The Roles of CCL19 and CCL21 Chemokines in Osteoclast Migration and Bone Resorption
파골세포 이동과 골흡수에 대한 CCL19 및 CCL21 케모카인의 역할

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dc.description학위논문 (박사)-- 서울대학교 대학원 : 치의과학과, 2017. 2. 김홍희.-
dc.description.abstractOBJECTIVE: Bone resorption is a severe problem in inflammatory diseases such as periodontitis and rheumatoid arthritis (RA). Osteoclasts are responsible for bone resorption and CCL19 and CCL21 act as chemokines for several cell types. The purpose of this study was to investigate the role of CCL19 and CCL21 in bone resorption by osteoclasts.

METHODS: The levels of CCL19, CCL21 and CCR7 in RA and osteoarthritis patient samples were measured using ELISA. Data deposited to gene expression omnibus was also analyzed. The expression levels of these molecules and differentiation markers of osteoclasts were measured by real time polymerase chain reaction (PCR), western blotting or flow cytometry. Osteoclast differentiation was evaluated by tartate-resistant acid phosphatase staining and osteoclast migration was assessed with transwell assay and Oris migration assay kits. Resorption activity was performed with calcium phosphate-coated dishes or dentin slices, which were analyzed by von Kossa staining or confocal microscopy, respectively. Involvement of CCR7 was demonstrated with the small interference RNA system and contribution of GTPase Rho was investigated by the Rho pull-down assay. Collagen transplantation model was used to examine the in vivo effects of these chemokines.

RESULTS: The expression levels of CCL19, CCL21 and CCR7 were higher in RA patient samples compared to OA or normal samples. Bone marrow macrophages and osteoclasts expressed more CCR7 in response to the stimulation of TNF, IL-1 and LPS. CCL19 and CCL21 promoted the migration and resorption activity of both bone marrow macrophages and osteoclasts. Knock-down of CCR7 significantly reduced the CCL19- and CCL21-induced migration. Moreover, CCL19 and CCL21 stimulated small GTPase Rho and its downstream molecule, ROCK. Rho inhibitors suppressed both the migration and resorption activity of BMMs and osteoclasts. It demonstrates that the increase of migration and bone resorption by CCL19 and CCL21 was mediated by the CCR7/Rho axis. Collagen transplantation study showed that CCL19 and CCL21 can promote bone resorption in vivo.

CONCLUSION: This study indicates that under inflammatory conditions, osteoclast precursors increase the CCR7 expression. CCL19 and CCL21 are up-regulated in RA patients and enhance the osteoclast migration and resorption activity, resulting in severe bone destruction. This study suggests that neutralizing antibody for CCL19 and CCL21 or CCR7 knock-down are potential therapeutic strategies for periodontitis and RA.
dc.description.tableofcontentsI. Introduction 1
1. Roles of osteoblasts and osteoclasts in bone remodeling 1
2. Rheumatoid arithritis and osteoclasts 5
3. Osteoclast migration and bone resorption 8
4. Roles of CCL19/CCL21 and CCR7 in rheumatoid arithritis 13
5. Purpose of this study 17

II. Materials and Methods 19
1. Reagents 19
2. Bone marrow-derived macrophage (BMM) generation 19
3. Osteoclast differentiation 20
4. Enzyme-linked immunosorbent assay (ELISA) 20
5. Quantification of mRNA 21
6. Western blotting analysis 21
7. TRAP staining 22
8. Cytotoxicity assay 22
9. Flow cytometry 23
10. Small interference RNA transfection 23
11. Cell migration assay 24
12. Rho pull-down assay 25
13. von Kossa staining 25
14. Dentin resorption and confocal microscopy 25
15. Confocal fluorescence microscopy 26
16. In vivo mouse calvariae resorption assay 26
17. Statistical analysis 27
18. Institutional Review Board (IRB) 27

III. Results 28
1. Increased expression of CCR7 increased in synovial macrophages of RA patients 28
2. Expression levels of CCL19, CCL21 and CCR7 increased in synovial tissues of RA patients 31
3. Increased mRNA expression level of CCR7 in response to inflammatory stimuli 34
4. Increased protein expression level of CCR7 in response to inflammatory stimuli 37
5. Effects of CCL19 and CCL21 on osteoclast differentiation 40
6. Regulation of osteoclast migration activity by CCL19 and CCL21 44
7. CCL19 and CCL21 increase osteoclast migration through CCR7 47
8. CCL19 and CCL21 increase osteoclast resorption activity 49
9. CCL19 and CCL21 increase osteoclast actin ring density 53
10. CCL19 and CCL21 stimulate osteoclasts via Rho 55
11. Rho inhibitors suppress CCL19- and CCL21-induced osteoclast migration and resorption activity 58
12. CCL19 and CCL21 increase mouse calvariae resorption 61
13. LPS accentuates the CCL19-induced calvariae resorption 64

IV. Discussion 66
V. References 75
국문초록 89
dc.format.extent3352627 bytes-
dc.publisher서울대학교 대학원-
dc.subjectRheumatoid arthritis-
dc.titleThe Roles of CCL19 and CCL21 Chemokines in Osteoclast Migration and Bone Resorption-
dc.title.alternative파골세포 이동과 골흡수에 대한 CCL19 및 CCL21 케모카인의 역할-
dc.contributor.AlternativeAuthorJiyeon Lee-
dc.contributor.affiliation치의학대학원 치의과학과-
Appears in Collections:
College of Dentistry/School of Dentistry (치과대학/치의학대학원)Dept. of Dental Science(치의과학과)Theses (Ph.D. / Sc.D._치의과학과)
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