Regulation of RANKL-induced Signaling in Osteoclast Differentiation

다국어 초록 (Multilingual Abstract)

Bone homeostasis is regulated by balanced actions of bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoclasts are derived from monocyte/macrophage lineage precursors in bone marrow through a process of cellular differentiation that is induced by receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) signaling. Osteoclasts are multinucleated giant cells that have unique morphological characteristics allowing them to resorb bone matrix. Accelerated bone destruction by osteoclasts might cause several metabolic bone-related diseases, such as osteoporosis and inflammatory bone loss. Therefore, the regulation of osteoclast differentiation and activation is the main therapeutic target for bone diseases. Here, I report the regulation of RANKL-induced signaling in osteoclast differentiation by a novel negative-feedback regulator or a chemical inhibitor.
In the present study, WD40-repeat-containing protein 23 (WDR23) was identified to be a novel negative-feedback regulator of RANKL-induced osteoclastogenesis. Tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) is an early adaptor protein essential for RANKL signaling. TRAF6-deficient mice exhibit a severe osteopetrotic phenotype and are defective in osteoclast formation. Because of the essential role of TRAF6 in osteoclastogenesis, TRAF6 can be the target of negative regulator to prevent excessive osteoclast differentiation. In this study, WDR23 was identified as a TRAF6-binding partner. The protein stability of TRAF6 was reduced by WDR23 through autophagy-mediated degradation system. In particular, WDR23 played the negative role in TRAF6-mediated RANKL signaling for osteoclast differentiation. WDR23 expression was induced by RANKL during osteoclastogenesis. The overexpression of WDR23 in bone marrow-derived macrophages (BMMs) inhibited the osteoclast formation. Next, it was showed that the reduction of TRAF6 protein stability causes the inhibition of RANKL-induced signal activation in WDR23-overexpressing BMMs. Consequently, the induction of NFATc1 was down-regulated in osteoclasts derived from WDR23-overexpressing BMMs, leading to inhibition of the gene expression for osteoclast specific markers. Moreover, the effects of WDR23 on TRAF6-mediated RANKL signaling was confirmed using WDR23 knockdown BMMs. Thus, these findings suggest that WDR23 is a critical for the regulation of osteoclastogenesis by modulating TRAF6 and might have an important role for proper bone remodeling.
Secondly, it was showed that D-chiro-inositol negatively regulates RANKL-induced osteoclastogenesis by down-regulating NFATc1. D-Pinitol (3-O-methyl-D-chiro-inositol) is a prominent component of dietary legumes and is actively converted to D-chiro-inositol, which is a putative insulin-like mediator. In addition, D-pinitol/D-chiro-inositol has recently shown the potential as an anti-inflammatory agent by inhibiting NF-κB signaling or proinflammatory cytokine expression. However, the cellular and molecular targets of D-pinitol/D-chiro-inositol remain poorly understood. In this study, the effect of D-chiro-inositol on osteoclast differentiation was analyzed. It was demonstrated that D-chiro-inositol acts as an inhibitor of RANKL-induced osteoclastogenesis. The formation of multinucleated osteoclasts by cell–cell fusion was reduced by the treatment with D-chiro-inositol in a dose-dependent manner. The activation of NF-κB, p38 and JNK pathways downstream of RANKL signaling was inhibited by the treatment with D-chiro-inositol. Finally, it was demonstrated that D-chiro-inositol inhibits the expression of several osteoclastogenic genes by down-regulating NFATc1. Hence, these results show that D-chiro-inositol might be a good candidate to treat inflammatory bone-related diseases or secondary osteoporosis in diabetes mellitus.
Taken all together, these two studies provide a new insight into the regulation of RANKL-induced signaling in osteoclast differentiation and show a potential as therapeutic candidates for bone-related diseases.

목차 (Table of Contents)

• GENERAL INFORMATION 1
• 1. RESEARCH BACKGROUND 3
• 1-1. The osteoimmune system 3
• 1-2. Molecular understanding of osteoclastogenesis 6
• 1-3. Negative regulation of osteoclastogenesis 11
• 2. REFERENCES 14
• CHAPTER I. WDR23 is a Novel Negative-Feedback Regulator of RANKL-induced Osteoclastogenesis 17
• 1. INTRODUCTION 19
• 2. MATERIALS AND METHODS 25
• 2-1. Reagents and mice 25
• 2-2. Plasmids 25
• 2-3. Cell culture 26
• 2-4. Protein screening and analysis 27
• 2-5. In vitro binding assay and immunoblotting 27
• 2-6. Subcellular localization 28
• 2-7. Luciferase reporter assay 28
• 2-8. Retrovirus preparation and osteoclast formation 28
• 2-9. Analysis of osteoclasts 29
• 2-10. Observation of RANKL-induced signaling pathway 30
• 2-11. Semi-quantitative RT-PCR and real-time PCR analysis 30
• 2-12. Statistical analysis 30
• 3. RESULTS 33
• 3-1. WDR23 binds to TRAF6 through the first and second WD40 repeats in the cytoplasm 33
• 3-2. WDR23 inhibits TRAF6-induced NF-κB activation by reducing protein stability of TRAF6 38
• 3-3. WDR23 reduces the protein stability of TRAF6 through autophagy-dependent degradation system 42
• 3-4. WDR23 expression is induced by RANKL during osteoclastogenesis 47
• 3-5. WDR23 negatively regulates RANKL-induced osteoclastogenesis by reducing protein stability of TRAF6 49
• 4. DISCUSSION 57
• 5. REFERENCES 60
• CHAPTER II. D-Chiro-inositol Negatively Regulates the Formation of Multinucleated Osteoclasts by Down-Regulating NFATc1 65
• 1. INTRODUCTION 67
• 2. MATERIALS AND METHODS 71
• 2-1. Reagents and mice 71
• 2-2. Cell culture, osteoclastogenesis, and cytotoxicity assay 71
• 2-3. Osteoblast differentiation and activation 72
• 2-4. Analysis of OCs 72
• 2-5. Cell fusion assay 73
• 2-6. Pit formation and OC survival assays 73
• 2-7. Retrovirus preparation and OC formation 74
• 2-8. Luciferase reporter assay and immunoblotting 74
• 2-9. Statistical analysis 75
• 3. RESULTS 77
• 3-1. D-Chiro-inositol inhibits OC differentiation 77
• 3-2. D-Chiro-inositol had no effect on cell proliferation and bone forming activity of OBs 83
• 3-3. D-Chiro-inositol inhibits the early stage of OC differentiation 87
• 3-4. D-Chiro-inositol inhibits the fusion of OC precursors 91
• 3-5. D-Chiro-inositol inhibits gene expression of OC markers through NFATc1 down-regulation 95
• 4. DISCUSSION 103
• 5. REFERENCES 107
• Summary (in Korea) 113
• Acknowledgements 117