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Functional characterization of activin A-activin receptor IIB pathway in osteoarthritis

초록/요약

Osteoarthritis (OA) is a degenerative joint disease involving various pathogenic factors. To date, the treatment of OA has been limited to pain relief through drugs, physiotherapy, and surgical intervention. However, methods to completely stop disease progression or to regenerate damaged cartilage are yet to be developed. Here, I studied two aspects concerning the prevention and treatment of OA by identifying the genes, proteins, and natural products that induce cartilage degeneration or regulate cartilage regeneration. In part I, I identified activin receptor IIB (encoded by ACVR2B) as a therapeutic target and a novel receptor that plays a crucial role in the pathogenesis of OA. The pathogenesis of OA is a result of the interaction of various pathogenic cytokines and growth factors with their cognate receptors; however, the actual pathogenic receptors/ligands involved remains unclear. In vitro and in vivo analyses demonstrated that activin receptor IIB inhibition attenuated osteoarthritic cartilage destruction by regulating the expression of matrix metallopeptidase 3 (Mmp3), Mmp13, and cyclooxygenase 2 (Cox2). Furthermore, a soluble form of activin receptor IIB fusion protein (sACVR2B-Fc) protected against osteoarthritic cartilage destruction. Among the activin receptor IIB ligands, activin A significantly regulates catabolic factor expression and cartilage destruction in OA pathogenesis. NOX4 and AP-1 were identified as essential mediators and transcription factors involved in activin receptor IIB -activin A expression, thereby amplifying the expression of catabolic factors. In part II, I demonstrated the role of 3’-sialyllactose as a prophylactic agent in osteoarthritic cartilage destruction. 3’-sialyllactose has specific physiological functions in various tissues; however, its effects on osteoarthritic development remain unknown. In in vitro and in vivo studies, 3’-sialyllactose restored the synthesis of Col2a1 and accumulation of a sulfated proteoglycan, a critical factor for cartilage regeneration in osteoarthritic development. Additionally, in vitro, biochemical and histological analysis demonstrated that 3’-sialyllactose was inhibited by the expression of MMP3, MMP13, and Cox2 induced by IL-1β, IL-6, IL-17, and TNF-α, thereby indicating a role in the attenuation of cartilage degradation. In conclusion, the activin receptor IIB-induced catabolic cascade was shown to play a critical role in the progression of OA, and 3’-sialyllactose protected against osteoarthritic cartilage degradation. These results suggest that activin receptor IIB ligand traps may have therapeutic implications in OA pathogenesis, and 3’-sialyllactose may be considered a naturally-occurring therapeutic compound for the prevention of OA. These results indicate that the soluble form (sACVR2B-Fc) and 3’-sialyllactose may serve as potential targets and could be manipulated to ameliorate the progression of osteoarthritic cartilage degradation.

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목차

II. Introduction 1
1. Osteoarthritis 1
2. Cartilage and osteoarthritis 2
3. Treatment of osteoarthritis 3
4. Activin receptor IIB (ACVR2B) and ligands 5
5. 3'-Sialylllactose 6
6. Aim of this study 8
II. Material and Methods 9
Primary articular mouse chondrocytes culture 9
Human OA cartilage 10
RT-PCR and qRT-PCR 10
Immunoprecipitation analysis and Western blot 12
Experimental OA in mice 13
Histology and immunohistochemistry 14
Collagenase activity and PGE2 assay 15
Detection of ROS 16
In silico analysis, microarray, and Ingenuity Pathway Analysis (IPA) 16
Viability analysis 18
LS-MS experiments 18
Statistical analysis 19
III. Results 20
Part I_Therapeutic target 20
Identification of novel pathogenic receptor in osteoarthritic development 20
Activin receptor IIB increases in human and mouse OA cartilage 26
Inhibited Activin receptor IIB attenuate cartilage destruction in OA development 26
Increased activin A, an Activin receptor IIB ligand, promotes OA pathogenesis 31
Activin A depletion inhibits cartilage damage by inhibiting catabolic factors 36
NOX4 mediated activin A regulates catabolic factor 39
NOX4 is required for activin A induced OA pathogenesis in mice 44
Mechanism underlying activin A-mediated NOX4-induced catabolic factor expression 49
Systemic activin receptor IIB inhibition attenuates cartilage destruction in OA development 52
Part II_ Prophylactic agent 57
3'-sialyllactose increased Col2a1 synthesis in chondrocytes 57
3′-sialyllactose blocks proinflammatory cytokines induced Mmp3, Mmp13 and Cox2 expression 62
Both activation of Sox9 and inhibition of NF-κB are regulated by 3'-sialyllactose in chondrocytes 67
Oral administration of 3'-sialyllactose protects cartilage destruction in the OA model 69
3'-sialyllactose modulates ERK and NF-κB signaling pathways for anabolic and catabolic factor expression 73
IV. Discussion 75
V. Reference 88
국문 초록 104

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