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The molecular property of Ninjurin1 and its role in bone development : 뼈 발생 과정에서 Ninjurin1 단백질의 기능 및 분자적 특성 연구
DC Field | Value | Language |
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dc.contributor.advisor | 김규원 | - |
dc.contributor.author | Sung-Jin Bae | - |
dc.date.accessioned | 2017-07-13T16:39:42Z | - |
dc.date.available | 2017-07-13T16:39:42Z | - |
dc.date.issued | 2017-02 | - |
dc.identifier.other | 000000140804 | - |
dc.identifier.uri | https://hdl.handle.net/10371/120149 | - |
dc.description | 학위논문 (박사)-- 서울대학교 대학원 : 약학과, 2017. 2. 김규원. | - |
dc.description.abstract | Osteoclasts (OCs) are bone-resorbing cells that originate from hematopoietic stem cells and develop through the fusion of mononuclear myeloid precursors. Dysregulation of OC development causes skeletal disorders such as osteopetrosis, osteoporosis, and rheumatoid arthritis. Although the molecular mechanisms underlying osteoclastogenesis have been well established, the means by which OCs maintain their survival during OC development remain unknown. In the present study, it was found that Ninjurin1 (Ninj1), a cell surface protein, supports OC development by enhancing the survival of prefusion OCs (preOCs). Ninj1 expression was dynamically regulated during osteoclastogenesis. In addition, Ninj1-/- mice exhibit mild osteopetrosis owing to impaired OC development. However, typical markers of OC differentiation such as Nfatc1, c-Fos, integrinβ3, Oscar, and Calcr were unaffected by Ninj1 deficiency. Furthermore, other important events of OC development such as transmigration, fusion, and actin ring formation were also not impaired by the absence of Ninj1. Instead, Ninj1 deficiency increased Caspase-9-dependent intrinsic apoptotic cell death in preOCs. Furthermore, overexpression of Ninj1 enhanced the survival of mouse macrophage/preOC RAW264.7 cells in osteoclastogenic culture, which suggests that Ninj1 is important for the survival of preOCs. Finally, analysis of publicly available microarray datasets revealed a potent correlation between high NINJ1 expression and bone disorders in humans. Regarding to Ninj1 molecular property, it was revealed that Ninj1 assembles into a homomeric protein complex composed of two to six monomeric Ninj1 molecules, and the intracellular region of Ninj1 encompassing Leu101 to Ala110 is important for Ninj1 assembly. Furthermore, Ninj1 is an N-glycosylated protein, and the N-glycosylation enhances homomer formation and regulates protein stability and plasma membrane sorting. These findings suggest that Ninj1 plays an important role in bone homeostasis by enhancing the survival of preOCs and might represent a potent therapeutic target for destructive bone disorders. Moreover, targeting of N-glycosylation in Ninj1 might be a regulatory strategy for modulation of Ninj1 function(s). | - |
dc.description.tableofcontents | INTRODUCTION 1
1. Bone and osteoblast 1 2. Osteoclast 4 3. Ninjurin1 8 4. Homomeric protein complex 11 PURPOSE OF THIS STUDY 12 MATERIALS AND METHODS 14 1. Ethics statement 14 2. Animals 14 3. Radiologic analysis 15 4. Plasmid construction, transfection and retroviral infection 15 5. Reagents 17 6. In vitro osteoclastogenesis 18 7. Cell culture 19 8. TRAP activity staining and measurement of TRAP activity 20 9. FACS analysis 21 10. Confocal microscopy 23 11. Proliferation assay 25 12. Protein cross-linking with formaldehyde 25 13. Immunoblot assay 26 14. RNA isolation and qRT-PCR 26 15. Microarray analysis 27 16. Statistical analysis 28 RESULTS 32 1. OCs express high amount of Ninj1 and deletion of Ninj1 induces bone abnormality in mice 32 2. Ninj1 deficiency induces mild osteopetrosis in mice 35 3. Ninj1 is expressed in OCs, not in OBs 40 4. Ninj1 expression is dynamically regulated during osteoclastogenesis 42 5. Ninj1 deficiency reduces multinucleated OCs 45 6. Ninj1 is dispensable for OC differentiation 50 7. Ninj1 deletion augments development of OPCs 54 8. Ninj1-deficient OC precursor cells develop normally from OPCs. 56 9. Ninj1 is expendable for preOC migration 59 10. Macrophage fusion is enhanced by Ninj1 deficiency 61 11. Ninj1 deletion reduces mature OC area 67 12. Lack of Ninj1 attenuates TRAP activity in cultured media 69 13. Ninj1 is important for maintenance of cell population during preOC stage 72 14. Ninj1 enhances preOC survival during osteoclastogenesis 74 15. Ninj1 deficiency induces Caspase-9-dependent intrinsic apoptosis in preOCs 78 16. A high level of Ninj1 improves preOC survival 83 17. High NINJ1 expression correlates with human bone disorders 86 18. Ninj1 might be a co-stimulatory molecule and/or co-receptor for ITAMmediated signals 89 19. Ninj1 possesses homophilic binding affinity 92 20. Ninj1 is a cis-interacting protein 95 21. Ninj1 assembles into a homomeric protein complex 99 22. Intracellular region of Ninj1 from Leu101 to Ala110 is required for Ninj1 assembly 103 23. Ninj1 is an N-linked glycosylated protein 107 24. N-glycosylation stabilizes the Ninj1 homomeric complex 112 25. N-glycosylation is important for Ninj1 trafficking and stability 116 DISCUSSION 126 REFERENCES 138 요약 (국문초록) 150 | - |
dc.format | application/pdf | - |
dc.format.extent | 5439168 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | Ninjurin1 | - |
dc.subject | OC | - |
dc.subject | apoptosis | - |
dc.subject | osteopetrosis | - |
dc.subject | bone destructive disorder | - |
dc.subject | protein complex | - |
dc.subject | homomer | - |
dc.subject | N-glycosylation | - |
dc.subject.ddc | 615 | - |
dc.title | The molecular property of Ninjurin1 and its role in bone development | - |
dc.title.alternative | 뼈 발생 과정에서 Ninjurin1 단백질의 기능 및 분자적 특성 연구 | - |
dc.type | Thesis | - |
dc.contributor.AlternativeAuthor | 배성진 | - |
dc.description.degree | Doctor | - |
dc.citation.pages | xiii, 152 | - |
dc.contributor.affiliation | 약학대학 약학과 | - |
dc.date.awarded | 2017-02 | - |
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