AB0088 Deficient Autophagy Induces Premature Senescence in Aging and Osteoarthritis

BackgroundOsteoarthritis (OA) is characterized by insufficient extracellular matrix synthesis and articular cartilage degradation. Autophagy is essential to maintain chondrocyte homeostasis by regulating the intracellular macromolecule and organelle turnover (1). Previous findings indicated that autophagy is defective in Aging and OA articular cartilage (1,2), but the specific target/-s that regulates this mechanism and affect cartilage integrity are still unknown.ObjectivesThe objective of study is to identify relevant targets regulating autophagy in Aging and OA by proteomics.MethodsPrimary human chondrocytes from human donors were transfected with siRNA for Atg5 (100 nM, 72 hours), a key autophagy marker, to block the autophagy pathway. To identify the key proteins responding to defective autophagy, we performed a quantitative proteomics analysis of autophagy-deficient human chondrocytes using labeling iTRAQ (isobaric tags for relative and absolute quantitation) coupled with on-line 2D LC/MS/MS. Protein identification and quantification were performed using Protein Pilot Software v4.0 (ABSciex). Each MS/MS spectrum was searched in the Uniprot/Swissprot database for Homo sapiens. To confirm the candidate targets identified by the proteomic screening, inmortalized human chondrocytes (Tc28a2), human cartilage from healthy, aged and osteoarthritis human patients and mouse knee joints from young and old mice were employed to perform Western Blot and Histology analysis, respectively. The candidate targets were: Atg5 and LC3 for constitutive autophagy, p62, as a defective autophagy marker, Lamin A/C as an aging marker.ResultsFrom the total of 599 proteins found, 21 were significantly altered (p