Background/Purpose:   Due to exogenous stresses chondrocytes mitochondrial dysfunction is known to occur in osteoarthritis (OA) and amplifies the inflammatory cytokine induced cartilage degradation. Autophagy is an evolutionarily conserved mechanism that plays a central role in mitochondrial quality control. Here we investigated (a) whether IL-1β induce mitochondrial damage; and (b) whether the damaged mitochondria are cleared by autophagy in human OA chondrocytes under pathological conditions.

Methods:  Primary human OA chondrocytes were isolated by enzymatic digestion of the undamaged cartilage obtained from OA patients who underwent total knee arthroplasty at Crystal Clinic, Akron, Ohio. Chondrocytes were treated with IL-1β (5ng/ml) followed by total RNA isolation for qPCR or lysate preparation for Western blotting. Mitochondrial damage and dysfunction was determined by JC-1 staining of chondrocytes followed by flow cytometry. Mitochondrial ROS levels as an indicator of mitochondrial damage and dysfunction were determined by MitoSOX red dye assay. Rapamycin was used to activate autophagy in OA chondrocytes and autophagic flux was confirmed by immunoblotting and immunofluorescence staining of LC3 protein. siRNA mediated knockdown of ATG5 or small inhibitors 3MA, Bafilomycin A1, chloroquine and ammonium chloride were used to inhibit autophagy in OA chondrocytes. Targeting of mitochondria by mitophagy was assessed by colocalization of mitochondria with parkin or p62 or LC3 protein or lysosomes. siRNA mediated knockdown of PARK2 was used to block mitophagy. Activation of Parkin and ubiquitination of Mfn1 and Mfn2 was assessed by immunoblotting.

Results:  OA chondrocytes treated with IL-1β showed increased expression of COX-2, iNOS, IL6 and MMPs and decreased expression of COL2A1 and aggrecan. IL-1β treated OA chondrocytes also had decreased mitochondrial potential and several fold higher mitochondrial ROS production compared to controls and concomitant enhanced autophagic flux evidenced by increased LC3-II formation. Immunofluorescence staining of endogenous LC3 as well as of transiently overexpressed LC3-GFP protein also demonstrated increased autophagic flux in OA chondrocytes with damaged mitochondria. Autophagy inhibition augmented the IL-1β induced mitochondrial dysfunction, ROS production and OA like gene expression and apoptosis in OA chondrocytes while autophagy activation by rapamycin blocked the mitochondrial dysfunction and OA like gene expression. Immunofluorescence staining of mitochondria (using Mitotracker red) and of parkin or p62 protein or LC3 protein and lysosomes showed increased colocalization in IL-1β treated OA chondrocytes indicating active mitophagy. siRNA mediated knockdown of PARK2 blocked mitophagy and clearance of mitofusins and damaged mitochondria resulting in enhanced mitochondrial dysfunction and OA like gene expression in IL-1b-stimulated OA chondrocytes.

Conclusion:  Our data directly demonstrate a link between the diminished autophagy and sustenance of mitochondrial dysfunction in OA and highlight the importance of the development of strategies to activate autophagy for better management of OA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/autophagic-clearance-of-dysfunctional-mitochondria-requires-parkin-in-human-chondrocytes/