K. Homan (Sapporo, JP)
Hokkaido University Orthopaedic SurgeryPresenter Of 1 Presentation
10.4.8 - Identification of M9 high-mannose glycan regulating hypertrophy in articular cartilage
Abstract
Purpose
Ectopic initiation of chondrocyte hypertrophy in articular chondrocytes involves cartilage degeneration and osteoarthritis (OA) progression. However, the mechanism by which chondrocytes lead to hypertrophic differentiation has not been clarified. Glycans modulate many biological processes for cell differentiation, hence, we conducted a comprehensive analysis of the glycophenotype during the chondrocyte hypertrophy process.
Methods and Materials
The hypertrophic changes of primary mouse chondrocytes were induced by adding insulin (10 ug / mL). The alterations of glycans at day 0, 10 and 20 were comprehensively investigated by glycoblotting, total glycome, and cluster analysis. Based on the results of the total glycome and cluster analysis, we decided to target glycan on the list of upregulated glycan structures. We added glycosidase enzyme that cleaves a structure of target glycan to primary chondrocytes and cartilage explants ex vivo and evaluated them by qPCR, dye-binding assay of proteoglycan, and histological staining.
Results
A total of 151 glycan structures were significantly changed by insulin stimulation. All glycans were divided into four groups by cluster analysis. High-mannose type N-glycans (M5, 6, 7, 8, 9) were located at the top of the expression level by hierarchical cluster analysis (Fig. 1). Unlike other high-mannose type N-glycans, only the amount of M9 decreased with hypertrophy. Artificial enzymatic cleavage of M9 (α-mannosidase) in chondrocytes increased the mRNA expression of Adamts5, Col10a1, and Indian hedgehog. Moreover, enzymatic cleavage of M9 for the femoral head cartilage increased the numbers of type X collagen-positive cells in the surface layer and the release of proteoglycans (Fig. 2).
Conclusion
Our results presented the dynamic alteration of glycans in the process of chondrocyte hypertrophy. Enzymatic cleavage of M9 whose expression was reduced in the chondrocyte glycome induced hypertrophy of chondrocytes, suggesting that the alterations of high-mannose type N-glycans would regulate chondrocyte hypertrophy.