Ubiquitin Ligase MDM2 Regulates Chondrocyte Damage, Ferroptosis, and Oxidative Stress by Modulating the Ubiquitination Level of CDKN1A.

BACKGROUND

Osteoarthritis (OA) is a common degenerative disease involving pathological changes in joint tissues, which seriously affects the quality of life of patients. It was reported that both Cyclin dependent kinase inhibitor 1A (CDKN1A) and ubiquitinating enzyme MDM2 exhibited abnormal expression in OA. However, it is currently unclear whether there is a specific regulatory mechanism between the two.

METHODS

Firstly, C28/I2 cells were treated with IL-1β to construct an in vitro cell model of OA, while qRT-PCR and western blot were used to detect the mRNA and protein levels of CDKN1A and MDM2. C28/I2 cell viability, apoptosis, and the release of inflammatory factors were measured by CCK-8, flow cytometry, and ELISA kits. In addition, the levels of Fe2+, glutathione (GSH), reactive oxygen species (ROS), and Malondialdehyde (MDA) were evaluated by corresponding kits. Subsequently, the relationship between MDM2 and CDKN1A was predicted by the UbiBrowser website, and the ubiquitination level of CDKN1A and the interaction between them were verified by western blot and Co-IP technology. Cycloheximide (CHX) exposure was used to assess mRNA stability.

RESULTS

CDKN1A was downregulated in OA cartilage tissues and IL-1β induced C28/I2 cells, while overexpression of CDKN1A enhanced C28/I2 cell activity and inhibited cell apoptosis. Meanwhile, the release of IL-6 and TNF-α as well as ferroptosis and oxidative stress, were also hindered by CDKN1A overexpression. MDM2 was highly expressed in OA patients and in IL-1β induced C28/I2 cells and mediated the ubiquitination modification of CDKN1A. Most importantly, MDM2 knockdown alleviated IL-1β-induced chondrocyte damage via upregulating CDKN1A.

CONCLUSION

MDM2 downregulated the level of CDKN1A in chondrocytes by mediating the ubiquitination modification of CDKN1A, leading to impaired chondrocyte viability and inducing ferroptosis and oxidative stress.