DDX5 Inhibits Hyaline Cartilage Fibrosis and Degradation in Osteoarthritis via Alternative Splicing and G-Quadruplex Unwinding

The Novel Mechanism of DDX5 in Inhibiting Hyaline Cartilage Fibrosis and Degradation in Osteoarthritis

Background

Osteoarthritis (OA) is a chronic degenerative disease characterized mainly by the degradation, degeneration, and osteophyte formation of joint cartilage. Hyaline cartilage fibrosis is often considered the end-stage lesion of OA, leading to changes in the extracellular matrix (ECM). However, the mechanisms underlying hyaline cartilage fibrosis are not yet fully understood. Recent single-cell sequencing studies have shown that fibrocartilage-like chondrocytes play a crucial role during the progression of OA. Previous studies have indicated that the loss of RNA helicase DEAD-box RNA helicase 5 (DDX5) leads to cartilage degradation and exacerbates the fibrous phenotype. However, the specific regulatory mechanisms need further research.

Research Source and Publication Information

The article titled “Ddx5 inhibits hyaline cartilage fibrosis and degradation in osteoarthritis via alternative splicing and G-quadruplex unwinding” was written by multiple authors, including Qianqian Liu, mainly from Nanjing University Medical School and other institutions. The paper was published on May 17, 2024, in the journal “Nature Aging.” The article’s online publication link is https://doi.org/10.1038/s43587-024-00624-0.

Research Procedure

Research Steps

The study comprises several major steps: 1. Discovery of the expression of the DDX5 gene in OA: - Using RNA sequencing analysis of human patient samples and animal models, it was found that DDX5 is significantly downregulated during the progression of OA. 2. Construction of DDX5 deficiency mouse models: - Utilizing DMM (destabilization of the medial meniscus) and naturally aging DDX5 deficiency mouse models, it was found that DDX5 deficiency exacerbates cartilage lesions. - Constructing conditional DDX5 knockout mice using inducible recombinase and observing changes in cartilage structure using SO&FG staining. 3. RNA-seq and proteomics analysis: - Performing RNA sequencing and quantitative proteomics analysis in DDX5 knockdown ATDC5 chondrocyte cell lines revealed upregulation of multiple related genes, especially those associated with fibrosis and inflammation. 4. Functional validation: - Examining the effects of DDX5 overexpression on the mouse DMM model, it was found that expressing DDX5 via adeno-associated virus (AAV2) significantly alleviates OA progression.

Experimental Methods and Techniques

Main experimental methods include: - RNA-seq: Used to screen for DDX5 related differential alternative splicing events. - Tandem mass tag (TMT) labeling-based quantitative proteomic analysis: Used to quantify protein expression levels. - RNA immunoprecipitation PCR (RIP-PCR): Confirming the direct interaction of DDX5 with target RNA. - 1H-NMR and CD spectroscopy: Studying the structure and unwinding mechanism of G-quadruplex (G4).

Main Findings

Discovery Area

Through a series of experiments, it was found that DDX5 is significantly downregulated during the progression of OA. Its deficiency leads to: - Upregulation of genes related to fibrosis (e.g., Col1, ACTA2); - Upregulation of cartilage-degrading enzymes (e.g., MMP13, NOS2); - Decreased production of Col2 due to the inability to unwind the G4 structure of the Col2 promoter.

Data Support

RNA-seq analysis revealed that DDX5 knockdown significantly upregulates genes related to inflammation and fibrosis, specifically weakening the production of Fn1 containing exon 25 and Plod2 containing exon 14, thus promoting the expression of fibrosis and degradation genes. NMR experiments showed that DDX5 could unwind the G4 structure in the promoter region of Col2, thereby promoting its expression.

Conclusion and Significance

This study is the first to reveal the dual inhibitory role of DDX5 in cartilage degradation and fibrosis, proposing that DDX5 maintains cartilage health through the regulation of Fn1 and Plod2 splicing and unwinding G4. This provides a new direction for OA treatment by potentially upregulating DDX5 to significantly delay the processes of cartilage fibrosis and degradation.

Research Highlights

• New Mechanism Discovery: For the first time, it was found that DDX5 inhibits cartilage fibrosis and degradation via G4 unwinding and alternative splicing pathways.
• Comprehensive Technical Application: Utilizing RNA-seq, proteomics, and NMR technology for an in-depth analysis of the function and mechanism of DDX5.
• Therapeutic Potential: Providing a new therapeutic target for OA, i.e., protecting cartilage and delaying disease progression by upregulating DDX5 expression.

Further Application of the Research

Future research could focus on: - Further exploring how to specifically regulate DDX5 expression; - Evaluating DDX5-related therapeutic strategies in other cartilage-related diseases; - Developing therapeutic drugs or gene therapy approaches based on DDX5 and its regulatory mechanisms.