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dc.contributor.advisor李秀榮-
dc.contributor.author이나경-
dc.creator이나경-
dc.date.accessioned2016-08-26T03:08:20Z-
dc.date.available2016-08-26T03:08:20Z-
dc.date.issued2004-
dc.identifier.otherOAK-000000009676-
dc.identifier.urihttps://dspace.ewha.ac.kr/handle/2015.oak/205433-
dc.identifier.urihttp://dcollection.ewha.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000009676-
dc.description.abstractSignaling by receptor activator of NF-kB ligand (RANKL) is essential for differentiation of bone marrow monocytes/macrophage lineage cells (BMMs) into osteoclasts. Although RANK, TRAF6, and NF-kB are critical molecules for osteoclast differentiaton, the mechanisms by which RANKL regulates osteoclastogenesis are unclear. In this thesis, I have examined the roles of reactive oxygen species (ROS), Rac1 GTPase, and schlafen2 during RANKL-induced osteoclast differentiation. RANKL stimulation of BMMs transiently increases the intracellular level of ROS through a signaling cascade involving TNF receptor-associated factor (TRAF) 6 and an NADPH oxidase1 / Nox1. A deficiency in TRAF6 or expression of a dominant interfering mutant of TRAF6 blocks RANKL-mediated ROS production. Not only an application of N-acetylcysteine (NAC), the introduction of catalase and PrxⅡ but also blocking the activity of an NADPH oxidase with diphenylene iodonium (DPI) and apocynin inhibit RANKL responses of BMMs, including ROS production, activation of c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK), and osteoclast differentiation. Furthermore, experiments using siRNA show that Nox1, among homologous of NADPH oxidase (Nox2) is a critical mediator of the RANKL-mediated ROS generation and the activation of JNK and p38 MAP kinases, thus regulating osteoclast differentiation. These results indicate that RANKL-mediated ROS production roles a critical mediator of osteoclasts differentiation. RANKL selectively activates Rac1, but not Rac2 in osteoclast precursors. Expression of a dominant interfering mutant of TNF receptor-associated factor (TRAF) 6 blocks RANKL-mediated Rac1 activation, indicating that Rac1 lies downstream of TRAF6. Osteoclast precursors expressing a dominant-negative Rac1N17 are defective in RANKL-induced IKK activation and IkBa degradation resulting in inhibition of NF-kB-dependent reporter gene activity and NF-kB DNA binding. Rac1 acts upstream of TAK1 to induce NF-kB activation and is required for the normal differentiation of osteoclast precursors, indicating that Rac1 may represent a key regulator for differentiation of osteoclasts functioning downstream of TRAF6. I examined proteins that are upregulated by RANKL during osteoclasts differentiation using a proteomic tool and found that schlafen2 (slfn2) was induced by RANKL in BMMs. Slfn2 mainly exist in cytoplasm and is expressed in a Rac1-dependent manner. Mutagenic analysis shows that slfn2 has a functional NES in N-terminus and can export nucleus via CRM-1 pathway. The overexpression of slfn2 increases the expression of c-Jun, thus enhances its activation when treated with RANKL. Slfn2 siRNA inhibits the expression of NFATc1, as well as c-Jun, thus suppressing osteoclasts differentiation. These results show that slfn2 regulates osteoclasts differentiation by increasing the activation of c-Jun that regulates the expression of NFATc1. Taken together, these findings indicate that RANKL signaling involves TRAF6→Rac→NADPH oxidase (Nox1)→ROS signaling cascades in the regulation of osteoclastogenesis and RANKL-induced slfn2 is a critical mediator of osteoclasts differentiation.;RANK ligand (RANKL) 에 의한 신호전달은 골수에서 유래한 단핵구/포식세포로부터의 파골세포로의 분화에 필수적이다. RANK, TRAF6, and NF-kB 가 파골세포 분화에 중요한 단백질임은 잘 밝혀져 있으나, RANKL에 의한 파골세포 분화의 신호전달 기전 및 신호 조절 단백질에 대해서는 명확하지 않다. 본 논문에서는 RANKL에 의한 파골세포 분화시, 활성산소종, Rac1 GTPase와 신규 조절 단백질인 schlafen의 역할을 조사하였다. 파골전구세포에 RANKL 처리는 TRAF6와 NADPH oxidase (Nox1) 을 경유하는 신호전달 경로를 통해 일시적으로 세포내의 활성산소종을 증가시킨다. TRAF6의 결여나 TRAF6의 dominant mutant form의 발현은 RANKL에 의한 활성산소종의 생성을 막는다. 활성산소종 제거 약물인 NAC의 처리나, catalase와 PrxⅡ의 발현 뿐 아니라 NADPH oxidase의 활성화를 막는 DPI와 apocynin의 처리는 파골 전구세포에서 RANKL에 의해 일어나는 활성산소종의 생성, JNK, p38과 ERK MAP kinases의 활성과 파골세포 분화를 억제한다. 또한 siRNA를 이용한 실험은 NADPH oxidase (Nox1)이 RANKL에 의한 활성산소종의 생성과 JNK, p38 MAP kinases의 활성에 따른 파골세포 분화에 중요한 매개체임을 보여준다. 이러한 결과들은 활성산소종이 파골세포분화에 중요한 역할을 한다는 것을 제시한다. RANKL는 파골 전구세포에서 Rac1을 특이적으로 활성화시킨다. TRAF6의 dominant mutant form의 발현은 RANKL에 의한 Rac1 활성화를 막는다. 이는 Rac1이 TRAF6의 하위기전에 있음을 의미한다. Rac1의 dominant mutant form을 발현하는 파골전구세포는 RANKL에 의한 IKK 활성과 IkB-a의 분해가 일어나지 않아 NF-kB에 의존적인 reporter 유전자 활성과 NF-kB의 DNA로의 결합을 억제한다. Rac1은 NF-kB를 활성화시키는 TAK1의 상위기전에 있으며 파골전구세포로부터의 파골세포로의 분화에 필요하다. 이는 Rac1이 TRAF6의 하위기전에서 기능을 하는, 파골세포의 분화를 조절하는 중요한 매개체임을 보여준다. 파골세포 분화 과정 동안 RANKL에 의해 증가하는 단백질들을 찾기 위해proteomic방법을 수행하였고, 그 결과 schlafen2 (slfn2) 가 RANKL에 의해 유도됨을 확인하였다. Slfn2는 주로 세포질에 존재하며 Rac1에 의존적으로 발현된다. Mutagenic analysis는 slfn2가 주로 N-turminus에 기능적인 nucleus export signal (NES) 를 갖고 있어 CRM-1에 의존적으로 핵을 벗어남을 보여준다. Slfn2의 과발현은 c-Jun의 발현을 증가시킴으로써 RANKL와 함께 c-Jun의 활성화를 증폭시킨다. 또한 slfn2 siRNA는 c-Jun 뿐 아니라 NFATc1의 발현을 방해함으로써 파골세포 분화를 억제한다. 이러한 결과들은 slfn2가 NFATc1의 발현을 조절하는 c-Jun의 활성화를 증가시킴으로써 파골세포의 분화를 조절함을 제시한다. 종합적으로, 이러한 결과들은 RANKL에 의한 파골세포분화는 TRAF6→Rac1→NADPH oxidase (Nox1)→ROS 신호전달체계와 관련이 있으며, RANKL에 의해 유도되는 slfn2가 파골세포분화의 중요한 매개체임을 제시한다.-
dc.description.tableofcontentsContents Abstract = ⅰ Contents = ⅲ List of Figures = ⅶ Chapter 1 = 1 INTRODUCTION = 1 1-1. Balancing bone metabolism = 1 1-2. RANKL, OPG, and RANK = 1 1-3. Osteoclastogenesis = 3 1-4. Signaling by the RANK-RANKL = 5 1-5. Two ITAM-mediated costimulatory pathways = 7 REFERENCES = 10 Chapter 2 Crucial roles for reactive oxygen species and Rac1 GTPase in RANKL-induced osteoclast differentiation = 21 1. INTRODUCTION = 21 2. MATERIALS and METHODS = 25 2-1. Reagents and plasmids = 25 2-2. Isolation and culture of bone marrow precursors = 26 2-3. Assay of intracellular ROS = 26 2-4. Cell stimulation, transfection, and analysis = 26 2-5. Construction of 293 cells expressing RANK = 27 2-6. Adenoviral infection = 27 2-7. RT-PCR, siRNA preparation, and transfection = 28 2-8. Retrovirus preparation = 29 2-9. Osteoclast differentiation = 29 2-10. Rac activity assay = 30 2-11. Immune complex kinase assay of the endogenous IKK complex = 30 2-12. Luciferase reporter assay = 31 2-13. Electrophoretic mobility shift assay (EMSA) = 31 3. RESULTS = 32 3-1. RANKL generates ROS in BMMs = 32 3-2. Requirement of TRAF6 in RANKL-induced ROS production = 32 3-3. In vitro NAC blocks RANKL-induced ROS production, MAP kinases activation, and osteoclastogenesis = 35 3-4. Blockade of osteoclastogenesis by ectopic expression of catalase and peroxiredoxinⅡ(PrxⅡ) = 38 3-5. NADPH oxidase inhibitors block RANKL-induced ROS production, MAP kinases activation, and osteoclastogenesis = 38 3-6. Blockade of RANKL-induced ROS production, MAP kinases activation, and osteoclastogenesis by NADPH oxidase1 (Nox1) siRNA = 43 3-7. RANKL stimulation of BMMs activates Rac1 via TRAF6 = 48 3-8. Rac1 regulates RANKL-mediated NF-kB activation = 49 3-9. Rac1 regulates NF-kB transactivation through TAK1 = 54 3-10. Rac1 regulates osteoclasts differentiation = 54 4. DISCUSSION = 59 REFERENCES = 63 Chapter 3 RANKL-induced slfn2 is essential for osteoclast differentiation = 70 1. INTRODUCTION = 70 2. MATERIALS and METHODS = 72 2-1. Reagents and plasmids = 72 2-2. Isolation and culture of bone marrow monocyte/macrophates = 72 2-3. Two-dimensional gel electrophoresis and mass spectrometric analysis = 72 2-4. Northern blot and reverse transcription polymerase chain reaction (RT-PCR) analysis = 73 2-5. Cell culture, transfection, and Western blot analysis = 74 2-6. Mutagenesis, immunostain, and fluorescence microscopy = 74 2-7. RNA interference, retroviral gene transfer, and in vitro osteoclastogenesis = 75 2-8. Luciferase assay = 76 3. RESULTS = 77 3-1. Identification of slfn2 selectively induced by RANKL during osteoclast differentiation = 77 3-2. Expression of slfn2 by RANKL during osteoclast differentiaton = 77 3-3. Intracellular distribution of slfn2 = 79 3-4. Rac1-dependent expression of slfn2 = 82 3-5. Requirement of slfn2 in c-Jun expression and activation = 86 3-6. Suppression of c-Jun and NFATcl by slfn2 siRNA in BMMs = 89 3-7. Effects of sln2 siRNA on soteoclastogenesis = 89 4. DISCUSSION = 95 REFERENCES = 97 Chapter 4 = 103 DISCUSSION = 103 REFERENCES = 114 논문 개요 = 120 Appendix = 122-
dc.formatapplication/pdf-
dc.format.extent5364392 bytes-
dc.languageeng-
dc.publisher이화여자대학교 대학원-
dc.titleSignaling Mechanisms in The Regulation of Osteoclastogenesis-
dc.typeDoctoral Thesis-
dc.title.subtitleInvolvements of Reactive Oxygen Species, Rac1 GTPase, and Schlafen2-
dc.format.pageⅶ, 122 p.-
dc.identifier.thesisdegreeDoctor-
dc.identifier.major대학원 분자생명과학부-
dc.date.awarded2005. 2-
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