Analyses of induction of autophagic response related to the chondrocyte cell-death in osteroarthritis.

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Project I. Analyses of relationship between autophagy and osteroarthritis induced by intra-articular injection of MIA and ACLT in rats : Ultrastructure of a chondrocyte after MIA injection indicated appearance of vacuoles at 12 hours and the loss of organelles and presence of abundant vacuoles at 2 weeks (2w). Western blotting (WB) analyses demonstrated a significant increase in LC3 II form at 12 hours after MIA injection and an increase in Atg5-Atg12 form from 2w in cartilage after MIA injection. On the other hand, an increase of LC3 II form and Atg5-Atg12 form in cartilage extracts of the osteoarthritis induced by ACLT were little.Project II. Analyses of autophagy in chondrocytes of the rat epiphyseal growth plate : Ultrastructure of a chondrocyte in the growth plate showed presence of autophagic vacuoles, especially at 4 weeks-ages. WB analyses demonstrated a significant increase in LC3 II form at growth spurt (2w) and an increase in Atg5-Atg12 form at the development of secondary ossification center, respectively (6w, 2w). The expressions of autophagy-related genes showed significant increase in growth plate at 2w.Project III. Analyses of autophagy and ER-stress in chondrocytes of the human osteoarthritis : Ultrastructure of a chondrocyte in the Grade-3 showed presence of abundant vacuoles. WB analyses demonstrated a significant increase in LC3 II form in Grade-3. The expression levels of LC3, ATG5 and ATG7, was higher in Grade-3 cartilages of the osteoarthritis than in those of the controls. The expression of CHOP and processed-XBPI was higher in Grade-3. After immunohistochemical staining with anti-Ub antibody, the chondrocytes in the osteoarthritis were strongly stained. These results indicate that the degenerated chondrocytes demonstrate induction of autophagy and the endoplasmic reticulum stress. On the basis of our results, we propose that induction of autophagic response in chondrocytes has relevance to degeneration of cartilage tissues and growth plate.