KLF9, Epigenetic Silenced by DNMT1, Promotes ERK-Mediated Ferroptosis of Osteoarthritic Chondrocytes Through Transcriptionally Regulating CYP1B1

Abstract

Ferroptosis plays a crucial role in the pathogenesis of osteoarthritis (OA), and inhibition of chondrocyte ferroptosis effectively alleviates OA progression. Krüppel-like factor 9 (KLF9) is demonstrated to be upregulated in OA, but its molecular mechanism remains unclear. The study aimed to investigate the role of KLF9 in OA progression. Primary chondrocytes were treated with IL-1β to establish an OA cell model, and showed that KLF9 was highly expressed in IL-1β-incubated chondrocytes. Knockdown of KLF9 alleviated IL-1β-induced chondrocyte degeneration. In addition, chondrocytes treated with IL-1β showed a decreased methylation proportion in the KLF9 gene promoter. DNA methyltransferase 1 (DNMT1) directly bound to the KLF9 promoter, and overexpression of DNMT1 inhibited KLF9 expression by promoting its promoter methylation in chondrocytes. Subsequently, KLF9 shRNA and pcDNA-CYP1B1 were individually or altogether transfected into chondrocytes. KLF9 shRNA inhibited Cytochrome P450 1B1 (CYP1B1) expression in chondrocytes, and pcDNA-CYP1B1 abrogated the inhibitory effect of KLF9 shRNA on IL-1β-induced chondrocyte ferroptosis. Moreover, Ferrostatin-1 (Fer-1, an inhibitor of ferroptosis) reversed the promotion of pcDNA-CYP1B1 on IL-1β-induced chondrocyte ferroptosis. Finally, in vivo experiments showed that KLF9 shRNA significantly suppressed the cartilage tissue damage, ferroptosis, and the IHC scores of KLF9 and CYP1B1 in rats. In conclusion, our results suggested that KLF9, epigenetic silenced by DNMT1, promoted extracellular signal-regulated kinase (ERK)-mediated ferroptosis of OA chondrocytes through transcriptionally regulating CYP1B1. Thus, KLF9 is expected to be a new target for the treatment of OA.