RhoA deficiency in chondrocyte inhibits cartilage fibrosis and ameliorates osteoarthritis progression via SOX4/MMP2 axis

Background Cartilage fibrosis plays a critical role in the onset and progression of osteoarthritis (OA), and although RhoA is a well-known small GTPase that regulates cytoskeletal reorganization, its role in OA progression remains inadequately explored. Methods In this study, we first screened public scRNA-seq datasets for genes enriched in fibrocartilage chondrocytes and found that RHOA is significantly upregulated in fibrocartilage chondrocytes within OA cartilage obtained from patients undergoing total knee arthroplasty. Then, we induced post-traumatic OA in 8-week-old male C57BL/6J mice by destabilization of the medial meniscus and generated chondrocyte-specific Rhoa deletion using Col2a1 -CreERT- Rhoa -flox/flox mice. After that, cartilage damage was graded by Safranin-O/Fast Green, Toluidine blue, and Micro-CT, and molecular changes were validated by Immunofluorescence and Western blot, leading to the identification—by integrated single-cell and bulk RNA-seq—of a β-catenin/SOX4/MMP2 axis downstream of RhoA. Finally, we injected AAV- Sox4 or AAV- Mmp2 intra-articularly to rescue the loss of RhoA function. Results The results showed that conditional knockout of Rhoa in chondrocytes resulted in a marked reduction in cartilage fibrosis and a concurrent decrease in extracellular matrix degradation in OA mice. Mechanistically, the integrated single-cell and bulk tissue transcriptomic analyses indicated that RhoA promotes the chondrocyte transition to a fibrotic phenotype through the novel β-catenin/SOX4/MMP2 pathway, while notably, intra-articular delivery of adeno-associated viral vectors overexpressing Sox4 or Mmp2 reversed the phenotypes of Rhoa -deficient mice. Conclusion These findings position RHOA as a central regulator of chondrocyte fibrosis and as a promising therapeutic target for OA treatment. The translational potential of this article These findings highlight that RhoA may represent a therapeutic target for ameliorating cartilage fibrosis and the OA process via targeted gene intervention.