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Role of GDF15/MAPK14 Axis in Chondrocyte Senescence as a Novel Senomorphic Agent in Osteoarthritis.

ABSTRACT: Osteoarthritis (OA) is most prevalent in older individuals and exerts a heavy social and economic burden. However, an effective and noninvasive approach to OA treatment is currently not available. Chondrocyte senescence has recently been proposed as a key pathogenic mechanism in the etiology of OA. Furthermore, senescent chondrocytes (SnCCs) can release various proinflammatory cytokines, proteolytic enzymes, and other substances known as the senescence-associated secretory phenotype (SASP), allowing them to connect with surrounding cells and induce senesce. Studies have shown that the pharmacological elimination of SnCCs slows the progression of OA and promotes regeneration. Growth differentiation factor 15 (GDF15), a member of the tumor growth factor (TGF) superfamily, has recently been identified as a possible aging biomarker and has been linked to a variety of clinical conditions, including coronary artery disease, diabetes, and multiple cancer types. Thus, we obtained data from a publicly available single-cell sequencing RNA database and observed that GDF15, a critical protein in cellular senescence, is highly expressed in early OA. In addition, GDF15 is implicated in the senescence and modulation of MAPK14 in OA. Tissue and synovial fluid samples obtained from OA patients showed overexpression of GDF15. Next, we treated C20A4 cell lines with interleukin (IL)-1β with or without shGDF15 then removed the conditioned medium, and cultured C20A4 and HUVEC cell lines with the aforementioned media. We observed that C20A4 cells treated with IL-1β exhibited increased GDF15 secretion and that chondrocytes cultured with media derived from IL-1β-treated C20A4 exhibited senescence. HUVEC cell migration and tube formation were enhanced after culturing with IL-1β-treated chondrocyte media; however, decreased HUVEC cell migration and tube formation were noted in HUVEC cells cultured with GDF15-loss media. We tested the potential of inhibiting GDF15 by using a GDF15 neutralizing antibody, GDF15-nAb. GDF15-nAb exerted a similar effect, resulting in the molecular silencing of GDF15 in vivo and in vitro. Our results reveal that GDF15 is a driver of SnCCs and can contribute to OA progression by inducing angiogenesis.

SUBMITTER: Weng PW

PROVIDER: S-EPMC9266723 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundOsteoarthritis (OA) is a chronic degenerative joint disease that primarily affects middle-aged and elderly individuals. The decline in chondrocyte function plays a crucial role in the development of OA. Inflammasome-mediated chondrocyte pyroptosis is implicated in matrix degradation and cartilage degeneration in OA patients. Guanylate binding protein 5 (GBP5), a member of the GTPase family induced by Interferon-γ (IFN-γ), significantly influences cellular inflammatory responses, including intracellular inflammasome activation and cytokine release. However, the role of GBP5 in chondrocyte pyroptosis and OA progression remains unclear.MethodsIn this study, we used tumor necrosis factor-α (TNF-α) to induce inflammation and created an OA mouse model with surgically-induced destabilization of the medial meniscus (DMM). We isolated and cultured primary chondrocytes from the knee joints of suckling C57 mice. TNF-α-stimulated primary chondrocytes served as an in vitro model for OA and underwent RNA sequencing. Chondrocytes were transfected with GBP5-overexpression plasmids and small interfering RNA and were subsequently treated with TNF-α. We assessed the expression of cartilage matrix components (COL2A1 and aggrecan), catabolic factors (MMP9 and MMP13), and NLRP3 inflammasome pathway genes (NLRP3, Caspase1, GSDMD, Pro-IL-1β, and Pro-Caspase1) using RT-qPCR and Western blotting. We analyzed the expression of GBP5, NLRP3, and Caspase1 in the cartilage of DMM-induced post-traumatic OA mice and human OA patients. Immunohistochemistry (IHC) was used to detect the expression of GBP5, NLRP3 and GSDMD in cartilage specimens from OA patients and mouse DMM models. Chondrocyte pyroptosis was assessed using flow cytometry, and the levels of interleukin-1β (IL-1β) and interleukin-18 (IL-18) were measured with ELISA. We conducted double luciferase reporter gene and chromatin immunoprecipitation (ChIP) assays to confirm the relationship between IRF1 and GBP5.ResultsGBP5 expression increased in TNF-α-induced chondrocytes, as revealed by RNA sequencing. GBP5 inhibited COL2A1 and aggrecan expression while promoting the expression of MMP9, MMP13, NLRP3, Caspase1, GSDMD, Pro-IL-1β, and Pro-Caspase1. GBP5 expression also increased in the cartilage of DMM-induced post-traumatic OA mice and human OA patients. Knockout of GBP5 reduced chondrocyte injury in OA mice. GBP5 promoted chondrocyte pyroptosis and the production of IL-1β and IL-18. Additionally, we found that IRF1 bound to the promoter region of GBP5, enhancing its expression. After co-transfected with ad-IRF1 and siGBP5, the expression of pyroptosis-related genes was significantly decreased compared with ad-IRF1 group.ConclusionsThe IRF1/GBP5 axis enhances extracellular matrix (ECM) degradation and promotes pyroptosis during OA development, through the NLRP3 inflammasome signaling pathway.The translational potential of this articleThis study underscores the significance of the IRF1/GBP5 axis in NLRP3 inflammasome-mediated chondrocyte pyroptosis and osteoarthritic chondrocyte injury. Modulating IRF1 and GBP5 expression could serve as a novel therapeutic target for OA.

Project description:Mechanical stress damage and insufficient self-repair can contribute to osteoarthritis (OA) in the affected joint. As the effects of stress on chondrocyte metabolism can regulate cartilage homeostasis, the specific stress-response condition is therefore a key to the generation of an OA disease model. We aimed to produce a specific stress- and cell-based OA model after evaluating the metabolic responses of chondrocytes in response to a series of static and cyclic compression stressors. A static load exceeding 40 psi initiated extracellular matrix (ECM) degradation through a decrease in the sulphated-glycosaminoglycan (GAG) content, upregulation of catabolic matrix metalloproteinase (MMP)-13 encoding gene expression, and downregulation of the ECM-related aggrecan and type II collagen encoding genes within 24?h. Indicators of pro-inflammatory events and oxidative stress were found to correlate with increased IL-6 expression and reactive oxygen species (ROS) production, respectively. However, chondrocytes stimulated by moderate cyclic loading (30-40 psi) exhibited increased ECM-related gene expression without significant changes in catabolic and pro-inflammatory gene expression. BMP-7 expression increased at cyclic loading levels above 30-60 psi. These results demonstrated that static compression exceeding 60 psi is sufficient to produce OA-like chondrocytes that exhibit signs of ECM degradation and inflammation. These OA-like chondrocytes could therefore be used as a novel cell-based drug screening system. Impact statement The lack of an effective treatment for osteoarthritis (OA) reflects the great need for alternative therapies and drug discovery. Disease models can be used for early-stage compound screening and disease studies. Chondrocytes are solely responsible for the maintenance of the articular cartilage extracellular matrix. Our strategy involved the generation of a cell-based model of OA, a more readily studied disease. Instead of using animal cartilage explants, we incorporated isolated porcine chondrocytes with hydrogel to form three-dimensional assemblies. We could identify the specific magnitude-dependent metabolic responses of chondrocytes by applying a series of static and cyclic compression, and therefore successfully generated a novel OA-like cell-based model for drug screening.

Project description:ObjectivesTo investigate the potential role of Ribosomal protein L35 (RPL35) in regulating chondrocyte catabolic metabolism and to examine whether osteoarthritis (OA) progression can be delayed by overexpressing RPL35 in a mouse compression loading model.MethodsRNA sequencing analysis was performed on chondrocytes treated with or without 20 % elongation strain loading for 24 h. Experimental OA in mice was induced by destabilization of the medial meniscus and compression loading. Mice were randomly assigned to a sham group, an intra-articular adenovirus-mediated overexpression of the negative group, and an intra-articular adenovirus-mediated overexpression of the RPL35 operated group. The Osteoarthritis Research Society International score was used to evaluate cartilage degeneration. Immunostaining and western blot analyses were conducted to detect relative protein levels. Primary mouse chondrocytes were treated with 20 % elongation strain loading for 24 h to investigate the role of RPL35 in modulating chondrocyte catabolic metabolism and regulating cellular senescence in chondrocytes.ResultsThe protein expression of RPL35 in mouse chondrocytes was significantly reduced when excessive mechanical loading was applied, while elevated protein levels of RPL35 protected articular chondrocytes from degeneration. In addition, the RPL35 knockdown alone induced chondrocyte senescence, decreased the expression of anabolic markers, and increased the expression of catabolic markers in vitro in part through the hedgehog (Hh) pathway.ConclusionsThese findings demonstrated a functional pathway important for OA development and identified intra-articular injection of RPL35 as a potential therapy for OA prevention and treatment.The translational potential of this articleIt is necessary to develop new targeted drugs for OA due to the limitations of conventional pharmacotherapy. Our study explores and demonstrates the protective effect of RPL35 against excessive mechanical stress in OA models in vivo and in vitro in animals. These findings might provide novel insights into OA pathogenesis and show its translational potential for OA therapy.

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Role of GDF15/MAPK14 Axis in Chondrocyte Senescence as a Novel Senomorphic Agent in Osteoarthritis.

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