Ubiquitin‐Specific Peptidase 16 Alleviates Chondrocyte Damage in Osteoarthritis Through Deubiquitinating and Stabilizing POU Class 2 Homeobox 1

Osteoarthritis (OA), marked by articular cartilage degeneration, severely impairs joint function. Ubiquitin-specific peptidase 16 (USP16) is crucial for regulating cellular protein stability and signaling pathways. This study seeks to elucidate USP16's impact and mechanism on chondrocyte injury in OA. The expression of USP16 in OA was analyzed using the Gene Expression Omnibus (GEO) database. An in vitro model using IL-1β to induce OA-like conditions. Real-time quantitative PCR (RT-qPCR) and western blot were used to detect gene expression in tissues and cells. The cellular oxidative stress levels were assessed using cell metabolism kits specifically targeted at reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD). Enzyme-linked immunosorbent (ELISA) assays were conducted to detect the levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in cell culture supernatants and serum, thereby evaluating the inflammatory response state. The apoptotic status of the cells was detected by flow cytometry. The Ubibrowser website was used to predict proteins that interact with USP16. Co-immunoprecipitation (CoIP) and GST pull-down experiments were conducted to validate the interaction between USP16 and POU class 2 homeobox 1 (POU2F1). The cycloheximide (CHX) chase assay was utilized to determine the half-life of proteins. Mouse models of OA were established for in vivo validation, and the degree of cartilage degeneration was assessed through hematoxylin-eosin (HE) staining. USP16 expression was found to be reduced in OA patients and in HACCs induced by IL-1β. The induction of IL-1β led to an increase in oxidative stress levels in human articular chondrocyte cells (HACCs), accelerated the inflammatory response and the rate of apoptosis within HACCs, and inhibited the normal expression of chondrogenesis-related proteins in HACCs. However, overexpressing USP16 was able to alleviate these adverse effects. Furthermore, USP16 interacted with POU2F1 and stabilized its expression by means of deubiquitination. Notably, when POU2F1 was knocked down, the protective effect of overexpressing USP16 on IL-1β-induced HACCs was significantly weakened. In vivo experiments demonstrated that overexpressing USP16 effectively inhibited the degenerative process of mouse cartilage, significantly lowered the Osteoarthritis Research Society International (OARSI) and Mankin scores in mice, decreased oxidative stress levels, and suppressed inflammatory responses. USP16 regulates chondrocyte damage in OA by exerting its deubiquitination role to stabilize the POU2F1 protein, providing a potential target and theoretical foundation for OA treatment.