BMI1 activated by ZBTB17 stabilizes SMAD2 to promote chondrocyte anabolism and alleviate osteoarthritis

INTRODUCTION: Osteoarthritis (OA) is the most prevalent degenerative joint disorder worldwide, characterized by impaired anabolic activity and excessive catabolic responses in chondrocytes. However, the precise mechanisms governing chondrocyte homeostasis during OA progression remain unclear. OBJECTIVES: This study aimed to elucidate the role of polycomb protein BMI1 in maintaining chondrocyte homeostasis, identify its upstream transcriptional regulator and downstream signaling cascade, and evaluate its therapeutic potential in OA. METHODS: BMI1 expression was assessed in articular cartilage from OA patients and destabilization of the medial meniscus (DMM) mouse models. Transcriptional regulation of BMI1 by zinc finger and BTB domain-containing protein 17 (ZBTB17) was analyzed using western blotting, chromatin immunoprecipitation and dual-luciferase reporter assays. In vivo effects of BMI1 were evaluated by intra-articular injection of rAAV6-Bmi1. In vitro BMI1 overexpression and inhibition were achieved using Bmi1-overexpressing plasmid and PTC209, respectively. The BMI1-SMAD2 interaction was investigated using nuclear-cytoplasmic fractionation, co-immunoprecipitation, ubiquitination assays, cycloheximide chase assays, and immunofluorescence. Therapeutic efficacy of BMI1 was evaluated using a three-dimensional hydrogel-based delivery system in DMM mice. RESULTS: BMI1 expression was significantly downregulated in human and murine OA cartilage and negatively correlated with disease severity. ZBTB17 directly activated BMI1 transcription by binding to its promoter. BMI1 overexpression alleviated OA progression by enhancing chondrocyte anabolism and antioxidant capacity through upregulating SMAD2 and promoting its nuclear localization. Mechanistically, BMI1 interacted with SMAD2 to increase its stability by inhibiting SMURF2-mediated K48-linked ubiquitination while facilitating K63-linked ubiquitination at Lys157 to facilitate SMAD2 nuclear translocation. DKK1 was identified as a principal downstream target of the BMI1-SMAD2 axis. Finally, we confirmed that local delivery of BMI1 mdified stromal cells via a three-dimensional hydrogel significantly attenuated cartilage degeneration in vivo. CONCLUSION: Our findings identify a novel ZBTB17-BMI1-SMAD2-DKK1 axis essential for chondrocyte homeostasis and highlight BMI1 as a promising therapeutic target for osteoarthritis.