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

IF 2 4区医学 Q2 RHEUMATOLOGY

International Journal of Rheumatic Diseases Pub Date : 2025-08-16 DOI:10.1111/1756-185x.70384

Kaihong Gui, Xiaosong Li, Junlai Song, Ao Li, Jun Fan, Lin Huang

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引用次数: 0

Abstract

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.

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泛素连接酶MDM2通过调节CDKN1A的泛素化水平调节软骨细胞损伤、铁凋亡和氧化应激

骨关节炎（Osteoarthritis， OA）是一种常见的涉及关节组织病理改变的退行性疾病，严重影响患者的生活质量。据报道，细胞周期蛋白依赖性激酶抑制剂1A （CDKN1A）和泛素化酶MDM2在OA中均表现出异常表达。然而，目前尚不清楚两者之间是否存在特定的监管机制。方法首先用IL-1β处理C28/I2细胞，建立OA体外细胞模型，采用qRT-PCR和western blot检测CDKN1A和MDM2 mRNA和蛋白水平。采用CCK-8、流式细胞术和ELISA试剂盒检测C28/I2细胞活力、凋亡和炎症因子释放。此外，采用相应的试剂盒检测各组Fe2+、谷胱甘肽（GSH）、活性氧（ROS）和丙二醛（MDA）水平。随后，通过UbiBrowser网站预测MDM2和CDKN1A之间的关系，并通过western blot和Co-IP技术验证CDKN1A的泛素化水平及其相互作用。使用环己亚胺（CHX）暴露来评估mRNA的稳定性。结果CDKN1A在OA软骨组织和IL-1β诱导的C28/I2细胞中表达下调，而CDKN1A过表达可增强C28/I2细胞活性，抑制细胞凋亡。同时，IL-6和TNF-α的释放以及铁下垂和氧化应激也受到CDKN1A过表达的阻碍。MDM2在OA患者和IL-1β诱导的C28/I2细胞中高表达，并介导CDKN1A的泛素化修饰。最重要的是，MDM2敲低可通过上调CDKN1A来减轻il -1β诱导的软骨细胞损伤。结论MDM2通过介导CDKN1A泛素化修饰下调软骨细胞CDKN1A水平，导致软骨细胞活力受损，诱导铁凋亡和氧化应激。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

International Journal of Rheumatic Diseases RHEUMATOLOGY-

CiteScore

3.70

自引率

4.00%

发文量

362

审稿时长

1 months

期刊介绍： The International Journal of Rheumatic Diseases (formerly APLAR Journal of Rheumatology) is the official journal of the Asia Pacific League of Associations for Rheumatology. The Journal accepts original articles on clinical or experimental research pertinent to the rheumatic diseases, work on connective tissue diseases and other immune and allergic disorders. The acceptance criteria for all papers are the quality and originality of the research and its significance to our readership. Except where otherwise stated, manuscripts are peer reviewed by two anonymous reviewers and the Editor.