Fig. 13. Knockdown of STAT3 expression using siRNA abrogates TNFα-induced capillary-like tube formation of BM-MSCs. 43
Fig. 14. TNFα activates MAPK proteins. 46
Fig. 15. TNFα activates JAK1, STAT1 and STAT3. 47
Fig. 16. Inhibition of p38 MAPK signaling pathway abrogates TNFα-induced JAK1, STAT1 and STAT3 protein activation. 48
Fig. 17. Inhibition of p38 MAPK signaling pathway abrogates TNFα-induced TIE2 expression. 49
Fig. 18. Neutralization of TNFR using anti-TNFR antibody abrogates TNFα- induced TIE2 expression. 50
Fig. 19. Inhibition of p38 MAPK signaling pathway abrogates TNFα-induced capillary-like tube formation of BM-MSCs. 52
Fig. 20. Summary of this study. 56