Talatibaike Maimaitijuma1, Jia-Hong Yu2, Ya-Li Ren3, Xin Yang1, Heng Liu1, Zhi-Chao Meng1, Rui Wang1, Yun-Peng Cui1, Hao Wu1, Li-Ping Pan1, Yang Jiao1, Ying-Yu Chen2, Yong-Ping Cao4. 1. Orthopedic Department, Peking University First Hospital, Beijing, China. 2. Key Laboratory of Medical Immunology, Ministry of Health, Peking University Health Science Center, Beijing, China. 3. Department of Electron Microscopy, Peking University First Hospital, Beijing, China. 4. Orthopedic Department, Peking University First Hospital, Beijing, China. Electronic address: freehorse66@163.com.
Abstract
AIM: Osteoarthritis (OA) is the main cause of disability and joint replacement surgery in the elderly. As a crucial cell survival mechanism, autophagy has been reported to decrease in OA. PHF23 is a new autophagy inhibitor which was first reported by us previously. This study aimed to explore the anti-autophagic mechanism of PHF23 to make it a possible therapeutic target of OA. MAIN METHOD: Lentiviral vectors specific to PHF23 were used on chondrocytes (C28/I2) to establish PHF23 overexpressed or knockdown stable cell strains. Interleukin (IL)-1β (10 ng/mL) and chloroquine (CQ, 25 uM) were used as an inducer of OA and inhibitor of lysosome, respectively. Autophagy was evaluated by autophagosome formation using transmission electron microscopy (TEM) and western blot analysis of P62 and LC3B on different groups of cells. Effects of PHF23 on OA were evaluated by collagen II immunofluorescent staining and western blot analysis of OA-associated proteins MMP13 and ADAMTS5. Effects of PHF23 on AMPK and mTOR/S6K pathways and mitophagy were determined by western blot analysis. KEY FINDINGS: Knockdown of PHF23 enhanced IL-1β-induced autophagy, while overexpression of PHF23 exerted the opposite effect. Knockdown of PHF23 protected chondrocytes against IL-1β-induced OA by decreasing the levels of OA-associated proteins and increasing expression of Collagen II. Knockdown of PHF23 also increased mitophagy level and altered the phosphorylation levels of AMPK, mTOR, and S6K. SIGNIFICANCE: PHF23 downregulates autophagy, mitophagy in IL-1β-induced OA-like chondrocytes and alters the activities of AMPK and mTOR/S6K, which suggests that PHF23 may be a possible therapeutic target for OA.
AIM: Osteoarthritis (OA) is the main cause of disability and joint replacement surgery in the elderly. As a crucial cell survival mechanism, autophagy has been reported to decrease in OA. PHF23 is a new autophagy inhibitor which was first reported by us previously. This study aimed to explore the anti-autophagic mechanism of PHF23 to make it a possible therapeutic target of OA. MAIN METHOD: Lentiviral vectors specific to PHF23 were used on chondrocytes (C28/I2) to establish PHF23 overexpressed or knockdown stable cell strains. Interleukin (IL)-1β (10 ng/mL) and chloroquine (CQ, 25 uM) were used as an inducer of OA and inhibitor of lysosome, respectively. Autophagy was evaluated by autophagosome formation using transmission electron microscopy (TEM) and western blot analysis of P62 and LC3B on different groups of cells. Effects of PHF23 on OA were evaluated by collagen II immunofluorescent staining and western blot analysis of OA-associated proteins MMP13 and ADAMTS5. Effects of PHF23 on AMPK and mTOR/S6K pathways and mitophagy were determined by western blot analysis. KEY FINDINGS: Knockdown of PHF23 enhanced IL-1β-induced autophagy, while overexpression of PHF23 exerted the opposite effect. Knockdown of PHF23 protected chondrocytes against IL-1β-induced OA by decreasing the levels of OA-associated proteins and increasing expression of Collagen II. Knockdown of PHF23 also increased mitophagy level and altered the phosphorylation levels of AMPK, mTOR, and S6K. SIGNIFICANCE: PHF23 downregulates autophagy, mitophagy in IL-1β-induced OA-like chondrocytes and alters the activities of AMPK and mTOR/S6K, which suggests that PHF23 may be a possible therapeutic target for OA.