Kruppel-Like Factor 5 Acetylation Upregulates Runx2 Expression and Accelerates the Dedifferentiation Process of Chondrocyte in Monolayer Culture

Abstract

This study aimed to investigate the role of KLF5 (Kruppel-like factor 5) in the abnormal dedifferentiation of chondrocytes (CHs) observed in osteoarthritis (OA) and monolayer culture conditions. Human primary CHs were cultured in a monolayer for 14 days, and various analyses were conducted. The area and aspect ratio of the cells were measured, and protein expression levels of KLF5, acetylated KLF5 (Ac-KLF5), and Runx2 were assessed using western blotting. Additionally, mRNA levels of chondrogenic genes (type II collagen, Col-II), hypertrophic genes (type X collagen, Col-X), and MMP-13 were determined through RT-PCR. The effects of TGF-β and Am80 supplementation on KLF5 acetylation and Runx2 expression were examined, and siRNA was used to silence Runx2 gene expression. Results showed that CHs exhibited dedifferentiation after 7 days of culture, characterized by increased cell size, larger aspect ratio, elevated Ac-KLF5 levels, decreased Col-II expression, and increased Col-X and MMP-13 expression. TGF-β treatment enhanced Ac-KLF5 and Runx2 expression, thereby accelerating dedifferentiation within 3 days. In contrast, Am80 suppressed Ac-KLF5 and Runx2 expression, leading to delayed dedifferentiation over 14 days. Silencing Runx2 mitigated KLF5 acetylation-induced CH dedifferentiation without affecting Ac-KLF5 levels. In conclusion, KLF5 acetylation promotes Runx2 expression and subsequently contributes to CH dedifferentiation during monolayer culture.