Excessive sulfur oxidation in endoplasmic reticulum drives an inflammatory reaction of chondrocytes in aging mice
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus Musculus
SUBMITTER: 
Kun Chen
PROVIDER: PXD037302 | iProX | Wed Oct 12 00:00:00 BST 2022
REPOSITORIES: iProX
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Project description:Background: Chondrocyte homeostasis is vital for maintaining the extracellular matrix (ECM) and overall cartilage health. In osteoarthritis (OA), oxidative stress, often caused by redox imbalances, can disrupt chondrocyte homeostasis, leading to cartilage degradation. Hydrogen peroxide (H2O2), a reactive oxygen species (ROS), is a key mediator of oxidative stress and contributes to chondrocyte apoptosis and ECM degradation. Previous studies have explored individual protein responses to oxidative stress; however, a comprehensive proteomic analysis in chondrocytes has not been conducted. In this study, we aimed to assess the global proteomic alterations in chondrocytes exposed to H2O2 using a shotgun proteomics approach, which enables the detection of a broader spectrum of proteomic changes. Methods: Chondrocytes were treated with H2O2 for 1, 4, and 16 h followed by protein extraction and processing, including denaturation, alkylation, and trypsin digestion. The peptides were then acidified, desalted, dried, and resuspended for LC-MS/MS. Proteomics data were analyzed using MaxQuant software to identify and quantify proteins. Secretome analysis was performed to examine protein secretion changes under oxidative stress. The statistical significance of all proteomics and secretome data was assessed using a two-tailed Student’s t-test in the Perseus software. Complementary methods, including quantitative PCR, western blotting, and immunofluorescence, were employed to validate the proteomic analysis. Results: Our findings revealed that oxidative stress primarily affected the endoplasmic reticulum (ER), causing notable alterations in the expression of ER-associated proteins, redox-responsive enzymes, chaperones, and sialyltransferases. These changes increased intracellular accumulation of ECM proteins and decreased secretion into the extracellular environment, indicating impaired protein trafficking and secretion. Additionally, immune-related pathways were activated in the long term, with a short-term upregulation of inflammatory markers, such as interleukin (IL)-6) and IL-18, although the levels of matrix metalloproteinases (MMPs) remained stable, indicating a complex inflammatory response and stress responses that disrupt ECM homeostasis. Conclusions: Our study demonstrates a detailed proteomic view of the stress response of H2O2-treated chondrocytes, highlighting the significant changes in ER function, cytoskeletal remodeling, protein secretion, and immune responses. These changes suggest that oxidative stress impacts ECM balance and contributes to OA progression, offering new insights into the molecular mechanisms underlying oxidative stress in OA.
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Project description:Osteoarthritis is a prevalent joint disease in the aging population. The hallmark of osteoarthritis is the degeneration of the joint cartilage, characterized by changes in chondrocytes including mitochondrial dysfunction. However, the precise mechanisms of how this affects chondrocyte homeostasis and whether such processes can be explored as therapeutic targets for osteoarthritis remain unclear. Here, we show that impaired mitochondrial function and disrupted cartilage matrix metabolism due to loss of mitofusin-2 (MFN2) expression in chondrocytes leads to the development of osteoarthritis. Sirtuin-3 (SIRT3), a key regulator of mitochondrial function, plays a critical role in modulating MFN2 to restore mitochondrial dynamics, reduce fragmentation, and preserve mitochondrial function in chondrocytes. Specifically, SIRT3 directly deacetylates and indirectly deubiquitinates MFN2, preventing its degradation. MFN2-mediated mitochondrial–endoplasmic reticulum (ER) junctions support cellular homeostasis, alleviate ER stress, and maintain mitochondrial calcium ion balance, which collectively mitigate chondrocyte senescence. Extracellular vesicles engineered with MFN2 mRNA effectively prevented cartilage degeneration and restored mobility in osteoarthritic mice. These findings suggest that targeting MFN2 is a promising strategy to prevent cartilage degeneration and alleviate progression of osteoarthritis.
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